The mechanism of microsomal and mitochondrial nitroreductase. Electron spin resonance evidence for nitroaromatic free radical intermediates. Electron spin resonance spectra are observed during the enzymatic reduction of many nitrophenyl derivatives by rat hepatic microsomes or mitochondria. The spectra indicate that nitroaromatic anion radicals are present and are freely rotating in aqueous solution at a steady-state concentration of 0.1-6 muM. The rate of formation of p-nitrobenzoate (NBZO) dianion radical in microsomal incubates is consistent with the radical being an obligate intermediate in the reduction of NBZO to p-aminobenzoic acid. A model system consisting of NBZO, NADPH, and FMN, but no heme-containing compounds, also reduced NBZO to the NBZO dianion free radical. The steady-state concentration of the anion radicals in microsomal systems is not altered by CO. This observation, together with the results from the model system, suggests that the formation of nitroaromatic anion radicals is mediated through a flavine and not cytochrome P-450. The oxidation of the anion radical intermediate by O2 to the parent nitro compound is proposed to account for the well-known O2 inhibition of microsomal nitroreductase.


Iodohexestrols. II. Characterization of the binding and estrogenic activity of iodinated hexestrol derivatives, in vitro and in vivo. The affinity of ortho-iodinated hexestrols for the estrogen binding protein from rat uterus, determined by competitive binding assay, decreases with progressive iodine substitution; 3-iodohexestrol (I-Hex) has a binding affinity 42% that of estradiol. Analysis of [3-H]-I-Hex binding in rat uterine cytosol by sucrose density gradient centrifugation shows both an estrogen-specific binding component (8 S) and a more abundant component (4 S) that is not estrogen specific. Scatchard analysis indicates that this latter binding is of high affinity (Kd equals to 3.7-8.3 times 10- minus-9 M) but is not uterine specific. Polyacrylamide gel electrophoresis shows that most of the [3-H]-I-Hex binding activity in serum and uterine cytosol is distinct from and anodic to the principal protein component (albumin), and that is comigrates with [14-C]thyroxine binding activity. In in vitro incubation of rat uteri, I-Hex can block the specific uptake of [3-H]estradiol into the nuclear fraction; it itself causes a translocation of estrogen-specific binding capacity (as measured by exchange) from cytoplasm to nuclei, and can induce the synthesis of an estrogen-specific uterine protein, all under conditions where it is not metabolically deiodinated to hexestrol. The uterotrophic activities of the iodohexestrols are in most cases comparable to that expected on the basis of their competitive binding affinities. However, selective, estrogen-specific uptake of [3-H]-I-Hex into rat uterus, either in vitro or in vivo, cannot be demonstrated.


Steroidal 21-diazo ketones: photogenerated corticosteroid receptor labels. The 21-diazo derivatives of 9 alpha-fluoro- and 9 alpha-bromo-21 deoxycorticosterone, 21-deoxycorticosterone, and progesterone were synthesized for use as photoaffinity labels for corticosteroid receptors. In the isolated toad bladder system, 9 alpha-bromo-21-diazo-21-deoxycorticosterone was as active as d-aldosterone and more active than 9 alpha-fluoro-cortisol in augmenting active Na+ transport. The activities of 21-diazoprogesterone and progesterone were equal; both were much less potent than d-aldosterone, however. These results indicate that the 21-diazo derivatives had significant functional activity in the toad bladder system. The rat kidney slice system was used to estimate the relative affinities of the diazo steroids for aldosterone receptor sites by competition experiments. At 100-fold excess of competitor to [3-H]aldosterone, the order of affinities was 9 alpha-fluoro-21-diazo-21-deoxycorticosterone greater than 9 alpha-bromo-21-diazo-21-deoxycorticosterone greater than 21-diazoprogesterone. Moreover, 9 alpha-bromo-21-diazo-21-deoxycorticosterone reduced binding of [3-H]aldosterone to cytoplasmic and nuclear forms of the receptor proportionately. On the basis of competition for [3-H]corticosterone binding, presumably to corticosteroid-binding globulin (CBG), the order of affinities was 21-diazo-21-deoxycorticosterone greater than 21-diazoprogesterone greater than 9 alpha-bromo-21-diazo-21-deoxycorticosterone. These findings indicate that 21-diazo steroids may be suitable as photogenerated affinity labels for mineralocorticoid receptors. The tritiated derivative, [1,2-3-H]-9 alpha-bromo-21-diazo-21-deoxycorticosterone (specific activity 25 Ci/mol) was synthesized and used in model experiments on photogenerated covalent binding to rat plasma proteins. Irradiation with uv light resulted in binding of [1,2-3-H]-9 alpha-bromo-21-diazo-21-deoxycorticosterone to plasma proteins, that was resistant to extraction with methylene dichloride and did not exchange with unlabeled corticosterone. The diazocorticosteroids, therefore, may have the requisite functional and selectivity properties for photoaffinity labeling of corticosteroid-binding proteins. Further studies are needed, however, to assure that photogenerated labeling with these steroids was site specific.


Physicochemical characterization of Rhesus low density lipoproteins. The serum low density lipoprotein (LDL; p 1.019-1.050 g/ml) of the normal Macaca mulatta monkey (rhesus), kept on a low-fat Purina primate chow diet, was isolated by ultracentrifugal flotation, and its physicochemical properties were compared with those previously reported for human LDL. Rhesus LDL was found to be chemically similar to human LDL. The values for the sedimentation (S25, w-O) and diffusion (D25,w-O) coefficients were 7.09 S and 2.50 times 10- minus-7 cm-2 sec- minus-1, respectively. The intrinsic viscosity was 3.40 ml g- minus-1. The partial specific volume of rhesus LDL, determined in an Anton Paar precision density meter, was 0.960 ml g- minus-1. Molecular weights, calculated from a combination of S-O and D-O and of S-O and [n], were in agreement with the weight-average molecular weight, Mw, of 2.29 times 10-6 obtained by high-speed sedimentation equilibrium. In addition, a Z-average molecular weight, Mz, of 2.73 times 10-6 was calculated because curvature in the graphs of log c vs. r-2 indicated that rhesus LDL was heterogeneous. From the frictional ratio of 1.02, a maximum hydration of 0.1 g of H2O/g of lipoprotein was obtained. On electron micrographs, rhesus LDL appeared spherical with a mean diameter of 196 A, which was substantiated by hydrodynamic analysis.


The specificity of different classes of ethylating agents toward various sites of HeLa cell DNA in vitro and in vivo. The sites and extent of ethyl products of neutral ethylation of HeLa cell DNA by [14-C]diethyl sulfate, [14-C]ethyl methanesulfonate, and [14-C]ethylnitrosourea have been determined in vitro and in vivo, and found to differ significantly depending on the ethylating agents. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of HeLa cell DNA in the following order: 7-ethylguanine greater than 3-ethyladenine greater than 1-ethyladenine, 7-ethyladenine greater than 3-ethylguanine, 3-ethylcytosine, O-6-ethylguanine. Ethyl bases accounted for 84-87% of the total ethyl groups associated with HeLa cell DNA. Ethylnitrosourea, in contrast, has particular affinity for the O-6 position of guanine. It ethylates the bases of HeLa cell DNA in the following order: O-6-ethylguanine, 7-ethylguanine greater than 3-ethyladenine greater than 3-ethylguanine, 3-ethylthymine greater than 1-ethyladenine, 7-ethyladenine, 3-ethylcytosine. Ethylation of the bases only accounts for 30% of the total ethylation in the case of ethylnitrosourea. The remaining 70% of the [14-C]ethyl groups, introduced in vivo and in vitro, are in the form of phosphotriesters which after perchloric acid hydrolysis are found as [14-CA1ethanol and [14-C]ethyl phosphate. In contrast, phosphotriesters amounted to only 8-20% of total ethylation in in vivo or in vitro diethyl sulfate and ethyl methanesulfonate treated HeLa cell DNA, and 25% of the total methylation in in vitro methylnitrosourea treated HeLa cell DNA. Alkylation at the N-7 and N-3 positions of purines in DNA destabilizes the glycosidic linkages. Part of 7-ethylguanine and 3-ethyladenine are found to be spontaneously released during the ethylation reaction. Incorporation of the 14-C of the alkylating agents into normal DNA bases of HeLa cells can be eliminated by performing the alkylations, in the presence of cytosine arabinoside, for 1 hr.


Different cyclic adenosine 3',5'-monophosphate requirements for induction of beta-galactosidase and tryptophanase. Effect of osmotic pressure on intracellular cyclic adenosine 3,5-monophosphate concentrations. In this study we have tried to answer the following questions: (1) is it possible for different catabolite-repressible genes, although submitted to the same control, to be expressed selectively depending upon the growth conditions, and (2) what is the effect of increasing the osmolarity of the medium on the intracellular level of cAMP? Two conditions were found to cause a continuous variation of intracellular cAMP levels during growth. With different strains, higher cAMP levels are required for induction of the tryptophanase gene than one required for induction of the lactose operon. cAMP has been provided externally in adenyl cyclase minus cells of a mutant that has been made permeable by EDTA treatment. Although external cAMP concentrations, 10 times higher than the usual intracellular levels, are required for induction of beta-galactosidase and tryptophanase, the difference of requirements of cAMP is maintained. An increase in the osmolarity of the medium by sucrose addition causes a fourfold decrease in the intracellular cAMP level. As a consequence this prevents the induction of tryptophanase whereas beta-galactosidase is still inducible. After pulse induction, a difference in the kinetics of expression of the tryptophanase and beta-galactosidase genes was found. Its relationship with the previous results is discussed.


The role of superoxide radical in the autoxidation of cytochrome c. The net rate of autoxidation of ferrocytochrome c was decreased by ferricytochrome c. Superoxide dismutase accelerated this autoxidation to a limit and overcame the inhibitory effect of ferricytochrome c. This was the case whether the autoxidationwas observed in the presence or in the absence of denaturants, such as alcohols orurea, and whether the superoxide dismutase used was the Cu-2+-Zn-2+ enzyme from bovine erythrocytes or the Mn-3+-enzyme from Escherichia coli. It can be deduced that the autoxidation of ferrocytochrome c, under a variety of conditions, geenerates O2 minus which can then dismute to H202 + O2 or can reduce ferricytochrome c back to ferrocytochrome c. Superoxide dismutase, by accelerating the dismutation of O2 minus, prevents the back reaction and thus exposes the true rate of reaction of ferrocytochrome c with molecular oxygen.


31-P nuclear magnetic resonance studies on serum low and high density lipoproteins: effect of paramagnetic ion. A paramagnetic quenching reagent, Mn-2+/EDTA (1:2.2), was developed for the purpose of investigating the phospholipid phosphate groupings of human serum low and high density lipoproteins through the quenching effect of the reagent on the 31-P nuclear magnetic resonance signals from these complexes. Systems investigated included native low and high density serum liproteins (LDL, HDL2, and HDL3), egg phosphatidylcholine vesicles together with appropriate phosphodiester model systems, diethyl phosphate in aqueous buffer, and phosphatidylcholine and sphingomyelin both in anhydrous methanol. The results of these studies indicated that ca. 50 percent of the phospholipid-phosphorus signal of LDL is quenched upon titration as compared to an 80-85 percent figure observed for HDL2 and HDL3. In all cases the spectral effects were totally reversible upon removalof the paramagnetic ion by dialysis. The results of the titration studies indicated a similar but not an identical behavior between HDL2 and HDL3. The results are consistent with model structures of HDL and LDL particles derived from low angle X-ray diffraction.


2'-O-methylated oligonucleotides in ribosomal 18S and 28S RNA of a mouse hepatoma, MH 134. Simple two-dimensional thin-layer chromatography was found to be useful for the separation of sugar methylated dinucleotides in RNA. Of the 16 possible sequences of the type Nm-Np, 15 were separated and all the sequences were determined. In a mouse hepatoma, MH 134, the levels of the sugar methylation in the 18S and 28S RNA molecules were 17-18 and 11-12 per 1000 nucleotides, respectively. Thus, 18s RNA contained approximately 35 2'-O-methylated dinucleotides and 28S RNA approximately 60 2'-O-methylated dinucleotides. The pattern of distribution was also distinct between these two molecules. Two 2'-O-methylated trinucleotides were identified in the 28S RNA with the sequences Um-Gm-Up and Um-Gm-psip. A unique 2'-O-methylated tetranucleotide was present also in the 28S RNA, the sequence of which was Am-Gm-Cm-Ap. The 5'-terminal nucleotides of both 18S and 28S RNA were obtained as nucleoside 3',5'-diphosphates (pNp) in the trinucleotide fraction of the RNase T2 digest. The 5'-termimi of 18S and 28S RNA were pUp and pCp, respectively, and found to be almost homogeneous.


Nuclear magnetic resonance studies of substrate interaction with cobalt substituted alcohol dehydrogenase from liver. The role of zinc in liver alcohol dehydrogenase has been studied by replacement of 1.3 and 3.5 of the four Zn(II) ions with Co(II) and measuring the effects of the paramagnetic Co(II) on the relaxation rates of the protons of water, ethanol, and isobutyramide. Water relaxation studies at 8, 24, 100, and 220 MHz indicate two classes of bound Co(II). The similar to 2 readily replaced Co(II) ions retain one fast exchanging water proton in their inner coordination spheres, while the similar to 2 slowly exchanging Co(II) ions coordinate no detectable water protons, indicating that the former replaced Zn(II) at the "catalytic sites" and the latter replaced Zn(II) at the "structural sites" detected crystallographically. Ethanol, acetaldehyde, and isobutyramide bind with appropriate affinities to the Co(II) substituted alcohol dehydrogenases decreasing the number of fast exchanging protons at the catalytic Co(II) site by greater than or equal to 54 percent. Coenzyme binding causes smaller changes in the water relaxation rate which may be due to local conformation changes. The paramagnetic effects of Co(II) at the catalytic site on the relaxation rates of the methyl protons of isobutyramide at 100 and 220 MHz indicate that this analog binds at a site 9.1 A from the catalytic Co(II). This distance decreases to 6.9 A when NADH is bound, and a Co(II) to methyne proton distance of 6.6 A is determined indicating a conformation change leading to the formation of a second sphere enzyme-Co(II)-isobutyramide complex in which a hydroxyl or water ligand intervenes between the metal and the substrate analog. Similar behavior is observed in the enzyme-ethanol complexes. The paramagnetic effects of Co(II), at the catalytic site, on the relaxation rates of the protons of ethanol at 100 and 220 MHz, indicate that this substrate bind at a site 12-14 A distant from the catalytic Co(II) but that this distancedecreases to 6.3 A in the abortive enzyme-NADH-ethanol complex. The role of the catalytic Co(II) thus appears to be the activation of a hydroxyl or water ligand which polarizes the aldehyde carbonyl group by hydrogen bonding. The role of the structural Co(II), which is more distant from isobutyramide (9-11 A), may be that of a template for protein conformation changes. By combining the present distances with those from previous magnetic resonance studies on the liver enzyme, the arrangement of coenzyme, metal, and substrate at the active site in solution can be constructed. This arrangement is consistent with that of ADP-ribose and zinc in the crystalline complex of liver alcohol dehydrogenase as determined by X-ray crystallography (Branden et al., (1973), Proc. Natl. Acad. Sci. U.S.A.70, 2439).


Flexibility of coupling and stoichiometry of ATP formation in intact chloroplasts. Since coupling between phosphorylation and electron transport cannot be measured directly in intact chloroplasts capable of high rates of photosynthesis, attempts were made to determine ATP/2 e ratios from the quamdum requirements of glycerate and phosphoglycerate reduction and from the extent of oxidation of added NADH via the malate shuttle during reduction of phosphoglycerate in light. These different approaches gave similar results. The quantum requirement of glycerate reduction, which needs 2 molecules of ATP per molecule of NADPH oxidized was found to be pH-dependent. 9-11 quanta were required at pH 7.6, and only about 6 at pH 7.0. The quantum requirement of phosphoglycerate reduction, which consumes ATP and NADPH in a 1/1 ratio, was about 4 both at pH 7.6 ant at 7.0. ATP/2 e ratios calculated from the quantum requirements and the extent of phosphoglycerate accumulation during glycerate reduction were usually between 1.2 and 1.4, occasionally higher, but they never approached 2. Although the chloroplast envelope is impermeable to pyridine nucleotides, illuminated chlrooplasts reduced added NAD via the malate shuttle in the absence of electron acceptors and also during the reduction of glycerate or CO2. When phosphoglycerate was added as the substrate, reduction of pyridine-nucleotides was replaced by oxidation and hydrogen was shuttled into the chloroplasts to be used for phosphoglycerate reduction even under light which was rate-limiting for reduction. This indicated formation of more ATP than NADPH by the electron transport chain. From the rates of oxidation of external NADH and of phosphoglycerate reduction at very low light intensities ATP/2e ratios were calculated to be between 1.1 and 1.4. Fully coupled chloroplasts reduced oxaloacetate in the light at rates reaching 80 and in some instances 130 mumoles times mg-1 chlorophyll times h-1 even though ATP is not consumed in this reaction. The energy transfer inhibitor phlorizin did not significantly suppress this reduction at concentrations which completely inhibited photosynthesis. Uncouplers stimulated oxaloacetate reduction by factors ranging from 1.5 to more than 10. Chloroplasts showing little uncoupler-induced stimulation of oxaloacetate reduction were highly active in photoreducing CO2. Measurements of light intensity dependence of quantum requirements for oxaloacetate reduction gave no indication for the existence of uncoupled or basal electron flow in intact chloroplasts. Rather reduction is brought about by loosely coupled electron transport. It is concluded that coupling of phosphorylation to electron transport in intact chloroplasts is flexible, not tight. Calculated ATP/2e ratios were obtained under con a decreENG


The iron electron-nuclear double resonance (ENDOR) of 4-Fe clusters in iron-sulfur proteins from Chromatium and Clostridium pasteurianum. Iron electron-nuclear double resonance (ENDOR) measurements were made of the 4-Fe clusters in oxidized Chromatium high-potential iron-sulfur protein, dithionite-reduced high-potential iron-sulfur protein in 80% dimethylsulphoxide, fully reduced Clostridium pasteurianum ferredoxin in aqueous solution and in 80% dimethylsulfoxide. The hyperfine couplings determined show that: i) the electron distribution in each case is nearly symmetric; ii) there are two types of iron in oxidized high potential iron-sulfur protein; iii) only one type of iron is observed in each fully reduced 4-Fe cluster; iv) the data also suggest a greater electron delocalization onto the ligands as compared to the 2-Fe ferredoxins.


Studies on the active transfer of reducing equivalents into mitochondria via the malate-aspartate shuttle. 1. The effects of mitochondrial energy states onthe extramitochondrial NADH/NAD ratio via a reconstituted malate-aspartate shuttle have been investigated. 2. The transfer of reducing equivalents into isolated mitochondria is stimulated by ATP and by electron transport. The effect of ATP is inhibited by oligomycin. The effect of electron transport is inhibited by uncouplers. 3. Uncoupling of the mitochondria is required for rapid transfer of reducing equivalents out of the mitochondria. 4. A glutamate-stimulated entry of aspartate into energized mitochondria suggests that the malate-aspartate shuttle is to some extent reversible even in a high energy state of the mitochondria. 5. It is concluded that the malate-aspartate shuttle contributes to the formation of the skewed redox situation across the inner mitochondrial membrane, which has a more reduced inside.


Mitochondria from human term placenta. I. Isolation and assay conditions for oxidative phosphorylation. Human term placental mitochondria were resolved by differential centrifugation into three fractions, heavy mitochondria, light mitochondria and a third, less dense fraction. Approximately equal amounts of mitochondrial protein were found in the three fractions. These mitochondrial preparations differed in physical properties. ATPase and "ADPase" content and oxidative capacities. Assay conditions were developed which permitted the polarographic measurement of respiration and coupled phosphorylation carried out by all three mitocondrial preparations despite the variable nucleotide-phosphate phosphatase activities present. With heavy mitochondria, rates of respiration were consistently higher than those previously reported for unfractionated placental mitochondria. Respiratory control ratios were comparable to those of mitochondria from other steroid hormone-producing endocrine tissues and ADP/O ratios approaching the theoretical maxima were obtained. Both lighter placental mitochondrial fractions displayed somewhat lower respiration rates and respiratory control but their primary defect was a selective uncoupling of the third site of energy conservation. Modification of isolation procedures were evaluated in terms of quantitative yield and functional activity of the three fractions.


Multiple molecular forms of phosphoprotein phosphatase. III. Phosphorylase phosphatase and phosphohistone phosphatase of rabbit liver. 1. Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) in the soluble fraction of rabbit liver which catalyzes the dephosphorylation of muscle phosphorylase a and phosphohistone (P-histone) was resolved into three active fractions by NaCl gradient elution from a DEAE-cellulose column (Fraction I, 11 and III in order of elution). They have different relative reaction rates for the two substrates and different degrees of stimulation by Mn-2+. Apparent Km values of Fraction I, II and III were 15, 20 and 16 muM for phosphorylase a, and 6.9, 5.3 and 4.4 muM for P-histone, respectively (with Mn-2+ in the assay mixture). 2. On sucrose density gradient centrifugation Fraction I and II were revealed to contain a major peak (7.0 S and 7.8 S, respectively) and a minor peak (4.0 S) of activity, while Fraction III contained only one peak (5.8 S). Freezing and thawing in the presence of 0.2 M mercaptoethanol dissociated all three fractions into subunits of similar molecular size (3.4 S), with concomitant enhancement of phosphorylase phosphatase activity. The Km values all became essentially the same (20 muM for phosphorylase a and 16 muM for P-histone). 3. The phosphorylase phosphatase and P-histone phosphatase activities could not be separated with any of the procedures described. Competition between the two phosphoprotein substrates was observed with some of the fractions.?


An interpretation of the three line EPR spectrum of nitric oxide hemeproteins and related model systems: the effect of the heme environment. The EPR spectra of the nitric oxide (NO) derivatives of structurally perturbed Fe (II) hemeproteins show various patterns, all of which are characterized by the conspicuous three-line hyperfine splitting due to 14NO, in contrast to that of the native proteins. For the purpose of obtaining structural information from these three line spectra, the model systems were studied, which consist of NO, heme (or TPP-Fe(II), where TTP means alpha, beta, gamma, delta-tetraphenylporphine) and the nitrogenous base, pyridine or quinoline, which, respectively, give the native type or the three line (perturbed type) EPR spectrum. The ring proton paramagnetic shift of quinoline in this system shows that it is not coordinated to NO-TPP-Fe(II) as a normal axial ligand, in contrast to pyridine which gives the shift pattern of the ordinary axial ligation. This observation suggests that in the NO-hemeproteins some perturbations of the protein structure cause the rupture or distortion of the bond between the imidazole nitrogen on the fifth coordination site and the heme iron, resulting in the three line spectrum. The EPR study of the model systems, the pentacoordinated complex, NO-heme and NO-TPP-Fe(II), in various media revealed that the pentacoordinated species indeed exhibits, depending upon its environment, a variety of spectra, which closely reproduce the three line patterns observed in the perturbed proteins and some related model systems. Such spectral variation can be attributed to the difference in the degree of quenching the internal motion and/or the structural heterogeneity caused by molecular environment.


An extended Scatchard analysis for competitively bound ligands and its application to cyclic adenosine-3',5'-monophosphate and cyclic 5'-amido-5'-deoxyadenosine-3', 5'-monophosphate binding to beef skeletal muscle protein. A method for determining the dissociation constants of ligands and ligand analogs is described. It is based on competition binding studies in the presence of an isotope-labeled, or otherwise measurable, ligand and suitable analog concentrations. The steps used are determination of (1) the maximal amount of radioactive ligand that can be bound, (2) the slopes and intercepts from Scatchard plots at different analog concentrations and (3) the values for the dissociation constants of radioactive ligand and ligand analog from replots of the reciprocals of the slopes and intercepts obtained from the Scatchard plots. Application of the method to a cyclic AMP-binding protein from beef muscle is demonstrated, yielding dissociation constants of 2.10-9 M for cyclic (3H) AMP and cyclic AMP, and 3.10-5 M for cyclic 5'-amido-5'-deoxyadenosine-3', 5'-monophosphate.


Hormone action at the membrane level. IV. Epinephrine binding to rat liver plasma membranes and rat epididymal fat cells. [3-H]Epinephrine binding to isolated purified rat liver plasma membranes is a reversible process. An initial peak in binding occurs at about 15 min and a plateau occurs by 50 min. Optimal binding occurred at a membrane protein concentration of 125mug. Rat liver plasma membranes stored at-70 degrees C up to 4 weeks showed no difference in epinephrine binding capacity as compared to control fresh membranes. Epinephrine binding to liver plasma membranes was decreased by 79% by phospholipase A2 (phosphatide acylhydrolase EC 3.1.1.4), 81% by phospholipase C (phosphatidylcholine choline phosphohydrolase EC 3.1.4.3) and 59% by phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4). Trypsin and pronase digestion of the membrane decreased epinephrine binding by 97 and 47% respectively. In the presence of 10-3M Mg-2+ ions, increasing concentrations of QTP decreased epinephrine binding to liver plasma membranes. A maximal effect was demonstrated with 10-5M GTP, representing an inhibition of 52% of the control. In a Mg-2+ -free system, epinephrine binding was unaffected by GTP. However, in a Mg-2+ -free system, increasing concentrations of ATP cause increasing inhibition of hormone binding. ATP at 10-3 M reduced epinephrine binding to 28% of the control. GRP (10-5 M) was shown to inhibit epinephrine uptake rather than epinephrine release from the membrane. [3-H]Epinephrine binding to isolated rat epididymal fat cells shows an initial peak within 5 min followed by a gradual rise which plateaus after 60 min. Epinephrine binding increased nearly linearly with increasing fat cell protein concentration (40-200 mug protein). GTP (10-5 M) and ATP (10-4 M) decreased epinephrine binding to rat epididymal fat cells by 41%. Nearly complete inhibition of binding was demonstrated with 10-2-10-3M ATP. Epinephrine analogs that contain two hydroxyl groups in the 3 and 4 position on the benzene ring act as inhibitors of [3-H]epinephrine binding to rat adipocytes. Alteration of the epinephrine side chain has relatively little influence on binding. Analogs in which one of the ring hydroxyl groups is missing or methylated are poor inhibitors of [3-H]epinephrine binding. Alpha-(phentolamine and phenoxybenzamine) and beta-(propranolol and dichorisoproterenol) adrenergic blocking agents were tested with respect to their ability to influence [3-H]epinephrine binding and their influence on epinephrine-stimulated lipolysis. Only dichloroisoproterenol significantly inhibited epinephrine binding (by 25%). The two beta-adrenergic blocking agents caused an inhibition of epinephrine-stimulated glycerol release, with propranolol being most effective. Phentolamine and phenoxybenzamine had no significant effect on the epinephrine stimulation of glycerol release by fat cells.


Sodium + potassium-activated ATPase of mammalian brain. Regulation of phosphatase activity. 1. The K+-nitrophenylphosphatase activity associated with mammalian brain (Na+ + K+)-ATPase displays K+ activation curves that have intermediary plateaus and maxima in the presence of less than saturating concentrations of Na+. Zero Na+ and saturating Na+ produce sigmoid K+-activation curves with low and high K+ affinities respectively. 2. ATP inhibits K+-activated nitrophenylphosphatase through both competitive and non-competitive mechanisms. ATP is synergistic with Na+ in the mechanism which converts the enzyme from low to high K+ affinity. 3. The Na+ and K+ interactions can be accounted for by equations which describe a model with separate regulatory sites for Na+ and K+ and with K+- requiring catalytic site which is only accessible in one of the two principal conformational stages of the enzyme. 4. The effects of ATP can be accounted for by the same model through interactions at a single nucleotide binding site. Inhibition which is competitive with K+ and non-competitive with substrate arises from stabilization of the inactive enzyme conformation. Inhibition which is non-competitive with K+ and competitive with substrate results from interactions with the active enzyme conformation. The synergism between Na+ and ATP appears to arise as a consequence of the formation of phosphoryl enzyme. 5. A model for (Na+ + K+)-ATPase is discussed which involves in-phase coupling of subunit interactions as suggested by these studies.


Interaction of Clostridium perfringens theta-haemolysin, a contaminant of commercial phospholipase C, with erythrocyte ghost membranes and lipid dispersions. A morphological study. Commercially available preparations of phospholipase C from Clostridium perfringens are commonly contaminated with theta haemolysin, one of a group of bacterial haemolysins called oxygen labile (O-labile) haemolysins. Treatment of erythrocyte ghosts and a mixed lipid dispersion containing cholesterol with commercially available phospholipase C in the absence of Ca-2+ and the presence of phosphate buffer and/or EDTA resulted in the formation and release of ring or arc-shaped structures. Highly purified phospholipase C, free of theta-haemolysin, produced no changes in the morphology of erythrocyte ghosts or lipid dispersions in the presence of phosphate or EDTA, but caused the formation of typical diglyceride droplets in the presence of Ca-2+ in the absence of these inhibitors. Ring structures, identical to those caused by commercial phospholipase C, were formed on addition of highly purified theta-haemolysin to erythrocyte ghost membranes, lipid dispersions containing cholesterol and cholesterol dispersions, but not on treatment of membranes from Micrococcus lysodeikticus. Heat-inactivated O-haemolysin (60 degrees C for 10 min) produced no such effects. The dimensions of rings and arcs displayed heterogeneity. The outside diameters in various preparations varied from approx. 27-58 nm with border thickness of 4.1-7.8 nm.


An EPR study of structural perturbations induced by methylindole in the protein and lipid regions of erythrocyte membranes. 3-Methylindole has been shown in previous work to cause pulmonary edema and emphysema in cattle and goats. In this paper, evidence is presented to show that 3-methylindole induces structural perturbations in bovine erythrocyte membranes. The structural perturbations which were induced as a function of 3-methylindole concentration in the membranes were measured by EPR using the attachment of maleimide spin label to the sulfhydryl groups of membrane proteins and by intercalation of methyl-5- doxylstearate, methyl-12-doxylstearate, and methyl-16-doxylstearate into the lipid region. The EPR spectra of the malemide spin-labeled membrane proteins became more immobilized as the concentration of 3-methyl-indole increased. The order parameter describing the EPR spectra of methyl-5-doxylstearate decreased from 0.69 to 0.55 as the concentration of 3-methylindole increased. The acyl chains in the region of the carbon 5 position were converted to a less ordered structure. The EPR-spectra of methyl-12-doxylstearate was a superposition representing at least three tumbling rates. As the concentration of 3-methylindole increased, the major fraction of the methyl-12-doxylstearate probes experienced an increase in tumbling rate and a smaller fraction is observed a strongly immobilized state. The EPR spectra of methyl-16-doxylstearate were not perceptibly changed in the presence of 3-methylindole. The concentration dependence suggests that 3-methylindole preferentially intercalates into the ordered region of the alkyl chains sampled by the methyl-5-doxylstearate. These results confirm that 3-methylindole induced structural changes at the molecular level.


Cellular binding sites for insulin in rat liver. A study of the sites of insulin binding in subcellular fractions of rat liver is reported. A method for the isolation of liver plasma membranes, which permits one to follow quantitatively the distribution of all the parameters of interest, was modified and applied to the study of the cellular topography of insulin binding. The insulin binding capacity did not follow closely the enzyme marker (5'-nucleotidase) for plasma membranes when differential centrifugation schemes were used, and the divergence from this marker was more prominent when separations were performed on discontinous sucrose gradients.  A significant amount of insulin binding capacity was always present in fractions with higher density than those containing the majority of 5'-nycleotidase. Results of studies on linear sucrose gradients have disclosed in some of the purified membrane fractions small but consistent differences in density of the insulin binding, and plasma membrane particles. It is suggested that there may be several types of intracellular membranes to which insulin can bind besides the plasma membranes.


Cyclic adenosine monophosphate receptor: effect of cyclic AMP analogues on DNA binding and proteolytic inactivation. The cyclic AMP receptor protein of Escherichia coli in the presence of cyclic AMP undergoes a conformational change resulting in an increased affinity for DNA and an increased susceptibility to attack by proteolytic enzymes resulting in loss of DNA binding capacity. Of several cyclic nucleotides tested only cyclic AMP and cyclic tubercidin monophosphate are able to effect the conformational transition in cyclic AMP receptor protein, prerequisite to proteolytic inactivation or DNA binding. Other analogues such as cyclic GMP or cyclic IMP which are competitive inhibitors of cyclic AMP do not support DNA binding or proteolytic inactivation.


Depression by NAD of x-ray-induced repair-type DNA synthesis in toluene-treated Bacillus subtilis. NAD prevents a DNA repair-type synthesis that is dependent on polymerase I in toluene-treated, X-irradiated Bacillus subtilis. In unirradiated preparations, NAD had little effect on an ATP-dependent, semiconservative synthesis but partially inhibited a repair-type synthesis. In a mutant lacking polymerase I (polA1-), the presence of NAD did not affect dTTP utilization in DNA synthesis. Nicotinamide mononucleotide (NMN) partially reverses the NAD inhibition of repair-type DNA synthesis. NADP and FAD were ineffective as substitutes for NAD. Since NAD is the cofactor for polynucleotide ligase in Bacillus subtilis and NMN is known to discharge AMP from the active AMP ligase complex, it is proposed that activation of DNA ligase reduces dTMP incorporation by reducing sites for, or limiting DNA polymerase I action.


On the role of tryptophan residues in the mechanism of action of glyceraldehyde-3phosphate dehydrogenase as tested by specific modification. A method is described to selectively modify one of the three tryptophan residues of the subunit of glyceraldehyde-3-phosphate dehydrogenase from yeast.  As modifying agent dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide was used.  The residue which is modified by the procedure described has been identified as Trp-193.  There are either one or two molecules of the modifying agent being added to this tryptophan side chain.  The modification apparently does not cause a detectable conformational change of the protein as judged from the methods employed.  However, the enzymatic activities in the dehydrogenase as well as in the esterase reactions are lost after the modification.  It could be established that the modification rendered the enzyme unable to bind the oxidized coenzyme.  Also the charge-transfer interaction between enzyme and coenzyme could no longer be observed.


Changes in cyclic AMP-dependent protein dinase activity in Tetrahymena pyriformis during the growth cycle. An adenosine 3':5'-monophosphate-dependent protein kinase II (ATP:protein phosphotransferase, EC 2.7.1.37) was partially purified from the cytosol fraction of an exponentially growing culture of Tetrahymena pyriformis.  Protein kinase II represented approximately 90% of the cytosolic protein kinase activity.  The enzyme had a high degree of substrate specificity for calf thymus and Tetrahymena histones as compared to casein, protamine and phosvitin.  The enzyme incorporated the terminal phosphate of ATP into serine and threonine residues of all the histone fractions.  The apparent Km of the enzyme for adenosine 3':5'-monophosphate (cyclic AMP) was 1-10-minus 8 M.  Protein kinase II was also activated by other cyclic nucleotides with apparent Km values in the range 2.k-10-minus 6 M.  Ther specific activity of the cyclic AMP-dependent protein kinase of Tetrahymena decreases markedly from initial high values during the transition from the lag to early log phase of growth.  This is followed by a shrp increase in the activity of the enzyme as the log phase of growth progresses.  The specific activity of the enzyme increases rapidly during the heat-induced synchronization of Tetrahymena cells.  The capacity for rapid phosphorylation of multiple classed of organelle-specific phosphoproteins and the level of cyclic AMP were maximal in Tetrahymena during the earliest phase of growth.  These results demonstrate that the cell cycle of Tetrahymena may be coordinated by marked variations in the level of cyclic AMP which in turn regulate the cyclic AMP-dependent protein kinase.


Investigation of the adenosine 3', 5'-cyclic phosphate binding site of pig brain histone kinase with the aid of some analogues of adenosine 3', 5'-cyclic phosphate. 2'-O-Chloroacetyl cyclic AMP, 2'-O-acrylyl cyclic AMP and N-6, 2'-O-diacrylyl cyclic AMP were synthesized by the reaction of cyclic AMP with chloroacetic and acrylic anhydrides, respectively.  Selective O-deacylation of N-6, 2'-O-diacrylyl cyclic AMP yielded N-6 -monoacrylyl cyclic AMP.  In the reaction of gamma-mercaptobutyric acid with 8-bromo cyclic AMP, 8-(gamma-carboxypropylthio) cyclic AMP was obtained.  The compounds synthesized and other cyclic AMP analogues (8-bromo cyclic AMP and adenosine 3', 5'-cyclic sulphate) were tested for ability to interact with the highly purified pig brain histone kinase.  All compounds under study were found to be activators of the enzyme.  The highest activating potency was manifested by 8-bromo cyclic AMP and 8-(gamma-carboxypropylthio) cyclic AMP; adenosine 3', 5'-cyclic sulphate was the least potent in this respect.  All compounds were shown to inhibit binding of cyclic [-3-H]AMP to histone kinase.  The inhibition was competitive with respect to cyclic AMP in all cases.  All compounds, except for 2'-O-chloroacetyl cyclic AMP may indicate the formation of a covalent bond between this analogue and the enzyme.  These findings suggest that an active site of the regulatory subunit of the histone kinase contains at least three specific areas responsible for cyclic AMP binding.


Cyclic nucleotide phosphodiesterase activities of pig coronary arteries. Most (85% or more) of the cyclic nucleotide phosphodiesterase (3' :5' -cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) activity of pig coronary arteries was found in the 40 000 times g supernatant fraction of homogenates of the intima plus media layer. Chromatography of the soluble fraction of this layer on DEAE-cellulose resolved two phosphodiesterase activities and a heat stable, non-dializable activator. Peak I activity had apparent Km values of 2-4 muM for cyclic GMP and 40-100 muM for cyclic AMP. Peak II activity was relatively specific for cyclic AMP and exhibited apparent negatively cooperative behavior. Peak I but not peak II activity could be stimulated 3-8-fold by the addition of the boiled activator fraction or a boiled crude supernatant fraction. Cyclic AMP hydrolysis by peak I or peak II was more rapid in the presence of Mn-2+ than Mg-2+, but the latter promoted hydrolysis of cyclic GMP by peak I more effectively than did Mn-2+ in the presence of activator. In the absence of added metals, ethylene bis(oxyethylenenitriol)tetra-acetic acid (EGTA) and EDTA both inhibited hydrolysis of cyclic AMP and cyclic GMP by phosphodiesterase activities in the supernatant fraction and in peak I, but EDTA produced more complete inhibition at lower concentrations than did EGTA. Imidazole (1 muM to 10 mM) had virtually no effect on the hydrolysis of cyclic AMP or cyclic GMP catalyzed by either of the two separated peaks or by total phosphodiesterase activities in crude supernatant or particulate fractions.


Purification and characteriazation of collagenase from guinea pig skin. Guinea pig skin col-agenase, isolated from culture medium of whole skin, was separated into two enzymatically active fractions. These two fractions have been purified extensively. Peak II fraction has been purified to homogeneity as examined by polyacrylamide gel electrophoresis. Their molecular weights are approximately 130 000 (peak I) and 40 000 (peak II). Both guinea pig skin collagenase fractions are capable of degrading the native collagen fibrils and are inhibited by serum, cysteine and EDTA. They appear to be glycoproteins. Guinea pig skin (peak II) and human skin collagenase were compared. They are both glycoproteins and have similar molecular size (Mr = 40 000). Immunodiffusion assay showed that no cross-reactivity was seen between the enzymes, indicating species specificity among collagenases.


Excitation-secretion coupling in exocrine glands.  Properties of cyclic AMP phosphodiesterase and adenylate cyclase from the submaxillary gland and pancreas. 1. The cyclic AMP phosphodiesterase in homogenates of the submaxillary gland and pancreas was found to be associated mainly with the 300,000 times g supernatant fraction.  A Lineweaver-Burk plot showed a high-affinity (Km app. = 1.6 muM) and a low-affinity (Km app. greater than 100muM) component for the cyclic AMP substrate.  The enzyme was magnesium dependent, and strongly inhibited by papaverine, theophylline and caffeine.  Cyclic GMP inhibited cyclic AMP phosphodiesterase, but only in concentrations greatly exceeding that of the cyclic AMP.  Calcium did not alter the activity of the enzyme.  The activity of the submaxillary cyclic AMP phosphodiesterase was not influenced by noradrenaline, dopamine, histamine, 5-hydroxytryptamine or gamma-amino butyric acid, and that of the pancreatic enzyme by acetylcholine, pancreozymin or secretin.  2. Adenylate cyclases from guinea-pig submaxillary gland and cat pancreas are particulate enzymes.  The highest specific activity was recovered from the 1500 times g pellet.  Guineo-pig submaxillary adenylate cyclase was activated by fluoride, noradrenaline, isoprenaline and adrenaline.  The noradrenaline activation was blocked by the beta-adrenoceptor blocker, propranolol, but not by the alphs-adrenoceptor blocker, phentolamine.  Neither acetylcholine nor carbachol had any effect on the adenylate cyclase activity.  The apparent Km value for the 10- minus 4 M noradrenaline activated adenylate cyclase activity was completely aboliched by 5 mM calcium.  Cat pancreatic adenylate cyclase was clearly and consistently activated by secretin, but not by pancreozymin or carbachol.


Protein kinases in brown adipose tissue of developing rats.  II. two soluble kinase activities and their affinities for nucleotide and protein substrates. A heat-stable, soluble component of brown adipose tissue from newborn rats was found to be readily phosphorylated by protein kinase of the same subcellular fraction.  The concentration of this component in brown fat decreased with the age of the animals.  A boiled crude microsomal preparation from rat liver was also phosphorylated by brown fat protein kinase.  The GTP-linked phosphorylation of the endogenous heat-stable protein was not stimulated by ATP (in contrast to phosphorylation of histone).  The maximum velocity of phosphorylation achieved with GTP was about 2.5 times higher than that with ATP as nucleotide substrate.  This difference was not due to ATPase activity in the assay.  With histone as the protein acceptor both activities were the same.  The affinity of protein kinase(s) for ATP was lower with the endogenous heat-stable brown-fat protein and with boiled microsomes (Km of 0.21 mM and 0.17 mM, respectively) than with histone (Km of 0.05 M). No detectable ATPase activity was present in either acceptor protein.  It is concluded that the 100 000 times g supernatant fraction from brown fat of infant rats contains two protein kinase activities.  One preferentially uses ATP and histone as substrates and the other uses endogenous heat-stable soluble proteins and either ATP or GTP.


Location of gluconeogenesis from phosphoenolpyruvate in cotyledons of Cucurbita pepo. 1. The aim of this work was to discover the location of the enzymes that convert phosphoenolpyruvate to fructose 6-phosphate during gluconeogenesis in fatty seeds.  Cotyledons of 5-day-old dark-grown seedlings of marrow (Cucurbita pepo) were used as experimental material.  2. Cotyledons were separated into palisade and mesophyll tissue.  Extracts of the two tissues had comparable activities of gluconeogenic enzymes.  3. Extracts of cotyledons were fractionated by density gradient centrifugation to yeild mitochondria and glyoxysomes, and by gel filtration to yield proplastids.  The isolated organelles retained their characteristic ultrastructure and appreciable amounts of marker enzymes.  The proportions of the total activities of phosphoglyceromutase and fructose-1, 6-diphosphatase recovered in the mitochondrial and glyoxysomal preparations were insignificant.  The same was true for the activities of phosphoglyceromutase and phosphopyruvate hydratase found in the proplastid preparations.  4. Extracts of a number of other gluconeigenic plant tissues were centrifuged at 2500 times g to yield particulate preparations.  None of these preparations contained a significant proportion of the total activity of phosphoglyceromutase.  5. It is suggested that gluconeogenesis from phosphoenolpyruvate in plants occurs in the cytoplasm.


Studies on the interaction between heparin and mouse bone collagenase. Mouse bone collagenase was found to be tightly bound to a heparin-substituted gel at low ionic strength.  The bond was reversible, however, and the collagenase could be elutted at high ionic strength.  In addition to providing a method for purifying the enzyme with high yield, the results suggest that the strong ionic bond between heparin and collagenase may partially explain the mechanism wherein heparin enhances the activity of mouse bone collagenase.


8-azaguanine and flavinogenesis in Eremothecium ashbyii. 8-Azaguanine (10- minus 4 M) supplementation in synthetic medium inhibited flavinogenesis in Eremothecium ashbyii to far greater extent (68per cent) than the growth (25 per cent).  That enzymes comprising the biosynthetic pathway of riboflavin are synthesized during early growth phase of the organism is supported by the data presented.  8-Azaguanine mediated inhibition in flavinogenesis was closely related with decreased levels of ribose-5'-phosphatase, ribose reductase and ribitol kinase, the enzymes involved in supplying ribitol for flavinogenesis.  Addition of guanine and not ribitol during early growth phase to 8-azaguanine-added cultures released the inhibition of riboflavin synthesis and restored the enzyme levels in the presence of the antimetabolite.


Stimulatory effect of gonadotropins on the synthesis of adenosine 3': 5'-cyclic monophosphate and progesterone by suspensions of rat ovarian interstitial cells. A cell suspension was prepared from immature rat ovaries by treatment with trypsin and collagenase.  The isolated cells were capable of converting [8-14-C]adenine to cyclic [-14-C]AMP and the rate of this conversion was stimulated in vitro by luteinizing hormone and human chorionic gonadotropine, but not by prolactin, norepinephrine, dopamine or albumin.  The accumulation of progesterone was also measured in these cells by radioimmunoassay.  In vitro addition of luteinizing hormone and human chorionic gonadotropine, but not by prolactin, norepinephrine, dopamine or albumin.  The accumulation of progesterone was also measured in these cells by radioimmunoassay.  In vitro addition of luteinizing hormone stimulated the accumulation of radioimmuno-assayable progesterone.  The conversion of [8-14-C]adenine to cyclic [-14-C]AMP showed a rapid increase during the first 30 min of the incubation period when luteinizing hormone was added to the incubation medium.  Progesterone accumulation in response to the same dose of luteinizing hormone showed a lag period for the first 30 min of incubation after which there was an increase up to 2 h.  The luteinizing hormone-induced progesterone accumulation was sensitive to puromycin, but there was no effect on the luteinizing hormone-induced increase in cyclic [-14-C]AMP formation from [8-14-C]-adenine.  Actinomycin D also inhibited the luteinizing hormone-induced progesterone accumulation in rat ovarian interstitial cell suspension is preceded by an increased accumulation of cyclic AMP and that the accumulation of steroid under the influence of luteinizing hormone involve processes sensitive to puromycin and antinomycin D.


Chemotaxis and binding of cyclic AMP in cellular slime molds. To obtain more information about how cyclic AMP mediates cell aggregation as found in some species of the cellular slime molds, we determined the maximal binding activity of cyclic AMP in different species under various environmental conditions. The binding of cyclic AMP is limited to amoebae using this cyclic nucleotide as chemotactic agent. Maximal binding activity proved to coincide with a maximal chemotactic response and to be related to the length of the period between the vegetative and the aggregative phase. Of the species studied, Dictyostelium discoideum has the highest cellular density of cyclic AMP receptors and is the most sensitive to cyclic AMP as attractant. At 15 degrees C, aggregation begins later, chemotaxis takes effect over a greater distance, and the maximal binding activity is higher than 22 degrees C. The number of cyclic AMP receptors is independent of temperature. The delay in the onset of aggregation and the increased chemotactic response in darkness is not due to a change in the maximal binding activity. The binding of cyclic AMP and its inactivation is discussed in the light of cell aggregation.


Regulation of glycogen metabolism in liver by the autonomic nervous system. VI. Possible mechanism of phosphorylase activation by the splanchnic nerve. The effects of autonomic-nerve stimulation on the activities of phosphorylase (EC 2.4.1.1), dephospho-phosphorylase kinase (EC 2.7.1.38) and phosphorylase phosphatase (EC 3.1.3.17), and on the concentration of adenosine 3', 5'-monophosphate in rabbit liver were investiaged. Results were compared with the effects of epinephrine and glucagon on these enzymes. 1. The acitivity of liver phosphorylase increased rapidly and markedly on electrical stimulation of the splanchnic nerve, or after intraportal administration of epinephrine or glucagon. The activity was not affected by vagal stimulation. 2. The activity of dephospho-phosphorylase kinase increased about 2--3-fold 1 min after injections of epinephrine and glucagon, glucagon causing more activation than epinephrine. The enzyme activity was not altered by splanchnic-nerve, or vagal stimulation. 3. Injections of epinephrine and glucagon caused marked elevation of liver adenosine 3', 5'-monophosphate within a few minutes. With epinephrine, the nucleotide concentration rose to a maximum after 1 min and amounted to about 3-fold increase, while with glucagon the maximum increase of approximately 8-fold increase was observed after 2 min. Stimulation of the splanchnic nerve for 10 min did not affect the adenosine 3', 5'-monophosphate level in the liver. Vagal stimulation also had no effect on the level. 4. The activity of phosphorylase phosphatase decreased promptly (within 30 s) and markedly on splanchnic-nerve stimulation, but did not change significantly on administration of epinephrine of glucagon. A small but insignificant increase in phosphatase activity wasobserved upon vagal stimulation. 5. The effect of Ca-2+ on purified dephospho-phosphorylase kinase was studied. The activity was found to depend partially on free Ca-2+ at low Ca-2+ concentrations (1-10-minus 7--1-10-minus 5 M). 6. These results suggest that the rise in hepatic phosphorylase content upon splanchnic-nerve stimulation, unlike that induced by epinephrine and glucagon, is not mediated by adenosine 3', 5'-monophosphate and subsequent activation of dephospho-phosphorylase kinase, but rather by inactivation of phosphorylase phosphatase. The possible existence of a new factor in this mechanism is discussed.


On the regulation of adenosine 3', 5'-monophosphate synthesis in bacteria. I. Effect of carbon source variation on cyclic AMP synthesis in Escherichia coli B/r. 1. The effect of carbon source variation in bacterial growth media on their growth rate, inducible enzyme and cyclic AMP synthesis was examined: an inverse relationship between the culture's growth rate and its differential rate of inducible enzyme (tryptophanase and beta-galactosidase), and cyclic AMP synthesis was found. 2. The effect of the culture's growth phase on its sensitivity or resistance to glucose catabolite repression was determined in the wild type and a catabolite insensitive mutant (ABDROI): the wild type's sensitivity to glucose repression was not affected, whereas the insensitivity of the mutant was found to be limited to its early logarithmic phase of growth. At late log, or stationary phase, the mutant was found to be sensitive to glucose repression. 3. Examination of the kinetics of glucose uptake by the mutant, using alpha-[1 4-C] methyl-glucoside showed evidence for two transport systems each with a different affinity to glucose. A low affinity transport system (apparent Km of 3.4-10-minus 5 M) which appears mostly at the early logarithmic phase of growth. A high affinity transport system (apparent Km of 1.2-10-minus 5 M) which appears mostly at the late log and stationary phases of growth. 4. The effect of the culture density variation on its sensitivity to glucose repression showed that sensitivity to glucose catabolic repression is primarily a reflection of the formation of an allosteric effector molecule between glucose and its specific transport molecule which in turn regulates the activity of the adenylate cyclase.


On the regulation of cyclic AMP level in bacteria. II. In vitro regulation of adenylate cyclase activity. Solubilization and reconstitution of a functional membrane-bound adenylate cyclase system responsive to regulation by glucose. 1. The in vitro regulation of the membrane bound adenylate cyclase of Escherichia coli B/r by a variety of carbohydrates and one mammalian hormone was examined. 2. The membrane bound adenylate cyclase was found responsive to regulation by the various growth substrates and to glucagon. 3. Solubilization of the bacterial membrane preparation by a procedure specific for the solubilization of the phosphotransferase enzyme E1 1 to its E1 1 A and E1 1 B subunits was found to be accompanied by the loss of the adenylate cyclase regulation by glucose. 4. Reconstitution of the membrane was found to result in a recovery of the regulative response of the adenylate cyclase to glucose. 5. A model for the intermediate steps in the interaction between glucose and phosphotransferase E1 1 and the adenylate cyclase is discussed.


Electrochemical regeneration of nicotinamide adenine dinucleotide. Reduced nicotinamide adenine dinucleotide (NADH) has been characterized electrochemically by solid electrode voltammetry and controlled potential electrolysis. Photometric and enzymatic assay showed that enzymatically active nicotinamide adenine dinucleotide (NAD-+) could be regenerated electrolytically from its reduced form without the use of so-called electron mediators. Complete regeneration of enzymatically active NAD can be expected in pyrophosphate buffers and phosphate buffers during the electrolysis. Advantages of electrochemical regeneration of coenzymes are discussed, especially with regard to immobilization of enzymes.


A plausible role for a membrane-bound cyclic AMP phosphodiesterase in cellular slime mold chemotaxis. 1. Kinetics of membrane-bound cyclic AMP phosphodiesterase of the cellular slime mold, Dictyostelium discoideum, were studied under two conditions: in the 27 000 times g sediment of cell homogenates (particle-bound phosphodiesterase) and in cell suspensions using external cyclic AMP as a substrate (cell-bound phosphodiesterase). Both methods revealed non-Michaelian kinetics with interaction coefficients less than 1. 2. The membrane-bound phosphodiesterase has a specificity different from that of the cyclic AMP receptor, also present at the cell surface. 3. The membrane-bound enzyme was solubilized by lithium 3, 5-diiodosalicylate and partially purified. In this state the non-linear kinetics were still retained; however, the enzyme was not inhibited by the D. discoideum inhibitor, unlike the cell-bound phosphodiesterase in vivo. This indicates that both enzymes share an inhibitor binding site and that this site is cryptic in the cell-bound state. 4. Production of periodic cyclic AMP pulses by centers, and their relay by other cells, is believed to occur during aggregation. It is suggested that the cell-bound enzyme determines a "time window" significantly smaller than the period of pulsing, and optimizes stimulation of the cyclic AMP receptors in chemotaxis and signal relaying.


The functional metabolism of vitamin D in chicks fed low-calcium and low-phosphorus diets. Radioactively labelled cholecalciferol was administered continuously to chicks that were fed normal, low-calcium and low-phosphorus diets. It has been possible to show that under such steady state conditions with regard to cholecalciferol, and mineral restriction, the animal reacts by increased accumulation of 1, 25-dihydroxycholecalciferol in the intestinal and the kidney cell, which was associated in the intestine with an increased calcium-binding activity. A similar accumulation of 1, 25-dihydroxycholecalciferol in bone was not noticed. It is proposed that the intestine and the kidney, but not bone, are the main target organs for cholecalciferol in the maintenance of calcium homeostasis, and that both calcium and phosphorus play a role in the regulation of the formation and subsequent function of 1, 25-dihydroxycholecalciferol.


Essential arginine residues in D-glyceraldehyde-3-phosphate dehydrogenase. Two arginyl residues per subunit of yeast D-glyceraldehyde-3-phoshphate dehydrogenase were modified by treatment with butanedione without significant changes in the compostion of other amino acid residues. The modified enzyme displays no dehydrogenase activity. It retains the capacity for interacting with the coenzyme NAD, but binds it less firmly than does the native enzyme. The molar absorbance of the enzyme-NAD complex is markedly reduced and the reactivity of the active-center SH groups is changed in the modified enzyme. The native and modified enzymes show identical fluorescence spectra, absorbance and CD spectra.


Application of affinity chromatography to the purification of collagenase for the isolation of insoluble elastin. ClostrIdium histolyticum collagenase (clostridiopeptidase A, EC 3.4.4.19) was purified by batchwise separation with DEAE-cellulose followed by affinity chromatography on a column of alkali-treated elastin. The N-terminal amino acid profile of elastin isolated from bovine ligamentum nuchae using this enzyme preparation was compared with that of a duplicate sample purified with a mixture of collagenase I and II (Yoshida, E, and Noda, H. (1965) Biochim. Biopsys. Acta 105, 562-574). An approx. three-fold decrease in the molar concentration of N-terminal residues and a considerable reduction in their number was obtained by using the former enzyme preparation.


Magnetic circular dichroism studies on microsomal aryl hydrocarbon hydroxylase: comparison with cytochrome b-5 and cytochrome P-450-cam. Magnetic circular dichroism spectra are reported for the visible and near ultraviolet spectral regions of liver microsomes from dimethylbenzanthracene-treated rats. The sequential addition of NADH, dithionite, and carbon monoxide enables us to determine contributions to the magnetic circular dichroism by cytochromes b-5 and P-450, which dominate the spectra. The magnetic circular dichroism of the microsomal preparation is compared with that of purified oxidized and reduced cytochrome -b-5 from pig liver and with the camphor-complexed and camphor-free oxidized, reduced, and reduced carbonmonoxy cytochrome P-450-cam from Pseudomonas putida. The magnetic circular dichroism spectra of the membrane bound cytochrome -b-5 are similar to those of the purified protein, indicating that little or no alteration in the environment of the heme occurs during the isolation procedure. The soluble bacterial cytochrome P-450 also appears to be a suitable model for microsomal P-450, although differences in the magnetic circular dichroism intensity are observed for the two enzymes. No effect of dimethylbenzanthracene on the magnetic circular dichroism spectra of induced compared to control rat microsomes could be observed.


Separation of respiratory reactions in Rhodospirillum rubrum: inhibition studies with 2-hydroxydiphenyl. 1. Respiration of chemotrophically and phototrophically grown Rhodospirillum rubrum is inhibited by 2-hydroxydiphenyl. 2. Membrane-bound NADH oxidase and NADH: cytochrome c reductase are inhibited also. The inhibitor constant for both reactions (Ki) is 0.075 plus or minus 0.012 mM. NADH dehydrogenase is not inhibited significantly. 3. The inhibition of succinate:cytochrome c reductase is associated for chemotrophic membranes with Ki equals 0.22 plus or minus 0.03 mM and for phototrophic membranes with Ki equals 0.49 plus or minus 0.09 mM. Succinate dehydrogenase is not affected by 2-hydroxydiphenyl. 4. Cytochrome oxidase is inhibited only slightly. 5. While NADH-dependent reactions in both phototrophic and chemotrophic membranes are inhibited maximally more than 95 percent, succinate-dependent reactions can be inhibited more than 95 percent only in chemotrophic membranes. In phototrophic membranes the maximum inhibition of succinate-dependent reactions is about 70 percent. 6. The type of inhibition in both cases 2 and 3 is non-competitive. 7. While the reduction of b-type cytochrome is inhibited by 2-hydroxydiphenyl, the degree of ubiquinone reduction is not influenced. The data suggest that the site of inhibition is localized between ubiquinone and cytochrome b. 8. Implications of these data for the respiratory electron transport system in R. rubrum are discussed.


Kinetic study of oxygen evolution parameters in Triswashed, reactivated chloroplasts. Tris-washed chloroplasts which have lost the ability to evolve oxygen can be reactivated by the procedure of Yamashita T., Tsuji, J. and Tomita G. (1971) Plant Cell Physiol. 12, 117-126) [7] to give 100 percent of the rate of control chloroplasts in continuous illumination. Furthermore, in flashing light the reactivated chloroplasts exhibit oxygen-yield oscillations of period four that are characteristic of the control. Similar kinetic parameters for intermediate steps in the water-splitting process are observed for the two preparations. We conclude that the reactivation procedure restores the native oxygen evolution mechanism to Tris-washed chloroplasts. A relatively rapid and reversible (0.5 s decay) light-induced component of EPR Signal II is observed upon inhibition of O2 evolution by Tris washing (Babcock G. T. and Sauer, K. (1975) Biochim. Biophys. Acta 376, 315-328) [10]. Reactivated chloroplasts are similar to untreated chloroplasts in that this Signal IItransient is not observed. Manganese, which is released by Tris treatment to the interior of the thylakoid membrane in an EPR-detectable state, is returned to an EPR-undetectable state by reactivation. The reactivation procedure does not require light to restore O2 evolution and EDTA has no effect on the extent of reactivation. These results are discussed in terms of possible mechanisms for manganese incorporation into photosynthetic membranes.


The tiron free radical as a sensitive indicator of chloroplastic photoautoxidation. Wheat chloroplasts photochemically reduced molecular oxygen, as a Hill oxidant in the Mehler reaction, to superoxide anion which then oxidized added 1,2-dihydroxybenzene-3,5-disulfonate to its semiquinone, a comparatively stable free radical at pH 7. The last mentioned reaction was rapid in aqueous solution, but the rate of formation of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone by the chloroplast system was calculated as T1 of 0.6 s. The Mehler reaction, or more specifically the univalent reduction of oxygen by Photosystem I, was rate-limiting so that the 1,2-dihydroxybenzene-3,5-disulfonate seniquinone was a useful spin probe for superoxide anion production at room temperature. The ESR signal of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was proportional to its steady state concentration and decayed in the dark with a T1/2 of 5-6 s. This oxygen-dependent signal was enhanced by mediation of chloroplastic oxygen reduction through methyl viologen. The superoxide anion scavengers ascorbate and L-epinephrine competitively obscured 1,2-dihydroxybenzene-3,5-disulfonate semiquinone formation, butadded superoxide dismutase was not as effective in this role. Partial inhibition by superoxide dismutase was achieved only by preincubation of Photosystem I enriched particles with ten times the endogenous concentration of superoxide dismutase. This and the persistence of a small amount of a 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) oxidizing species in the dark supports the concept of Tiron accessibility but not the superoxide dismutase accessibility of superoxide anion bound in its formative enzyme complex. Benzoquinone and naphthoquinone disulfonate also reacted with superoxide anion, and supported both the Hill reaction and the Mehler reaction as final oxidants of both water and superoxide anion.


Biochemical and biophysical studies on cytochrome c oxidase.  XX. Reaction with sulphide. 1. Upon addition of sulphide to oxidized cytochrome c oxidase, a low-spin heme sulphide compound is formed with an EPR signal at gx = 2.54, gy = 2.23 and gz = 1.87.  Concomitantly with the formation of this signal the EPR-detectable low-spin heme signal at g = 3 and the copper signal near g = 2 decrease in intensity, pointing to a partial reduction of the enzyme by sulphide.  2. The addition of sulphide to cytochrome c oxidase, previously reduced in the presence of azide or cyanide, brings about a disappearance of the azido-cytochrome c oxidase signal at gx = 2.9, gy = 2.2, and gz = 1.67 and a decrease of the signal at g = 3.6 of cyano-cytochrome c oxidase.  Concomitantly the sulphide-induced EPR signal is formed.  3. These observations demonstrate that azide, cyanide and sulphide are competitive for an oxidized binding site on cytochrome c oxidase.  Moreover, it is shown that the affinity of cyanide and sulphide for this site is greater than that of azide.


Asymmetry of an energy transducing membrane the location of cytochrome c2 in Rhodopseudomonas spheroides and Rhodopseudomonas capsulata. Monospecific antibodies have been prepared against cytochrome c2 from Rhodopseudomonas spheroides and Rhodopseudomonas capsulata, and against cytochrome c' from Rps. capsulata.  These antibodies precipitated their respective antigens, but did not cross react with a wide range of procaryotic or eucaryotic cytochromes, or with other bacterial proteins.  The cytochromes produced during aerobic growth were immunologically indistinguishable from those produced during photosynthetic growth.  Cytochrome c2 is located in vivo in the periplasmic space between the cell was and the cell membrane, and when chromatophores are prepared from whole cells the cytochrome becomes trapped inside these vesicles.  The implications of these results to energy coupling in the photosynthetic bacteria are discussed.


Metabolism of lipoproteins in nonhuman primates. Studies on the origin of low density lipoprotein apoprotein in the plasma of the squirrel monkey. The plasma of squirrel monkeys contains extremely low levels of very low density lipoproteins. The delipidated apoproteins from the different lipoprotein density classes of this species show a heterogeneity similar to that of man and the rat. The biosynthesis of the apoproteins of squirrel monkey lipoproteins was studied in fasted normal and Triton WR1339-treated animals. After intravenous injection of [3-H] leucine, maximal labeling of very low density lipoproteins occurred after 1 h, intermediate density lipoproteins (d 1.006--1.019) in 2 h, and low density lipoproteins after 3 h. At all times, however, low density lipoproteins had the greatest percentage of radioactivity. Polyacrylamide gel electrophoresis revealed that the apoprotein B moiety of very low density and intermediate density lipoproteins contained 62% and 81% of the total radioactivity in these lipoproteins whereas the fast-migrating peptides were minimally labeled. In monkeys injected with Triton WR1339, 70--80% of the radioactivity incorporated into d smaller than 1.063 lipoproteins was in very low density lipoproteins with only 10--15% in intermediate and low density lipoproteins. After injection of 3-H-labeled very low density lipoproteins and [14-C] leucine into Triton-treated monkeys, catabolism of 3-H-labeled very low density lipoprotein to intermediate and low density lipoproteins was small and was significantly less than corresponding values for the incorporation of [14-C] leucine. Thus, breakdown of very low density lipoproteins could not account for all the labeled apoprotein B present in the intermediate and low density lipoprotein fractions. The results indicate that most, but not all, of the newly synthesized apoprotein B enters plasma in very low density lipoproteins and that the low concentrations of this lipoprotein in squirrel monkey plasma are a consequence of its rapid turnover.


Lipoprotein-acid mucopolysaccharide complexes of human atherosclerotic lesions. Lipoprotein-acid mucopolysaccharide complexes occurring in different types of human atherosclerotic lesions were isolated and partially characterized. Complexes from fatty streaks and fibrous plaques were extracted from pooled tissues with 0.15 M NaCl, purified by gel filtration, and fractionated by ultracentrifugation. Both low and very low density lipoproteins were present; low density lipoprotein was predominant. The complexes were analyzed for uronic acid, cholesterol, phospholipid, and Ca-2+ contents; there was no significant difference in the relative molar ratios between complexes from fatty streaks and fibrous plaques. Acid mucopolysaccharides were isolated from the complexes and identified by electrophoresis and enzymatic studies. Chondroitin sulfate C and hyaluronic acid were found in complexes from fatty streaks and fibrous plaques. Heparin was detected only in fibrous plaques.


Fluorescence quenching in lecithin and lecithin/cholesterol liposomes by parmagenetic lipid analogues. Introduction of a new probe approach. 1. Perylene, whether incorporated into lecithin or lecithin/cholesterol (1:1) liposomes, exhibits identical fluorescence spectra, but fluorescence in the presence of cholesterol is enhanced by 30-50%. 2. The fluorescence of perylene in pure dipalmitoyllecithin vesicles increases sharply at the transition temperature (Tt equals 41 degrees C). No such fluorescence jump is observed in lecithin/cholesterol (1:1) micelles. 3. In lecithin liposomes maximal quenching of perylene fluorescence at 25 degrees C is effected by cholestane spin label (80%) followed by androstane spin label (70%), 5-nitroxide stearate (60%) and 16-nitroxide stearate (50%). 4. In liposomes containing 5  mol % cholesterol these differences are reduced; however, the sequence of quenching efficiencies is the same except for the nitroxide stearates, which interchange their positions. 5. 5. Paramagnetic quenching of perylene fluorescence is stable below 35 degrees C and above 45 degrees C, but decreases sharply about the phase-transition temperature of dipalmitoyllecithin. 6. In lecithin/cholesterol (1:1, molar ratio) lipsomes fluorescence quenching diminishes linearly, but only slightly, with increasing temperature. 7. Cholestane spin label and androstane spin label at concentrations of greater than 20 mol % themselves suppress the quenching discontinuity at Tt, indicating a cholesterol-like structural effect. 8. The quenching phenomena observed are attributed to a non-random accommodation of fluorophore and quencher molecules (co-clustering) below the phase transition and a statistical distribution of both impurities above Tt. 9. In the presence of cholesterol the clustering tendencies are reduced or even  eliminated; this is compatible with the concept that cholesterol fluidizes the phosphatide acyl chains below the transtion temperature.


Sialoglycopeptides from bovine milk fat globule membrane. Milk fat globule membrane was shown to contain sialic acid, all of which could be released without disruption of the fat globule. Sialoglycopeptides were cleaved from the surface of intact fat globules by Pronase and fractionated on Sephadex G-50. Further fractionation of the major sialoglycopeptide peak on DEAE-Sephadex gave two groups of sialoglycopeptides eluted with 0.1 M NaCl (Group A) and 0.5 M NaCl (Group B), respectively. Refractionation gave a major sialoglycopeptide from each of the two groups together with a total of three minor sialoglycopeptides. All five sialoglycopeptides eluted as single peaks using shallow salt gradients on DEAE-Sephadex and contained a hydrophilic peptide chain together with galactose, mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid. Glycopeptides of Group A but not Group B contained fucose. The major sialoglycopeptide of Group B released 35% of its hexose and hexosamine on treatment with alkaline borohydride leaving a sialoglycopeptide which had reduced serine and threonine and elevated alanine levels and in addition contained 2-aminobutyric acid. An oligosaccharide fraction containing N-acetylgalactosaminitol, galactose and sialic acid in a molar ratio of 1:1:2 was partially characterised from the clevage mixture. The major sialoglycopeptide of Group A had a more complex carbohydrate structure and showed no released carbohydrate on treatment with alkaline borohydride. The sialoglycopeptides of milk fat globule membrane show many similarities with those of erythrocyte membrane and have a potential use in comparative and structural studies.


Relationship between L-alanine and sodium ion transport in isolated renal tubules. 1. Rat renal tubules were isolated by incubation with collagenase. The Na+ concentration in the tubules at 37 degrees C was increased by additions of g-strophantin and L-alanine. The increase of Na+ in the presence of both g-strophantin and L-alanine was stronger than with either alone. 2. Radioactive sodium (22-Na), which was taken up by the tubules at 0 degrees C in K+-free medium, was more slowly washed out in the buffer with added g-strophantin than in the control buffer, but L-alanine had no effect. 3. At 0 degrees C incubation without K+, g-strophantin did not affect the 22-Na transport of the tubules. But under the same conditions, L-alanine increased Na+ uptake significantly, and in conjunction with it, L-alanine uptake was also increased. 4. The relationship between L-alanine uptake and intra- extracellular Na+ concentration gradients was linear. The ration of L-alanine to Na+ uptake at 0 degrees C was about 1:2. 5. In the incubation without K+ at 0 degrees C, L-alanine could be accumulated in tubules against the chemical concentration gradient (about 1.5-fold). 6. In the incubation without K+ at 37 degrees C, the L-alanine concentration in tubules after 5 min was already steady (Ci/Ce = 2.2), but with K+ it was not stabilized after 10 min. The ration Ci/Ce with K+ WAS HIGHER THAN WITHOUT K+. 7. G-Strophantin, p-hydroxymercuribenzoate, amiloride, and 2,4-dinitrophenol inhibited L-alanine uptake in the tubules and at the same time increased Na+ concentration. The relationship between the L-alanine uptakes inhibited by g-strophantin, amiloride and dinitrophenol, and the respective intra- extracellular Na+ concentration gradients was strikingly linear. But in the case of p-hydroxymercuribenzoate there was no correlation. 8. The results indicate that L-alanine transport into the renal tubules might be regulated mainly by the intra- extracellular Na+ concentration gradient and that inhibitors such as g-strophantin, amiloride, and dinitrophenol could have a secondary effect on the L-alanine transport which follows the change of Na+ concentration in cells. p-Hydroxymercuribenzoate might have an inhibiting effect on the binding of carrier with Na+ and/or L-alanine.


Microsomal (Na- +K+)-activated ATPase from frog skin epithelium. Cation activations and some effects of inhibitors. A method is described for the extraction of microsomal ouabain-sensitive (a- + K+)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na+ K+)-stimulated activity in the range of 30- 40 nmol - mg -1 - min -1 at 26 degrees C. This portion which is also ouabain sensitive, is about half of the total activity in media containing Mg2+, Na+ and K+. These preparations also contain Mg2+-dependent or Ca2+-dependent activities which are not additive and which are not significantly affected by ouabain, Na+, K+ or Li+. The activations of the ouabain-sensitive ATPase activity by Mg2+, Na+, and K+ are similar to those described in other tissues. It is found that Li+ does not substitute for Na+ as an activator but in high concentrations does produce partial activation in the presence of Na+ with no K+. These results are pertinent to the reported observations of ouabain-sensitive Li+ flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li+ on the sodium pump.


Enhancement of the activity of asparaginyl-tRNA synthetase by an activator from rabbit liver. Asparaginyl-tRNA synthetase has been partially purified from acetone powders of rabbit liver. (NH-4)-2SO-4 fractionation was followed by chromatography of the enzyme on DEAE-cellulose. An activator separated from the enzyme. Further chromatography on Sephadex G-100 and G-200 showed that the latter consisted of two components. The smaller of these (mol wt is approximately equal to 35 000) possessed enzyme transfer activity but the larger one (mol wt 80 000-90 000) required the addition of activator before any enzyme transfer activity was demonstrable. The activator itself was devoid of transfer activity. After chromatography of crude extracts of asparaginyl-tRNA synthetase on Sepharose 4B the activity in the enzyme peak, which was of considerably larger molecular weight than any of the fractions found after purification could be enhanced by addition of the activator. None of the fractions of the enzyme or of activator catalysed any extensive asparagine-dependent ATP-PP-i exchange, either in the presence or the absence of added tRNA.


Deoxyribonucleotide metabolism in Herpes simplex virus infected HeLa cells. The effect of Rolly No. 11 strain herpes simplex virus infection of HeLa cells in culture on deoxynucleotide metabolism and the level of various enzymes concerned with the biosynthesis of DNA has been investigated. Of 18 enzyme activities studied, thymidine kinase, DNA polymerase and deoxyribonuclease were markedly augmented, a finding in agreement with previous reports. Deoxycytidine kinase, ribonucleotide reductase, thymidylate kinase and deoxycytidylate deaminase activities, in contrast with previous reports, did not increase; the activities of the other enzymes studied, also did not increase. Whereas most of the radioactivity derived from [14-C] thymidine in the acid-soluble fraction of the uninfected cells was present as deoxythymidine triphosphate, that present in the infected cells was primarily in the form of deoxythymidine monophosphate. Thus, in the infected cell deoxythymidylate kinase is a rate-limiting enzyme in the biosynthesis of deoxythymidine triphosphate. A marked increase in the pools of the four naturally occurring deoxynucleoside triphosphates (dTTP, dCTP, dATP, dGTP) was found. The rate of formation of the virus-induced enzymes was determined, as were the various nucleoside triphosphate pools and the other phosphorylated derivatives of thymidine; a maximum was reached for all these csmponents between 6 to 8 h post infection. Although an apparent greater synthesis of DNA occurred in the uninefected cells, when the specific activity of the radioactive deoxythymidine triphosphate was taken into account, there was actually a greater rate of DNA synthesis in the infected cells, with the peak at 8 h post infection.


Difference in the action of ethyl and methyl methane sulfonates on DNA template activity for RNA synthesis in vitro. RNA produced in vitro from alkylated T7 DNA has been characterized by polyacrylamide gel electrophoresis. Methylation of T7 DNA by methyl methane sulfonate reduces RNA chain length. In contrast, ethylation of T7 DNA by ethyl methane sulfonate, while reducing RNA synthesis to the same extent, does not alter chain length.


A transfer RNA-dependent protein synthesizing system from Ehrlich ascites extracts. An homologous cell-free system from Ehrlich ascites cells was made tRNA dependent by passing both the ribosomes and the ammonium sulfate fraction through DEAE-cellulose columns in 0.30 M KCl. Protein synthesis was routinely stimulated 20-50 fold by the addition of 100 mug/ml of ascites cell tRNA in the presence of oviduct mRNA, ascites mRNA, or encephalomyocarditis RNA.


Control of platelet glycogenolysis; Activation of phosphorylase kinase by calcium. An apparent enigma during platelet aggregation is that increased glycogenolysis occurs despite a fall in cyclic AMP levels; Activation by a classical cascade is therefore unlikely, and an alternative stimulus for phosphorylase a formation was sought. It was found that low levels of Ca-2+ markedly activate phosphorylase b kinase from human platelets, with a Ka of 0i muM Ca-2+, which is similar to that for the skeletal muscle enzyme; The kinase activity is unstable, and on enzyme ageing is a 50% loss in activity with the Ka decreasing to 0.33 muM Ca-2+. In unstilulated platelets, phosphorylase a was 13.3% of toal measured activity, and glycogen synthetase I was 32.3%. Aggregation induced by ADP did not change the percentage of I synthetase, while increasing that for phosphorylase a. Dibutyryl cyclic AMP did, as expected, increase the percentage of both phosphorylated enzymes; These findings suggest that the natural activator of platelet glycogenolysis during aggregation is Ca-2+, which directly stimulates phosphorylase b kinase without altering glycogen synthetase activity. The cyclic AMP-dependent protein kinase does not appear to be involved;


A quantitative assay for concanavalin A- and Ricinus communis agglutinin-mediated agglutinations of rat ascites hepatoma cells. Relationship between concanavalin A binding and cell agglutination. A simple quantitative assay method was developed for the agglutination of rat ascites hepatoma cells mediated by Concanavalin A or Ricinus communis agglutinin. This method was based on the principle that the turbidity of a cell suspension is proportional to the sum of the cross-sectional area of cells and aggregatesmas predicted by the theoretical consideration, the turbidity decreased when cells were aggregated and the decrease was a function of the average number of the cells in aggregates. The agglutinability of the cells, judged by this method, showed a maximum value at a certain concentration of the agglutinin. By further addition of the agglutinin, the agglutinability slightly decreased from the maximum. These phenomena were observed both for Concanavalin A and Ricinus communis agglutinin. The binding and the agglutination experiments using [3-H]concanavalin A revealed that the binding to approx;0% of the total receptors caused a maximal agglutination. This suggested that the receptors responsible for the agglutination constitute only a small part of the total receptors on the surface.


ESR study of aqueous dispersions of beta-lactoglobulin and spin-labelled glyceryl monostearate. From the ESR spectra of aqueous dispersions of synthetic glyceryl monostearate (spin labelled at C-12) a critical micelle concentration of 30 mumol/l at room temperature was obtained, which agrees with that deduced from surface tension measurements. At monoglyceride concentrations smaller or larger than the critical micelle concentration, the monomers show increased motional restriction with increasing molar ratio of beta-lactoglobulin to monoglyceride up to a value of 10, as determined from calculated rotational correlation times; A similar progressive interaction was deduced from spectral changes observed on equimolar dispersions of beta-lactoglobulin and monoglyceride on raising the temperature to 55 degrees C at which the protein and monoglyceride coprecipitate. The relevance of these finding for non-labelled monoglyceride dispersions is indicated by the similarity of the pH-dependent flocculation behaviour of labelled and non-labelled monoglycerides, both in the absence and presence of beta-lactoglobulin; In addition, proton magnetic resonance and mechanical stability measurements suggest that spin-labelled glyceryl monosterate behaves analogously to non-labelled glyceryl monooleate.


Inhibition by cyclic AMP of lactose production in lactating guinea pig mammary gland slices. Addition of the cyclic AMP phosphodiesterase inhibitors theophylline (10- minus 2 M) or papaverine (10- minus 4 M) leads to a complete inhibition of lactose synthesis in incubated guinea pig mammary gland slices. Addition of 10- minus 5 M cyclic AMP or dibutyryl cyclic AMP results in 1 30-40% inhibition of the synthesis, which effect is not increased by applying higher concentrations of these compounds. A 30-40% inhibition can also be obtained with epinephrine (5 - 10- minus 5 M), or isoproterenol (10- minus 4 M), but the polypeptide hormones glucagon (10- minus 7 M), insulin (1 munit/ml) and relaxin (10 mug/ml) do not significantly affect lactose synthesis. Cytochalasin B (5 mug/ml) inhibits lactose production by 58and colchicine (10- minus 5 M) by 25%. These experiments suggest that an increase in the intracellular level of cyclic AMP either through its addition, through hormonal stimulation of its synthesis, or through inhibition of its intracellular breakdown, leads to an inhibition of lactose production in lactating mammary gland. This effect of cyclic AMP is similar to that of progesterone, which is known to inhibit lactation in vivo and the withdrawal of which at parturition has been postulated to initiate lactogenesis.


Adenosine 3'5'-m onophosphate dependent phosphorylation of ribosomes and ribosomal subunits from bovine corpus luteum. In a previous publication the purification and properties of two protein kinases (KI and KII) from a soluble fraction of bovine corpus luteum and the stimulation of the latter fol. Chem. 248,494-501). We have now studied the effects oc cyclic AMP and luteinizing hormone on ribosomal protein phosphorylation of corpus luteum by protein kinase II. Protein kinase II catalyzed the phosphorylation of ribosomes by transfer of terminal phosphate of ATP to ribosomal proteinsmextraction with hot trichloroacetic acid and non-aqueous solvent revealed that about 80% of total radioactivity incorporated remain associated with the protein residue. Radioactivity was identified in the phosphoserine and phosphothreonine residues of polypeptides by high voltage paper electrophoresis; The extent of phosphorylation was stimulated by cyclic AMP but not by luteinizing hormonemat least 9 proteins of 80-S ribosomes and 12 proteins of the 60-S ribosomal subunit were phosphorylated in the presence of cyclic AMP as resolved by urea polyacrylamide gel electrophoresis. However, only one major and four minor bands were phosphorylated in the ase of 40-S ribosomal subunit under the influence of cyclic AMP. The ribosomal protein phosphorylation catalyzed by protein kinase II is regulated by cyclic AMP wherease luteinizing hormone has no effect on ribosome phosphorylation.


Effect of adrenergic agents on alpha-amylase release and adenosine 3',5'-monophosphate accumulation in rat parotid tissue slices. The role of cyclic AMP in stimulus-secretion coupling with investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20-30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both alpha-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effectivema parallel order of potency and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fractionmthe results suggest that these drugs are acting on a parotid acinar cell through a beta1-adrenergic mechanismmat the lowest concentrations tested, each of the adrenergic agonists stimulated significant alpha-anylase release with no detectable stimulation of cyclic AMP accumulationmeven in the presence of theophylline, phenylephrine at several concentrations increased alpha-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intra-cellular concentration of cyclic AMP may not be necessary for stimulation of alpha-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of alpha-amylase release by isoproterenol. Stimulation of alpha-amylase release by phenylephrine was only partially blocked by either alpha- or beta-adrenergic blocking agents, whereas stimulation of adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolaminemphenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and alpha-amylase release by N-6,O-2'-dibutyryl adenosine 3',5'-monophosphate; These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using alpha-adrenergic blocking agents as tools for investigation of alpha- and beta-adrenergic antagonism.


Selenium - induced redistribution of cadmium binding to tissue proteins: a possible mechanism of protection against cadmium toxicity. The mechanisms involved in the protection by Se against Cd toxicity in the rat were investigated. Se was found to significantly increase the Cd content in the blood and the testis, while decreasing that in the liver and kidney. Se diverted almost all the Cd in the soluble fraction of the testis from low-molecular-weight (MW) proteins to larger ones. Since the soluble fraction was the major subcellular Cd-binding component, the diversion of Cd by Se appears to be a mechanism involved in the protection by this element against the Cd-induced testicular injury. The diversion in binding of the Cd in the soluble fraction to higher MW proteins was also observed in the kidney and liver, and may be a second mechanism involved in the protection of these organs against Cd by Se, in addition to the reductive effect of Se on the tissue Cd concentration. Se was also found in these higher MW Cd-binding proteins. Based on a similarity of MW of about 115,000, the Cd-binding, Se-containing proteins found in these organs appear to be similar. A diversion of Cd from lower MW proteins to larger ones by Se was also found in the plasma, but the Cd-binding, Se-containing proteins in plasma appear to be different from those found in the other organs since they have a larger MW.


Effects of sulfhydryl reagents on basal and vasopressin-stimulated Na+ transport in the toad bladder. The role of reactive SH groups (presumably in proteins) of the apical plasma membrane in transepithelial Na+ transport was studied in the isolated urinary bladder of the toad. On the basis of assays for TCA-soluble SH compounds (e.g., glutathione, methionine), PCMB, PCMPS, NTCB, and DTNB did not penetrate the intracellular compartment from the luminal media either in control or vasopressin-treated bladders. In contrast, PCMB from the serosal side and NEM from the luminal side titrated significant fractions of the TCA-soluble SH compounds. We conclude, therefore, the PCMB, PCMPS, NTCB, and DTNB are suitable reagents for studies on the physiological properties of apical plasma membrane SH groups. Titration of apical membrane SH groups with PCMPS, NTCB, and DTNB revealed heterogeneity in functional responses: PCMPS and NTCB elicited transient, 25-60% increases in SCC. In substrate-free media, pretreatment with these reagents inhibited the increase in SCC produced by vasopressin or cyclic AMP (+ theophylline). In glucose-enriched media, the responses to combinations of vasopressin and PCMPS or NTCB were additive, implying activation via parallel pathways. Simultaneous addition of vasopressin or cyclic AMP (+ theophylline) and NTCB resulted in marked synergism, presumably as a result of unmasking of SH groups by the the hormone (or the intermediate). These results suggest that basal Na+ transport is regulated in part by SH compounds in the apical membrane that are distinct, although not necessarily different in kind, from those involved in the response to vasopressin.


Co-ordination behaviour of nucleic acid bases against the Fe(I)(NO)-2 group in solution. I. Purines: ESR study. The binding sites of purine bases in the presence of the Fe(I)(NO)-2 group were investigated on the basis of the nuclear hyperfine structure of the electron spin resonance spectra. Selective isotopic substitution with 15-NO was used to clarify co-ordindination. Coupling constants and different types of complexes were determined by means of computer-simulated ESR spectra. Comparison was made with the binding sites of nucleotides and it was concluded that N-7, in the imidazole ring, is the preferred binding site. A structure with two base molecules bonded to the iron atom was proposed. Finally 8-azaguanine, an antitumoral agent, was studied and the special behaviour of this antimetabolite was demonstrated in the biologically-interesting pH values.


[Phospholipid composition of synaptosomes from the brains of normal rabbits and rabbits poisoned with phospho-organic cholinesterase inhibitors]. The authors studied quantitative and qualitative composition of phospholipids isolated by fractionation in the sucrose density gradient. The diacyl-aminophospholipid (APL) content of synaptosomes was lower than that of the brain homogenates. On the other hand, the plasmologen APL, phosphoinositide and phosphatidylcholine content was greater in the synaptosomes. Strong excitation of the CNS induced with organo phosphorus inhibitors resulted in a 25% decrease of diacyl APL contnet and a 17% elevation of plasmalogen APL content in the whole brain homogenate. The phospholipid composition of synaptosomes remained unchanged, however.


[The effect of ethymizole on the concentration of cyclic 3',5'-adenosine monophosphate in brain tissue]. It was found by the enzymatic method of cAMP determination that aethymizol (25 mg/kg) caused a more than double increase in cAMP in the rat brain tissue 20 minutes after the intraperitoneal injection. Changes in carbohydrate metabolism in the brain typical of cAMP increased, i.e. reduction in glycogen and an increase in glucose were revealed. The mechanism of aethymizol action was apparently connected with an increase in cAMP formation in the brain tissue.