Glial fibrillary acidic protein from bovine and rat brain. Degradation in tissues and homogenates. Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol wt. polypeptide in bovine tissues incubated at 24 degrees C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000-40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 degrees C. At pH 8.0 PROTEOLYSIS OF The glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.


Studies on the binding of haemoglobin by haptoglobin using electrofocusing and gradient electrophoresis. 1. Gel electrofocusing followed by gel gradient electrophoresis separated the haptoglobins and their complexes with haemoglobin into characteristic two-dimensional patterns of protein bands. 2. Molecular weights of 107 000, 139 000 and 168 000 were obtained for the three bands seen after a purified preparation of haptoglobin type 1 was partially saturated with haemoglobin. This indicated that free haptoglobin, the intermediate haptoglobin-haemoglobin complex containing one half-haemoglobin and the saturated complex with two half-haemoglobins were present. 3. The three proteins showed considerable microheterogeneity and gave a number of isoelectric points in the pH ranges 4.58-4.77, 5.20-5.40 and 5.74-5.93, free haptoglobin type 1 being the lowest group. These ranges were all 0.15-0.30pH units lower if other values were taken for the isoelectric points of markers used to calibrate the pH gradient. 4. All three proteins were present over a wide range of haemoglobin concentrations, from 0.5% to 92% of that required for saturation. This would be expected if both binding sites have similar affinities for haemoglobin.


Cytochrome P450cam and its complexes. Mössbauer parameters of the heme iron. Mössbauer spectroscopy has been used to study the heme iron in various states of cytochrome P450cam from the camphor-hydroxylating system of the bacterium Pseudomonas putida. Native, camphor-free P450cam contains low-spin ferric iron, part of which (approx. 50-70%) is converted to the high-spin ferric state upon addition of camphor. The Mössbauer spectra of the camphor-free enzyme (S equals 1/2) and of the high-spin component (S equals 5/2) of the camphor complex have been successfully simulated using a model based on crystal-field theory and simple convalency considerations. The native low-spin ferric state of P450cam forms a complex with 2-phenylimidazole, with small changes in the g values and Mössbauer spectra. These changes can be accounted for consistently in the crystal-field model referred to above. The addition of putidaredoxin to the camphor-complexed, oxidized P450cam decreases the intensity of the high-spin component and changes its quadrupole splitting. The reduced form of P450cam contrins high-spin ferrous iron, both in the presence and absence of camphor. The complex of reduced P450cam with molecular oxygen is diamagnetic and has a combination of quadrupole splitting and isomer shift that is unusual for a ferrous complex, but strongly resembles that of oxyhemoglobin. These results are compatible with the bound superoxide, Fe3+-O-2, model proposed for oxyhemoglobin (Weiss, J. J. (1964) Nature 202, 83-84). Reduced P450cam and its complexes, oxyP450cam-CO, are all found to be analogous in some respects to the corresponding hemoglobin complexes.


Cross partition and determination of net charge of the isoenzymes of enolase. Enolase from bakers' yeast was separated into three isoenzymes by countercurrent distribution. The isoenzymes were partitioned in aqueous polymer two-phase systems containing positively charged trimethylamino poly(ethylene glycol) or negatively charged poly(ethylene glycol) sulphonate. The plots of the partition coefficient of each isoenzyme versus pH in the two biphasic systems intersect at pH equal to the isoelectric point. From slopes of the plots, the net charge of the isoenzymes at pH 6.57 was determined to be +2, -3, and -8 respectively.


Avoidance of strongly chaotropic eluents for immunoaffinity chromatography by chemical modification of immobilized ligand. The need for chaotropic eluents in immunoaffinity chromatography is a consequence of the high affinities of antibodies towards their antigens. This affinity is decreased and elution of antiglucagon antibodies from a column of immobilized glucagon can be achieved under mild conditions when the steric complementarity to the antibody binding site is perturbed by selective chemical modification of the hormone. The effects of reaction with 2-hydroxy-5-nitrobenzyl bromide, tetranitromethane and hydrogen peroxide have been studied. Conversely, treatment of immobilized antibodies with 2-hydroxy-5-nitrobenzyl bromide facilitates the elution of glucagon during immunoaffinity chromatography. The general implications of these results are discussed.


Release and activation of a particulate bound acid phosphatase from Tetrahymena pyriformis. A sedimentable form of acid phosphatase (EC 3.1.3.2) from Tetrahymena pyriformis was found to be solubilized by Triton X-100. The total enzyme activity in the insoluble cell fraction increased almost 200% upon solubilization with Triton X-100 or Nonidet P-40. Removal of membrane lipids and Triton X-100 from the particulate wash solution with a chloroform extraction resulted in non-specific enzyme-protein aggregation which was reversible upon addition of Triton X-100. The results indicate that this acid phosphatase is an integral membrane protein. The pH optima for this particulate bound acid phosphatase was 3.5 with o-carboxyphenyl phosphate and 4.0 with p-nitrophenyl phosphate as substrates. The Km values of each substrate were 3.1 and 0.031 mM, respectively.


Partial purification and characterization of post-proline cleaving enzyme: enzymatic inactivation of neurohypophyseal hormones by kidney preparations of various species. The inactivation of the neurohypophyseal hormones arginine vasopressin and oxytocin, both 14C-labelled in the C-terminal glycine residue, by enzymes present in kidney homogenates of various species has been investigated, and some of the enzymes responsible have been partially purified and characterized. The Leu-Gly peptide bond of oxytocin is generally most effectively cleaved by kidney homogenates, although with certain species enzymic activity hydrolyzing the Pro-Leu bond is significant. Degradation of arginine vasopressin is slower than oxytocin in all species studied, and appears to occur by a different overall mechanism since cleavage of the Pro-Arg bond is more significant than hydrolysis of the Arg-Gly bond. The enzyme releasing glycinamide from oxytocin and the "Post-Proline Cleaving Enzyme", which releases C-terminal dipeptide from oxytocin and arginine vasopressin, were partially purified from lamb kidney by ammonium sulfate fractionation and column chromatography. The two enzymes are shown to be separate entities with different pH profiles. The prolyl peptidase activity released the C-terminal dipeptides from oxytocin and arginine vasopressin at similar rates and was inhibited by p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, L-1-tosylamido-2-phenylethylchloromethyl ketone, Co2+, Ca2+, and Zn2+, but significantly enhanced by dithiothreitol. The prolyl peptidase preparation cleaves proline-containing peptide substrates at the Pro-X bond. The rate of cleavage is dependent on the nature of residue X and with the conditions used there is no cleavage when X equals Pro; however, cleavage occurs when X is a D isomer: [Mpr1, D-Arg8] vasopressin is inactivated at a rate similar to [Mpr1, Arg8]- and [Mpr1, Lys8] vasopressin, suggesting that the known prolonged biological action of [Mpr1, D-Arg8] vasopressin is not due to resistance to the prolyl peptidase. In all characteristics tested the lamb kidney prolyl peptidase was identical to the post-proline cleaving enzyme isolated earlier from human uterus. In vivo experiments in the cat suggested that both the glycinamide-releasing enzyme and post-proline cleaving enzyme are present and effective in inactivating neurohypophyseal hormones in the intact animal.


Adenosine phosphyorylase activity as distinct from inosine-guanosine phosphorylase activity in Sarcoma 180 cells and rat liver. Adenosine phosphorylase (EC 2.4.2.-) activity present in Sarcoma 180 cells grown in culture and in rat liver, is shown to be distinct from inosine-guanosine phosphorylase by several criteria: (a) treatment of Sarcoma 180 cell extract with p-chloromercuribenzoate inhibited the two activities to a different extent, (b) adenine selectively protected the adenosine phosphorylase activity of Sarcoma 180 and rat liver extract against heat inactivation, while hypoxanthine selectively protected inosine-guanosine phosphorylase activity, (c) at nearly saturating substrate concentrations and using Sarcoma 180 extract, the rates of ribosylation of a mixture of adenine + hypoxanthine or adenine + guanine, but not of hypoxanthine + guanine, were found to be almost equal to the sum of their individual rates as measured separately, (d) inosine selectively inhibited the ribosylation of hypoxanthine and guanine catalysed by Sarcoma 180 and rat liver extract while 2-chloroadenosine selectively inhibited the ribosylation of adenine and N6-furfuryladenine, (e) pH vs. activity curves were similar with hypoxanthine or guanine as the substrate but they were markedly different from the curve with adenine as the substrate. The potential role of adenosine phosphorylase activity in vivo is discussed.


Purification and properties of an alpha-amylase inhibitor from wheat. Four inhibitors of alpha-amylase (EC 3.2.1.1) were separated from an alcohol extract of wheat by ion-exchange chromatography on DE52-cellulose. One inhibitor, which showed the greatest specificity for human salivary amylase relative to human pancreatic amylase, has been purified by the following steps: (a) alcohol fractionation (60--90%) of water extract (b) ion-exchange chromatography on QAE-Sephadex A-50; (c) re-chromatography on DE52-cellulose and (d) gel filtration on Sephadex G-50. The purified inhibitor is 100 times more specific for human salivary amylase than for human pancreatic amylase. It shows an electrophoretic mobility of 0.2 on disc gel electrophoresis and a molecular weight of about 21 000. This inhibitor contributes about 16% to the total salivary amylase inhibiting power of the wheat extract.


Purification and specificity of prolyl dipeptidase from bovine kidney. Prolyl dipeptidase (iminodipeptidase, L-prolyl-amino acid hydrolase, EC 3.4.13.8) was purified 180-fold from bovine kidney. The enzyme which was obtained in a 10% yield was completely separated from a number of known kidney peptidases including an enzyme of very similar substrate specificity, proline aminopeptidase (L-prolyl-peptide hydrolase, EC 3.4.11.5). The specific activity of the enzyme with L-prolylglycine as substrate is 1600 units of activity per mg protein. Optimum activity of the enzyme is at pH 8.75 and the molecular weight on gel filtration was estimated to be 100 000. The isoelectric point of the enzyme is pH 4.25. Studies of substrate specificity showed that the enzyme preferentially hydrolyzes dipeptides and dipeptidyl amides with L-proline or hydroxy-L-proline at the N-terminus. Longer chain substrates with N-terminal proline were not hydrolyzed.


Isolation and characterization of beta-glucosidase from the cytosol of rat kidney cortex. A procedure is described for the preparation of extensively purified beta-D-glucosidase (EC 3.2.1.21) from the cytosol fraction of rat kidney. The specific activity of the beta-glucosidase in the high speed supernatant (100 000 X g, 90 min) fraction of rat kidney homogenate is 700-fold greater than that in the same fraction from heart, skeletal muscle, lung, spleen, brain or liver. beta-Glucosidase activity co-chromatographs with beta-D-galactosidase, beta-D-fucosidase, alpha-L-arabinosidase and beta-D-xylosidase activities through the last four column steps of the purification and their specific activities are 0.26, 0.39, 0.028 and 0.017 relative to that of beta-glucosidase, respectively. The specific activity of the apparently homogeneous beta-glucosidase is 115 000 nmol of glucose released from 4-methylumbelliferyl-beta-D-glucopyranoside per mg protein per h. All five glycosidase activities possess similar pH dependency (pH optimum, 6--7) and heat lability, and co-migrate on polyacrylamide disc gels at pH 8.9 (RF, 0.67). beta-Glucosidase acitivity is inhibited competitively by glucono-(1 leads to 5)-lactone (KI, 0.61 mM) and non-competitively by a variety of sulfhydryl reagents including N-ethylmaleimide, p-chloromercuribenzoate, 5,5'-dithio-bis(2-nitrobenzoic acid), and iodoacetic acid. Although the enzyme will release glucose from p-nitrophenyl and 4-methylumbelliferyl derivatives of beta-D-glucose, it will not hydrolyze xylosyl-O-serine, beta-D-glucocerebroside, lactose, galactosylovalbumin or trehalose. The enzyme consists of a single polypeptide chain with a molecular weight of 50 000--58 000, has a sedimentation coefficient of 4.41 S and contains a relatively large number of acidic amino acids. A study of the distribution of beta-glucosidase activity in various regions of the dissected rat kidney indicates that the enzyme is probably contained in cells of the proximal convoluted tubule. The enzyme is also present in relatively large amounts in the villus cells, but not crypt cells, of the intestine. The physiological substrate and function of the enzyme are unknown.


Studies on (Na+ + K+)-activated ATPase. XXXVIII. A 100 000 molecular weight protein as the low-energy phosphorylated intermediate of the enzyme. Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg2+ and ouabain has been studied at 0 degrees C (pH 7.4) and 20 degrees C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20 degrees C within 2 min with 50--155 and 5.6--17 muM 32Pi, respectively, and at 0 degrees C within 75 s with 300--600 and 33--66 muM 32Pi, respectively. Maximal phosphorylation yields 146 pmol 32P - mg-1 protein. Without ouabain (20 degrees C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached. Preincubation of the native microsomes with Mg2+ and K+, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations. Phosphorylation of the native microsomes by ATP in the presence of Mg2+ and Na+ reaches 90 and 50% maximal levels within 15--30 s at 0 degrees C and pH 7.4 at concentrations of [gamma-32P]ATP of 5--32 and 0.5--3.5 muM, respectively. The maximal phosphorylation level is 149 pmol 32P-mg-1 protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate on site per active enzyme subunit of 135 000 molecular weight. From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0 degrees C and 20 degrees C standard free-energy changes of --5.4 and --6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol - degree K. This characterizes the reaction as a process driven by an entropy change. The intermediate formed by phosphorylation with Pi has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. Solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range of 4--7. The non-solubilized preparation has optimal stability at pH 2--4, the level of which is equal to that of detergent-solubilized intermediate. Sodium dodecyl sulfate gel electrophoresis of the microsomes at pH 3, following incorporation of 32Pi yields 11 protein bands, only one of which (mol. wt 100 000--106 000) carries the radioactive label. This protein has the same molecular weight as the protein, which is phosphorylated by ATP in the presence of Mg2+ and Na+.


Partial purification and properties of a chromatin-associated phosphoprotein kinase from rat liver nuclei. A phosphoprotein kinase (EC 2.7.1.37) KIVb, from rat liver nuclei, was purified 75-fold by phosphocellulose chromatography and gel filtration on Sephadex G-200. The enzyme, which has an apparent molecular weight of 55 000, phosphorylates casein and chromatin-bound nonhistone proteins more readily than histones or ribosomal proteins. It exhibits an absolute requirement for divalent cation with optimum activity at 15--20 mM Mg2+. Maximal kinase activity is achieved at 100 mM NaCl. The pH vs. activity curve is biphasic with optima at pH 6.5 and pH 8.0. The Km value for casein is 280 mug/ml and the Km for ATP is 6-10(-6) M. Kinase KIVb phosphorylates numerous nonhistone nuclear proteins as shown by electrophoretic analysis. The addition of kinase KIVb to reaction mixtures containing nonhistone proteins results in the phosphorylation of a spectrum of polypeptides similar to those that are phosphorylated by endogenous nuclear kinases. Nonhistone proteins bound to chromatin appear to be better substrates for KIVb than nonhistones dissociated from chromatin. A comparison of nuclear phosphoproteins phosphorylated either in the intact animal or in vitro (by the addition of kinase KIVb) indicates some differences and some similarities in the patterns of phosphorylation.


Purification and some properties of rat liver cysteine oxidase (cysteine dioxygenase). Cysteine oxidase (cysteine dioxygenase, EC 1.13.11.20) was purified approximately 1000-fold from rat liver. The purified enzyme (protein-B) was obtained as an inactive form, which was activated by anaerobic preincubation with L-cysteine. The active form of protein-B was inactivated during aerobic incubation to produce cysteine sulfinate. This inactivation of protein-B was protected by a distinct protein in rat liver cytoplasm, namely stabilizing protein (protein-A). The Ka and Km values for L-cysteine were 0.8-10(-3) M and 1.3-10(-3) M respectively. The enzyme was strongly inhibited by Cu+ and/or Fe2+ chelating agents but not by Cu2+ chelating agent. The optimum pH of enzyme reaction was 8.5-9.5 while that of enzyme activation was 6.8-9.5, with a broad peak.


Conformations of lysine-sensitive aspartokinase. 1. The technique of differential thermal and proteolytic inactivation has been employed as a conformational probe for the lysine-sensitive aspartokinase (EC 2.7.2.4) of Escherichia coli B. 2. L-Amino acid inhibitors of this enzyme each induce a characteristic enzyme conformation. This is evidenced by rates of thermal and proteolytic inactivation and Arrhenius activation energies for thermal inactivation which are characteristic of the amino acid present. 3. Phenylalanine and leucine binding are mutually exclusive as evidenced by competitive behavior in thermal inactivation experiments, suggesting a hydrophobic amino acid binding site with broad specificity. 4. The phenylalanine-dependent conformation and the leucine-dependent conformation differ considerably. In comparison with the native enzyme, the former is more labile to proteolysis by trypsin whereas the latter is more stable. First-order rate constants for thermal inactivation of the phenylalanine- and leucine-dependent conformations are, respectively, about one-half and one-tenth that of the native enzyme. 5. Items 3 and 4 taken together suggest that the conformations are ligand induced and do not arise via ligand stabilization of spontaneously arising conformers.


The sulphatase of ox liver. XIX. On the nature of the polymeric forms of sulphatase A present in dilute solutions. Weight-average elution volumes of sulphatase A (an arylsulphate sulphohydrolase, EC 3.1.6.1) from Sephadex G-200 have been determined as functions of protein concentration, pH, ionic strength and temperature. The results are used to calculate the apparent association equilibrium constants for tetramer formation and the associated standard-state thermodynamic parameters. While the apparent association constant decreased from 10(28) to 10(21) M-3 on increasing the pH from 4.5 to 5.6 at ionic strength 0.1, at any particular pH value studied it was relatively insensitive to temperature variation so that deltaH is close to zero and tetramer formation in solution is associated with a positive entropy change. At pH 5.0, increasing the ionic strength from 0.1 to 2 decreased the association constant by a factor of 100. Methylumbelliferone sulphate has no effect on the association of sulphatase A. The equilibrium results are used to define the degree of association of sulphatase A likely to encountered in experiments designed to elucidate its kinetic properties. In the liver lysosome, the tetramer is probably the dominant species. The monomer and tetramer of sulphatase A have similar, or identical, specific activities with nitrocatechol sulphate and 4-methylumbelliferone sulphate as substrates. With nitrocatechol sulphate, sulphatase A shows Michaelis kinetics under conditions where the monomer is the dominant species and non-Michaelis kinetics where the tetramer is dominant. There is apparently a negative cooperativity between the monomer units in the tetramer. In 2 mM sodium taurodeoxycholate and 0.035 M MnCl2, but not in 0.1 M NaCl, the tetramer shows Michaelis kinetics. This is not due to dissociation of the tetramer. The critical micellar concentration of sodium taurodeoxycholate is about 0.8 mM in both 0.1 M NaCl and 0.035 M McCl2 but the aggregation number is greater in the latter.


Purification and characterization of an extracellular exo-D-galacturonanase of Aspergillus niger. A D-galacturonanase (EC 3.2.1.67) catalyzing the degradation of D-galacturonans by terminal action pattern was purified from a culture filtrate of Aspergillus niger by a procedure including the salting-out with ammonium sulfate, precipitation by ethanol, chromatography on DEAE-cellulose, and gel chromatography on Sephadex G-100. The obtained preparation was slightly contaminated by an enzymically inactive protein fraction. Maximum activity and stability of the enzyme was observed at pH 5.2. The enzyme degrades digalacturonic acid, p-nitrophenyl-alpha-D-galactopyranuronide, as well as oligogalacturonides containing at the nonreducing end 4-deoxy-L-threo-hexa-4-enopyranosyluronate. It differs from all A. niger enzymes so far described which degrade D-galaturonans by the terminal action pattern, in not clearly preferring low-molecular substrates. It is therefore classified as an exo-D-galacturonanase.


The biosynthesis of multi-L-arginyl-poly(L-aspartic acid) in the filamentous cyanobacterium Anabaena cylindrica. The cyanobacteria produce multi-L-arginyl-poly (aspartic acid), a high molecular weight (Mr=25 000-125 000) branched polypeptide consisting of a poly(aspartic acid) core with L-arginyl residues peptide bonded to each free carboxyl group of the poly(aspartic acid). An enzyme which will elongate Arg-poly(Asp) has been isolated and purified 92-fold from the filamentous cyanobacterium Anabaena cylindrica. The enzyme incorporates arginine and aspartic acid into Arg-poly(Asp) in a reaction which requires ATP, KCl, MgCl2, and a sulfhydryl reagent. The enzymatic incorporation of arginine is dependent upon the presence of L-aspartic acid but not visa versa, a finding which suggests the order of amino acid addition to the branched polypeptide-aspartic acid is added to the core followed by the attachment of an arginine branch. The elongation of Arg-poly(Asp) in-vitro is insensitive to the addition of protein synthesis inhibitors and to the addition of nucleases. These findings support the notion previosly suggested from in-vivo studies that Arg-poly(Asp) is synthesized via a non-ribosomal route and also demonstrate that amino-acetylated transfer-RNAs play no part in at least one step of the biosynthetic mechanism.


Separation of subchloroplast membrane particles by counter-current distribution. Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity. The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique. Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks. The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicated that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae.


Stimulation of ATP synthesis in Halobacterium halobium R1 by light-induced or artifically created proton electrochemical potential gradients across the cell membrane. The relationship between proton movement and phosphorylation in Halo-bacterium halobium R1 has been investigated under anaerobic conditions. The light-induced changes in the bacteriorhodopsin are accompanied by proton movements across the membrane which result in pH changes in the suspending medium. The initial alkaline shift is shown to be closely paralleled by (and hence correlated with) ATP synthesis. Acidification of the medium in the presence of valinomycin, under conditions of low external potassium, brings about ATP synthesis in the dark.


Mechanism of active shrinkage in mitochondria. II. Coupling between strong electrolyte fluxes. 1. Addition of succinate to valinomycin-treated mitochondria incubated in KCL causes a large electrolyte penetration. The process depends on a steady supply of energy and involves a continuous net extrusion of protons. Rates of respiration and of electrolyte penetration proceed in a parallel manner. 2. A passive penetration of K+ salt of permeant anions occurs in respiratory-inhibited mitochondria after addition of valinomycin. Addition of succinate at the end of the passive swelling starts an active extrusion of anions and cations with restoration of the initial volume. The shrinkage is accompanied by a slow reuptake of protons. The initiation of the active shrinkage correlates with the degree of stretching of the inner membrane. The extrusion of electrolytes is inhibited by nigericin, while it is only slightly sensitive to variations of the valinomycin concentration larger than two orders of magnitude. 3. Passive swelling and active shrinkage occurs also when K+ is replaced by a large variety of organic cations. The rate of organic cation penetration is enhanced by tetraphenylboron, while the rate of electrolyte extrusion is insensitive to variation of the tetraphenylboron concentration. 4. Active shrinkage, either with K+ or organic cation salts, is inhibited by weak acids. The phosphate inhibition is removed by SH inhibitors. The active shrinkage is also inhibited by mersalyl to an extent of about 60%. 5. Three models of active shrinkage are discussed: (a) mechanoprotein, (b) electrogenic proton pump, and (c) proton-driven cation anion pump.


Inhibitory effect of p-nitrothiophenol in the light on the photosystem II activity of spinach chloroplasts. The treatment of spinach chloroplasts with p-nitrothiophenol in the light at acidic and neutral pH'S caused specific inhibition of the Photosystem II activity, whereas the same treatment in the dark did not affect the activity at all. The photosystem I activity was not inhibited by p-nitrothiophenol both in the light and in the dark. The inhibition was accompanied by changes of fluorescence from chloroplasts. As observed at room temperature, the 685-nm band was lowered by the p-nitrothiophenol treatment in the light and, at liquid nitrogen temperature, the relative height of the 695-nm band to the 685-nm band increased and the 695-nm band shifted to longer wavelengths. The action spectra for these effects of p-nitrothiophenol on the activity and fluorescence showed a peak at 670 nm with a red drop at longer wavelengths. It was concluded that the light absorbed by Photosystem II is responsible for the chemical modification of chloroplasts with p-nitrothiopehnol to causing the specific inhibition of Photosystem II.


Relations between the electrical potential, pH gradient, proton flux and phosphorylation in the photosynthetic membrane. The transmembrane electrical potential (deltaphi), the proton flux (H+), the rate of electron transport (e), the pH gradient (deltapH) and the rate of phosphorylation (ATP) were measured in chloroplasts of spinach. Photosynthesis was excited periodically with flashes of variable frequencies and intensities. A new method is described for determining the rate of electron transport and proton flux. Under conditions where the rate of electron transport and proton flux are not pH controlled the following correlations were found in the range 50 mV less than or equal to deltaphi less than or equal to 125 mV and 1.8 less than or equal to deltapH less than or equal to 2.7: (1) The pH gradient, deltapH, increases with H+ independently of Phout between 7-9. (2) The rate of phosphorylation, ATP, depends exponentially on deltapH (at constant deltaphi) and is independent of pHout between 7-9. (3) The rate of phosphorylation, ATP, depends also on deltaphi (at constant deltapH and at constant proton flux H+). (4) The proton flux via the ATPase pathway, Hp+, depends non-linearly on the ratio of the proton concentrations: Hp+ approximately (Hin+/Hout+)b, (b=2.3--2.6). The proton flux via the basal pathway, Hb+, depends linearly on the ratio of the proton concentrations: Hb+ approximately (Hin/Hout). (5) The ratio deltaH+/ATP (e/ATP, i.e. the ratio of the total proton flux, Hp+ + Hb+, and the rate of ATP formation, ATP, depends strongly on deltaphi and on deltapH. The ratio is deltaH+/ATP approximately 3 (e/ATP approximately 1.5) at deltapH 2.7 and deltaphi = 125 mV. (6) It is supposed that the reason for the dependence of deltaH+/ATP on deltaphi anddeltapH is the different functional dependence of the basal proton flux Hb+ and the phosphorylating proton flux Hp+ on deltapH and deltaphi. The calculation of deltaH+/ATP on the basis of this assumption is in fair agreement with the experimental values. Also the "threshold" effects can be explained in this way. (7) The ratio of deltaHp+/ATP, i.e. the ratio of the phosphorylating proton flux Hp+ and ATP, is deltaHp+/ATP APPROXIMATELY 2.4.


A possible mechanism of the generation of singlet molecular oxygen in nadph-dependent microsomal lipid peroxidation. A simplified system, consisting of NADPH, Fe3+-ADP, EDTA, liposomes, NADPH-cytochrome c reductase and Tris - HCl buffer (pH 6.8), has been employed in studies of the generation of singlet oxygen in NADPH-dependent microsomal lipid peroxidation. The light emitted by the system involves 1deltag type molecular oxygen identifiable by its characteristic emission spectrum and its behavior with beta-carotene. The generation of another excited species (a compound in the triplet state) could be demonstrated in this system by changes of light intensity and emission spectra which arise from photosensitizer (9,10-dibromoanthracene sulfonate, eosin, Rose-Bengal)-mediated energy transfers. Chemiluminescence in the visible region was markedly quenched by various radical trappers and by an inhibitor of NADPH-cytochrome c reductase, but not by superoxide dismutase. During the early stage of lipid peroxidation, the intensity of chemiluminescence was proportional to the square of the concentration of lipid peroxide. These characteristics suggest that singlet oxygen and a compound in the triplet state (probably a carbonyl compound) are generated by a self-reaction of lipid peroxy radicals.


Primary reactions of photosystem II at low pH. I. Prompt and delayed fluorescence. Prompt and delayed chlorophyll fluorescence have been studied in broken spinach chloroplasts at pH values down to 2.6. No direct effect of low pH on the primary charge separation in Photosystem II was observed. The irreversible inactivation of a secondary electron donor in a narrow pH range around pH 4.5 was demonstrated. At lower pH values the photooxidized form of a more primary electron donor, revealed by its efficient fluorescence quenching, was reduced with a half time of about 200 mus, 25% by another electron donor and 75% by back reaction with the reduced acceptor. The electron donation had a half time of 800 mus and was practically irreversible. The back reaction had a pH dependent half time: about 270 mus at pH 4 and increasing towards lower pH. The competition of both reactions resulted in a net efficiency of the charge separation at pH 4 of 25%, increasing towards lower pH.


Immunological similarity between NADH-cytochrome b5 reductase of erythrocytes and liver microsomes. In a number of animal species soluble NADH-cytochrome b5 reductase of erythrocytes was compared with membrane-bound NADH-cytochrome b5 reductase of liver microsomes by using an antibody to purified NADH-cytochrome b5 reductase from rat liver microsomes. The results obtained indicated clearly that they are immunologically very similar to each other. The data with erythrocyte ghosts suggested that cytochrome b5 and NADH-cytochrome b5 reductase are also present in the ghost.


Effect of NADP on light-induced cytochrome changes in membrane fragments from a blue-green alga. The effect of NADP+ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragements prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NDAP+. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH2 as the electron donor, NADP+ had no effect on the light-induced redox changes of cytochromes: with or without NADP+, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an alpha peak at 549nm. With 664 nm illumination and water as the electron donor, NADP+ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b559. Cytochrome b559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP+) for the electron flow from water.


Kinetic studies on cytochrome c oxidase by combined epr and reflectance spectroscopy after rapid freezing. 1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.


Some thermodynamic and kinetic properties of the primary photochemical reactants in a complex from a green photosynthetic bacterium. We have examined the bacteriochlorophyll reaction-center complex of Chlorobium limicola f. thiosulfatophilum, strain Tassajara. Our results indicate that the midpoint potential of the primary electron donor bacteriochlorophyll of the reaction center is +250 mV at pH 6.8, while that of cytochrome c-553 is +165 mV. There are two cytochrome c-553 hemes per reaction center, and the light-induced oxidation of each is biphasic (t1/2 of less than 5 mus and approximately 50 mus). We belive that this indicates a two state equilibrium with each cytochrome heme being either close to, or a little removed from, the reaction-center bacteriochlorophyll. We have also titrated the primary electron acceptor of the reaction center. Its equilibrium midpoint potential at pH 6.8 is below -450 mV. This is very much lower than the previous estimate for green bacteria, and also substantially lower than values obtained for purple bacteria. Such a low-potential primary acceptor would be thermodynamically capable of direct reduction of NAD+ via ferredoxin in a manner analagous to photosystem I in chloroplasts and blue-green algae.


Reaction of uracil and thymine derivatives with sodium bisulfite. Studies on the mechanism and reduction of the adduct. The rates and equilibria for the addition of sodium bisulfite to uracil, thymine, and their nucleosides have been studied for the pH range 3-9.5. The rate of addition for uracil is proportional to the concentration of sulfite ion and unionized uracil. The equilibrium constant (25 degrees C) for the reaction is (1.0 +/- 0.15) X 10(3) 1 - mol-1 for uracil and 0.62 +/- 0.03 1- mol-1 for thymine. A pH of 6-7, with a high bisulfite concentration is suggested for biochemical applications of the uracil reaction. The uracil reaction, which proceeds readily under physiological conditions and has a high equilibrium constant, may be a contributing cause of the biochemical effects of bisulfite and sulfur dioxide. Additional evidence on the structure of the thymine-bisulfite adduct has been obtained by nuclear magnetic resonance spectroscopy. This spectrum supports the assignment of structure as dihydrothymine-6-sulfonate. The uracil-bisulfite adduct is reduced by sodium borohydride to sodium 3-ureido-propanol-2-sulfonate. This reaction is suggested for the chemical modification of nucleic acids.


Intramembrane particle aggregation in erythrocyte ghosts. II. The influence of spectrin aggregation. Physicochemical properties of mixtures of spectrin and actin extracted from human erythrocyte ghosts have been correlated with ultrastructural changes observed in freeze-fractured erythrocyte membranes. (1) Extracted mixtures of spectrin and actin have a very low solubility (less than 30 mug/ml) near their isoelectric point, pH 4.8. These mixtures are also precipitated by low concentrations of Ca2+, Mg2+, polylysine or basic proteins. (2) All conditions which precipitate extracts of spectrin and actin also induce aggregation of the intramembrane particles in spectrin-depleted erythrocyte ghosts. Precipitation of the residual spectrin molecules into small patches on the cytoplasmic surface of the ghost membrane is thought to be the cause of particle aggregations, implying an association between the spectrin molecules and the intramembrane particles. (3) When fresh ghosts are exposed to conditions which precipitate extracts of spectrin and actin, only limited particle aggregation occurs. Instead, the contraction of the intact spectrin meshwork induced by the precipitation conditions compresses the lipid bilayer of the membrane, causing it to bleb off particle-free, protein-free vesicles. (4) The absence of protein in these lipid vesicles implies that all the proteins of the erythrocyte membrane are immobilized by association with either the spectrin meshwork or the intramembrane particles.


The rate of calcium uptake into sarcoplasmic reticulum of cardiac muscle and skeletal muscle. Effects of cyclic AMP-dependent protein kinase and phosphorylase b kinase. Calcium transport into sarcoplasmic reticulum fragments isolated from dog cardiac and mixed skeletal muscle (quadriceps) and from mixed fast (tibialis), pure fast (caudofemoralis) and pure slow (soleus) skeletal muscles from the cat was studied. Cyclic AMP-dependent protein kinase and phosphorylase b kinase stimulated the rate of calcium transport although some variability was observed. A specific protein kinase inhibitor prevented the effect of protein kinase but not of phosphorylase b kinase. The addition of cyclic AMP to the sarcoplasmic reticulum preparations in the absence of protein kinase had only a slight stimulatory effect despite the presence of endogenous protein kinase. Cyclic AMP-dependent protein kinase catalyzed the phosphorylation of several components present in the sarcoplasmic reticulum fragments; a 19000 to 21 000 dalton peak was phosphorylated with high specific activity in sarcoplasmic reticulum preparations isolated from heart and from slow skeletal muscle, but not from fast skeletal muscle. Phosphorylase b kinase phosphorylated a peak of molecular weight 95000 in all of the preparations. Cyclic AMP-dependent protein kinase-stimulated phosphorylation was optimum at pH 6.8; phosphorylase b kinase phosphorylation had a biphasic curve in cardiac and slow skeletal muscle with optima at pH 6.8 and 8.0. The addition of exogenous phosphorylase b kinase or protein kinase increased the endogenous level of phosphorylation 25-100%. All sarcoplasmic reticulum preparations contained varying amounts of adenylate cyclase, phosphorylase b and a (b:a = 30.1), "debrancher" enzyme and glycogen (0.3 mg/mg protein), as well as varying amounts of protein kinase and phosphorylase b kinase which were responsible for a significant endogenous phosphorylation. Thus, the two phosphorylating enzymes stimulated calcium uptake in the sarcoplasmic reticulum of a variety of muscles possessing different physiologic characteristics and different responses to drugs. In addition, the phosphorylation catalyzed by these enzymes occurred at two different protein moieties which make physiologic interpretation of the role of phosphorylation difficult. While the role phosphorylation in these mechanisms is complex, the presence of a glycogenolytic enzyme system may be an important link in this phenomenon. The sarcoplasmic reticulum represents a new substrate for phosphorylase b kinase.


[Microcolorimetric study of the association of trypsin with a pancreatic inhibitor]. Enthalpy of the association of trypsin with pancreatic inhibitor from bovine pancreas at 25 degrees C as a function of pH and ionic strength is estimated. The dependence of the enthalpy on pH is of an extremal character with a minimum at pH 7.6 (delta H degrees =-10.3 ccal/mole). The increase of ionic strength with the addition of LiCl, KCl and CsCl at pH 7.6 leads to be increase of delta H degrees. Possible mechanisms of enthlpy changes depending on medium conditions are considered.


[Partial purification and properties of protease from Torula thermophila]. 11-Fold purified protease preparation is isolated from cultural medium of Torula thermophila UzPT-1 by means of ammonium sulphate precipitation and gel chromatography through Sephadex G-100. Disc polyacrylamide gel electrophoresis revealed two portease components, one of them possessing proteolytic activity. pH interval for protease activity was found to be 3.5-12, the maximal activity was observed at pH 8.5-11, the highest enzyme resistance--at pH 6-8. The enzyme almost completely preserved its activity for 1 hour in distilled water at 60 degrees C. The temperature maximum of the enzyme activity was 70 degrees at pH 8. The enzyme may be referred to proteases of serine nature, because it is completely inactivated with diisopropylphosphofluoridate, but it retains the activity in the presence of chelating agents (EDTA, o-phenantroline, ditizone) and inhibitors of SH-groups (sodium p-chloromercuriumbenzoate, iodoacetic acid). The enzyme was not inactivated with phenylmethylsulphonylfluoride and the trypsin inhibitor from soybean. The protease studied most efficiently hydrolyzed caseine and hemoglobin, in a less degree--human serum albumin and fibrinogen and almost did not attack egg albumin. The enzyme undergoes association-dissociation under pH change during gel filtration through Sephadex.


[Various properties of the creatine transport system and the location of creatine kinase in skeletal muscle mitochondria]. Water and creatine contents were studied in rat skeletal muscle mitochondria after their 5 min. incubation in creatine solutions, pH 7.2 or 8.4. The content of water and creatine in mitochondria was found to be higher at pH 8.4, than at pH 72, the creatine content correlated with the water content. Structural creatine analogues, containing aminogroups with pKa greater than or equal to 9.5 or carboxyl groups, inhibited the infusion of creatine into mitochondria more strongly than substances having aminogroups with pKa less than 5. The penetrating form is creatine amphiion; the effect of pH on the permeability is probably due to the activation of the creatine transmitter. Rat skeletal muscle mitochondria contain creatine kinase at both sides of the inner membrane. This conclusion is based on the fact that under conditions, supplying the direct course of the creatine kinase reaction (the incubation medium contains Ca2+ and creatine; pH 7.8), ADP produces the stimulation of mitochondrial respiration up to the oxygen exhausting in a polarographic unit. Similarly, ADP irreversibly stimulates mitochondrial respiration in the presence of 1 mM EDTA, if EDTA and ADP are added after the preincubation of mitochondria in creatine-containing medium and after accumulating small amounts of Ca2+ by mitochondria.


[Isolation and properties of a homogeneous L-asparaginase preparation from Pseudomonas fluorescens AG]. Highly purified L-asparaginase having a specific activity of 500+/- +/-40 IU./mg protein is isolated from Pseudomonas fluorescens AG cells. The purification procedure includes isopropanol fractionation, gel filtration through Sephadex G-100, chromatography on hydroxylapatite and DEAE-cellulose columns. The asparaginase preparation is homogenous on the basis of polyacrylamide gel electrophoresis data. The pH optimum is found to be 8.0-9.0, isoelectric point and molecular weight are 4.5+/-0.05 and 70,000+/-5,000 respectively, Km for L-asparagine being-4.1-10(-4)M. The enzyme does not hydrolyse L-glutamine. The hydrolysis rate of D-glutamine is less than 1% of the deamydation rate of L-isomer. p-Chloro-mercurium benzoate at a concentration of 10(-4) M completely inhibits the asparaginase activity. Asparaginase from Ps. fluorescens AG possesses and antileucosic activity, inhibiting 3H-thymidine incorporation into DNA of Berkit lymphoma cells.


[Analysis of sheep blood serum haptoglobin]. Three types of haptoglobin (Hp), differing in the number of bands of Hp--Hb complex on polyacrylamide gel electrophoregramm are found in sheep blood serum. HpA, HpB and HpC fractions included one, two-three and six-eight bands respectively. A modified procedure for the HpC isolation is described. Effects of urea, sodium dodecylsulpate, beta-mercaptoethanol, pH and maleinization on the behaviour of HpC under polyacrylamide gel electrophoresis is studied. The data obtained suggest that the HpC molecule consists of two subunits (of alpha and beta types), bound with S--S bounds in an alphabeta-dimer, which form a whole HpC molecule for the expense of non-covalent bonds.


[Reaction ability and alkylation kinetics of sulfhydride groups of soluble succinate dehydrogenase]. Inhibition kinetics of succinate--an acceptor of oxidoreductase activity of soluble succinate dehydrogenase by N-ethylmaleimide is studied. The alkylation reaction is described by the kinetic equation of the first order, its stechiometric coefficient being 1. The binding of enzyme sulphhydride groups by p-chloromercuriumbenzoate blocks the enzyme alkylation and its inhibition by oxaloacetate. Succinate protects succinate dehydrogenase from the inhibitory effect of N-ethylmaleimide. The reaction of the enzyme with an alkylating agent in the presence of different substrate concentrations corresponds kinetically to the model, according to which a sulphhydride group acts in the active site of the enzyme. pKa of this group is 7.0 at 20degreesC. The dependency of the maximal substrate oxidation reaction rate and that of the enzyme alkylation rate on pH coinside at the pH range 5.8--7.8. The presence of anions in the alkylation medium decreases the reaction ability of the active site with respect to N-ethylmaleimide. A mechanism of the initial stage of succinate oxidation with the cooperation of the sulphhydride group of the enzyme active site is postulated.


[Effect of IAA on the photophosphorylation of pea isolated chloroplasts]. Effect of IAA (10(-10)-10(-3) M) on photophosphorylation, NADP reduction and the oxygen exchange is investigated. It is shown that low concentrations of IAA (10(-10)-10(-7) M) increase the photophosphorylation reaction and the flow of electrones to NADP under the phosphorylation conditions in the chloroplasts, and their effect on the O2 exchange is not the same in different types of photophosphorylation. It is supposed that the effect of IAA on the photophosphorylation is connected with H292 metabolism in chloroplasts and with catalase and peroxidase functions.


[Purification, molecular multiplicity and kinetic properties of "biosynthetic" L-threonine dehydratase from E. coli K-12]. "Biosynthetic" L-threonine dehydratase was purified to homogeneous state with yield 29% of total activity from E. coli K-12. The cells were disrupted by means of ultra sound. Nucleic acids and nucleoproteins were precipitated with protamine sulphate, the proteins were fractioned with (NH4)2SO4, by gel filtration through Sephadex G-25 followed by chromatography on DEAE-cellulose using stepways elution by changing the pH-values. The homogenity of the enzyme was shown by polyacrylamide gel disc electrophoresis in the presence of dodecylsulphate. The enzyme consists of equal subunits having a molecular weight about 57000. The polyacrylamide gel disc electrophoresis had shown that the native enzyme consists of a set of oligomeric forms. The multiplisity of molecular organization of the enzyme was relfected in complicated kinetic behavior: at pH greater than 9 on the plots of initial reaction rate (upsilon) versus initial substrate concentration ([S]0) there were four inflexion points (two intermediate plateaux) the position and deepness of which depended on enzyme concentration. Kinetic properties of the highly purified enzyme and the enzyme in crude cell extracts at pH 9.3 and 7.4 were identical. At pH 8,3 on the upsilon versus [S]0 plots appeared two inflexion points (one intermediate plateau), the position of which practically did not depend on enzyme concentration in the reaction mixture but strongly depended on the enzyme concentration in the stock solution. Repeated polyacrylamide gel disc electrophoresis of several oligomeric forms isolated by the first electrophoresis had shown that oligomeric forms underwent a slow polymerization. It is suggested that "biosynthetic" L-threonine dehydratase from E. coli K-12 is a set of multiple oligomeric forms having different kinetic parameters. Probably, each form of the enzyme has a "simple" kinetics characterized by hyperbolic or sigmoidal shape of upsilon versus [S]0 plots. The rate of equilibrium between the oligomeric forms is small in comparison with the enzyme reaction velosity, that lead to the complex kinetic curves appearing as a result of summing up the kinetics inherent to the individual forms.


[Purification and some properties of electrophoretic variants of 6-phosphogluconate dehydrogenase from rat erythrocytes]. Purification procedure of electrophoretic variants FF and SS of 6-phosphogluconate dehydrogenase (6-PGD) is described. The method includes (NH4)2SO4 fractionation and chromatography on DEAE- and CM-celluloses. Isoenzymes were purified about 5000 fold, and were found to be homogenous by disc electrophoresis in 7% polyacrylamide gel. It was found by comparative studies of activities of variants FF and SS, that pH optimum was 8.2 for variant FF and 8.8 for variant SS. Km for 6-phosphogluconate were found to be 17,5-10(-5) M for variants SS and FF respectively.


[Comparative study of glutamate dehydrogenases of Chlorella]. The kinetic properties of the constitutive double specific glutamate dehydrogenase (NAD(P)--GDH) and the inducible NADP-specific glutamate dehydrogenase (NADP--GDH) of Chlorella pyrenoidosa Pringsheim 82T (thermophilic strain) in a deaminating reaction have been studied. NAD(P)-GDH behaves in a deamination as a Michaelis-Menten enzyme. NADP-GDH displays some lag-period before a steady-state phase. The duration of this lag depends on a substrate concentration. Besides that, an effect of all the substrates on a heat inactivation of both GDH and a product inhibition have been studied. All the substrates except the reduced co-factors protect effectively GDH from the heat inactivation, especially the thermolabille NADP-GDH. On the contrary, NAD(P)-H promote the heat inactivation of both GDH. The product inhibition analysis shows that the inducible NADP-GDH acts in vivo as a synthetic enzyme. In the previous paper (V. R. Shatilov et all., 1974, Dokl. Acad. Nauk USSR, 216,223) it was shown for the constitutive GDH that p-CMB strongly inhibited a desamination and slightly (if any) affect an amination. It this paper it is shown that action of p-CMB on the amination depends on the presence of NAD+ (not NADP+ or L-glutamate). p-CMB and NAD+ affect tha amination in a strongly sunergetic manner. Some suggestions about the intracellular localization of chlorella GDH are made.


[Fibril formation in solutions of solubilized collagen]. Influence of the preparations of bacterial proteinases, protorisine and prototerrisine, was studied on the stability of the mature collagen of beef skin. The chemical composition of the tissue has been shown to be changed by these enzymes inconsiderably. The tissue treated by orisine and terrisine is completely dissolved in 0.5 M acetic acid (solubilized collagen). When the solutions of such collagen are heated to 37 degrees within the pH range from 4 to 10 at the ionic strength of 0.25 fibrils are formed. Under electron microscope fibres are cross-striated that is typical of native collagen fibres with periodicity of about 640 A. After chilling to 4 degrees, a part of fibrils is dissolved again. Nephlometry was used to study the rate of fibril formation as a function of pH and temperature values. A conclusion has been drawn that the mature collagne dissolved after incubation with bacterial proteinases is close to the acid-soluble collagen fraction in the ability to produce fibres upon heating.


PAH clearance, sodium excretion, and PAH extraction ratio in acidotic near-term lambs treated with hypertonic sodium bicarbonate. The electrolyte changes and renal hemodynamic adjustment to hypertonic sodium bicarbonate (NaHCO3) correction of a metabolic acidosis were studied in 4 neonatal lambs and in 2 controls. PAH clearance increased from 0.92 to 1.65 ml/min/kg (p less than 0.05),  urine flow from 0.37 to 0.61 ml/min/kg (p less than 0.05), and Na excretion from 8.4 to 23.7 muEq/min/kg (p less than 0.05) during the NaHCO3 infusion. These increases were transient and returned to pre-infusion levels following NaHCO3 infusion. Calculation of Na intake and output revealed a net retention of 5.1 mEq/kg in the study lambs which was reflected in a rise of serum Na and osmolarity (Osm) during the post-NaHCO3 -infusion period. The extraction ratio of sodium p-aminohippurate (EPAH) and its relationship to arterial pH were studied in 4 additional lambs. The EPAH did not change with metabolic acidosis but for unknown reasons, the infusion of NaHCO3 resulted in a temporary depression of EPAH (p less than 0.001).


On the proteolytic acitivity of ribosomes. The presence of a proteinase on polysomes, isolated from rat liver has been demonstrated. The proteinase was not removed from polysomes upon treatment of the latter with 0,5 M ammonium chloride. The pH optimum of the enzymes is at pH 7,0.


[Correction of plasma hemoglobin measurements in calculating a hemolysis index]. A "haemolysis index" may be defined from plasma haemoglobin concentration. This is useful in evaluating the consequences of the artificial kidneys and lungs (pumps and circuitry). Measurements must be taken repeatedly, and corrections made for variations in plasma volume from standard conditions (haematocrit or total haemoglobin) to obtain meaningful comparisons. The "haemolysis index" must take into account the number of passages through the circuit.


[Study of the osmotic behaviour of human platelets]. Observation of the behaviour of platelets in hypotonic media affords an approach to the evaluation of their fragility. It should be possible to apply such a test to various fields such as the study of rheological properties of platelets, the investigation of certain pathological cases and eventual alterations during storage. The curve obtained reflects a continuous distribution of platelet osmotic fragility.


Oxygen transport by haemoglobin. A comparison of whole blood, washed erythrocytes and haemoglobin solution. The properties of haemoglobin oxygen transport were compared under three different conditions: red cell in its natural medium, i.e. plasma (whole blood), washed red cell and haemoglobin A, the former suspended, the latter solved in an iso-osmotic tris buffer. The oxygen haemoglobin affinity (expressed as P50) and the respiratory Bohr effect variations were studied with modified media and unchanged pH and 2,3-diphosphoglycerate (2,3-DPG) concentration. Provided they are refered to intra-erythrocytic pH, none of these values were changed when varying environment. These results suggest that the three major ligands (H+ ions, 2,3-DPG and CO2) interaction with haemoglobin is largely predominant upon other factors which would interfere, and can completely account for oxygen transport by haemoglobin.


Kinetics of the disordered chain-to-beta transformation of poly(L-tyrosine) in aqueous solution. The kinetics of the transformation of poly(L-tyrosine) from the disordered chain to the intramolecular beta structure in aqueous solution has been studied. The reaction is induced by an isothermal pH jump and is followed by conventional circular dichroism methods. Upon application of curve-fitting procedures, it is found that the kinetics are poorly represented by a single first-order process, but a two-step sequential first-order equation is adequate. Sharp pH-dependent maxima in the phenomenological rate constants and in the fractional amplitude of the rapid step were found. It is proposed to attribute these phenomena to a transition in initial states which is shown to occur over the same pH range within the domain of the disordered-to-beta transition. No sigmoid transient curves were observed, indicating that no slow nucleation events are discernible in this system. These observations contrast strikingly with the mechanism elaborated for beta formation in (Lys)n [R. Hartman et al., J. Mol. Biol. 90 (1974) 415].


Thermodynamic investigations of proteins. I. Standard functions for proteins with lysozyme as an example. A direct method is proposed for obtaining thermodynamic standard functions for native and denatured proteins using experimental data from scanning calorimetry, isothermal calorimetry and potentiometric titrations. The possibility of this approach is demonstrated on the example of lysozyme in the range of pH 1.5-7.0 and temperature 0-100 degrees C. Tests for the validity of the obtained functions of enthalpy and entropy are presented in the form of cyclic processes using experimental data obtained from thermodynamically different pathways. The Gibbs function is checked by comparison with results of an independent method. The methodic problems in determining and checking standard functions for proteins are discussed in detail.


Thermodynamic investigations of proteins. III. Thermodynamic description of lysozyme. Standard functions of enthalpy, entropy and the Gibbs energy of native and denatured lysozyme in the range of 0-100 degrees C and pH 1.5-7.0 are represented in three-dimensional projections. The denaturational Gibbs energy change reaches 16 kcal mol-1 at conditions of maximal protein stability (0 degrees C, pH 4.5-7.0) and equals 14.5 kcal mol-1 at 25 degrees C and neutral pH. This result was found to be in agreement with the data reported from guanidine hydrochloride denaturation studies. Partial thermodynamic functions of the conformational and ionizational changes of the protein are obtained from entropy and Gibbs-energy changes in denaturation. The conformational partial entropy and Gibbs-energy change are found to be independent of pH. The pH-dependent partial ionizational entropy and Gibbs-energy changes are induced by normalization of the ionization behaviour of buried groups and cause a decrease of protein stability.


The self-association of adenosine-5'-triphosphate studied by circular dichroism at low ionic strengths. The self-association of adenosine-5'-triphosphate (ATP) was studied as a function of pH, additional counterions, concentration and temperature. Circular dichroism measurements were employed as a measure of the base-stacking. The self-association of ATP is pH dependent with the protonation of the adenine ring helping stabilize the association. Highly charged counterions alter this aggregation. At pH 2.8 and 20 degrees C, a dimerization constant of 88 M-1 is obtained, while an isodesmic model leads to an equilibrium constant of 158 M-1. With increasing pH, the association constants decrease. At pH 2.8 there is a very strong temperature dependence of the CD amplitude. These results indicate the existence of additional electrostatic stabilization for the stacking of the adenine rings. At acidic pHs, models are proposed to explain this high degree of stability and a calculation of the approximate electrostatic contribution to the aggregation shows it to be of the proper magnitude.


Studies on immobilized trypsin in high concentrations of organic solvents. Trypsin was covalently immobilized on porous glass in the presence and absence of a specific substrate and reacted in various organic solvents of different dielectric constants. Optimum solvent concentration, pH profile, Km(app), Vmax(app), productivity versus temperature, activity, and reaction rates were determined. Reaction rates of six lysyl dipeptides were compared. Crystalline trypsin was dansylated for studies by nanosecond fluorescence techniques to determine the effects of introducing high concentrations of organic solvents on the molecule. The results indicated that greater reaction rates were observed with dipeptides having more acidic carboxyl terminal groups. The data also indicated that greater reaction rates were observed in higher concentrations of solvents of lower dielectric constants. Nanosecond fluorescence spectroscopy of trypsin in high concentrations of a low dielectric constant solvent indicated major dehydration even though maximal enzyme activity was achieved under these conditions.


Enzyme immobilization on fibrin.

Enzyme immobilization on fibrin. The following conclusions can be drawn concerning the utilization of fibrin to immobilized enzyme systems. Fibrin can be used both as a powder or membrane, to covalently immobilize trypsin with retention of activity. Carbon-14 labeled trypsin can be used to estimate the amount of immobilized enzyme on a proteinaceous support. Significant amounts of noncovalently coupled (adsorbed) enzyme are present on the surface of the support. Esterase activity of the immobilized labeled trypsin was inversely proportional to the amount of attached enzyme. Optimum TAME hydrolysis occurred at pH 8-8.4. The storage stability of trypsin was enhanced. Inhibition of trypsin esterase activity occurred at substrate concentrations greater than 30mM.


[Neuro-muscular synapse ultrastructure in the Lambert-Eaton myasthenic syndrome]. A study was made of the ultrastructure of the neuro-muscular synapses in the patients with the myasthenic Lambert-Eaton syndrome. Most of the synapses displayed an increased content of synaptic vesicles in the axon terminals, and the anastomosing synaptic folds were increased in number and depth. Local destructive changes were found in the terminals of some synapses. The data obtained confirmed the fact that this syndrome was underlied by disorder of the transmitter release from the presynaptic structures.


Prostaglandins in pyometrial fluid from the cow, bitch and ferret. 1 Pyometra is a disorder of the uterus usually associated with bacterial infection plus obstruction. 2 Large quantities of fluid often collect in the uterus during this condition. 3 Pyometrial fluid obtained from three species was found to contain prostaglandin F2alpha, usually in large quantities. 4 Prostaglandin E2 was present in smaller quantities in five of the six samples. 5 These findings are discussed in relation to the known occurrence of prostaglandins in inflammatory fluid, and to the problem of infertility.


Assessment in the guinea-pig ileum and mouse vas deferens of benzomorphans which have strong antinociceptive activity but do not substitute for morphine in the dependent monkey. 1 Four benzomorphans which have potent antinociceptive activity in the hot-plate and writhing tests in the mouse but do not suppress or precipitate withdrawal symptoms in the morphine-dependent monkey, have been examined for their pharmacological actions in the guinea-pig ileum and mouse vas deferens. 2 In the guinea-pig ileum their agonist potencies are 1.5 to 400 times greater than that of normorphine of morphine whereas in the mouse vas deferens their potencies relative to morphine are 0.3 to 100. They exhibit no antagonist activity in either preparation. Benzomorphans which substitute for morphine in the morphine-dependent monkey do not show such differences between their relative potencies in the guinea-pig ileum and mouse vas diferens. 3 The relative potencies of the four benzomorphans to inhibit stereospecific [3H]-dihydromorphine binding by membrane fragments from rat brain, are more closely related to their relative agonist potencies in the mouse vas deferens than to those found in the guinea-pig ileum. 4 In order to antagonize the agonist actions of these benzomorphans, naloxone is required in concentrations which are 3 to 7 times higher than those needed for the antagonism of normorphine or morphine or of benzomorphans which suppress abstinence in morphine-dependent monkeys. 5 It may be possible to use the three assays, namely, ratio of relative agonist potency in mouse vas deferens to that in guinea-pig ileum, ratio of relative agonist potency to relative affinity to opiate receptors and the concentration of nalozone required for antagonism, for the prediction of the potential of new compounds to produce physical dependence.


The actions of flupenthixol upon 5-hydroxytryptamine-induced aggregation and the uptake of 5-hydroxytryptamine and dopamine by human blood platelets. The effects of the alpha- and beta-isomers of flupenthixol on 5-hydroxytryptamine (5-HT)-induced platelet aggregation and on 5-HT and dopamine uptake were investigated. Alpha-Flupenthixol was 185 times more potent than the beta-isomer as an inhibitor of platelet  aggregation. In contrast both isomers were equipotent as inhibitors of uptake of 5-HT and dopamine. The data suggest that 5-HT-induced aggregation and uptake are separate processes.


Evolution of functional respiratory disorders in different types of pneumoconiosis. Three homogeneous groups of patients with silicosis, coal workers' pneumoconiosis and arc welders' pneumoconiosis had been reexamined after an interval of six years. The same examinations were repeated on each occasion with the purpose of evaluating the evolution of radiographic and functional changes. The clinical course, roentgenographic findings and results of function tests differed in the three groups. In silicosis and coal workers' pneumoconiosis the roentgenographic changes showed distinct progression. This progression was less evident in coal workers' pneumoconiosis, but deterioration of pulmonary function was more pronounced than in silicosis, apparently due to emphysema. In pneumoconiosis of welders roentgenographic changes showed a clear tendency to regression and respiratory function was not impaired.


Developmental variations of tyrosine hydroxylase and acetylcholinesterase in embryonic and post-hatching chicken sympathetic ganglia. The developmental variations of tyrosine hydroxylase (TH) and of acetylcholinesterase (AChE) were studied in embryonic and post-hatching chicken sympathetic ganglia. Different levels of TH activity were found in two different flocks of White Leghorn chicken, which are probably dependent on genetic differences. These enzymatic differences, however, do not become apparent before hatching and may indicate a combined effect of genetic variation and functional demands. During the period of incubation, TH activity is characterized by a pronounced and steady increase from the twelfth day of incubation up to day 2 after hatching. This corresponds to a period of intense maturation of the sympathetic neuron. In the period following hatching, the 'fourth day fall phenomenon' previously described by us for DOPA decarboxylase (DDC), dopamine-beta-hydroxylase (DBH), and monoamine oxidase (MAO) is not seen in the TH curve. Instead, TH activity tends to remain constant between days 2 and 14 after hatching (ah). Both ganglionic protein and weight remain constant in this period, indicating a phase of general pause in protein synthesis. AChE activity increases steadily from the eighth until the twenty-first day of incubation. A sudden and significant drop in AChE activity was found at day 2 ah followed by a period of rapid increase at day 3 ah and a levelling of activity up to day 30 ah. Comparing the present variations to those observed in our previous studies on DBH, a temporal relationship between TH and DBH activity is observed during the phases of synaptogenesis and maturation but not during the phase of intense functional activity. Our results strongly suggest that before hatching in chick embryo sympathetic ganglia, the cholinergic presynaptic terminals play a role in regulating the development of the adrenergic neurons. In the period following hatching, however, the DBH and TH levels in cell bodies seem to be principally regulated by the functional activity. This results in depletion of DBH, but not TH, through liberation along with the neurotransmitter at the periphery. Depletion of DBH at the terminals may result in increased transport and thereby depletion in the cell body. This mechanism is probably responsible for the difference in the profiles of activity of DBH and TH in the cell bodies observed in the first week after hatching.


[Erythrocyte stroma included in polyacrylamide gel. Applications to affinity chromatography]. Stromata prepared with human or animal red blood cells are suspended in acrylamide solution. Gel as prepared for electrophoresis is dispersed, before use, and can be used in chromatographic columns for the retention of agglutinins. Lectins, e.g., where first absorbed and then eluted with solution of inhibitory sugar or with acid buffer. Some applications are mentioned.


[Value of the "gastric chamber", new technic performed ex vivo, for the study of the gastric mucosa of the rat]. The "gastric chamber" technique, performed in the anaesthetised rat, enables the study of gastric mucosal fragility induced by doses of phenylbutazone, which do not themselves cause ulceration or exulceration. The perfusion of buffered solution at pH 2-8 into the gastric chamber shows that prior oral administration of phenylbutazone 50 mg/kg increases the fragility of the mucosa. The optimal delay separating this administration from the time of experimentation is 6 hours. The effects seen are essentially vascular disorders.


[Dopamine of the caudate nucleus in Perodictious potto, Macaca mulatta and Macaca fascicularis]. The dopamine, dopac and tyrosinehydroxylase contents of the caudate nucleus in the prosimian Perodicticus potto and in the simii Macaca mulatta and M. fascicularis have been estimated. The results do not support the hypothesis according to which the sluggishness of the potto is somehow related to a low dopamine content of part of the extrapyramidal system as found in the Parkinson-syndrome.


[The effect of activator on the esterase activity of plasmin obtained by streptokinase]. In the esterasic dosage of plasminogen, the authors aim to determine the optimal ratio between the amount of streptokinase to add and of plasminogen itself. It is essential that the activator formation be reduced, which explains the dissociations obtained with other methods.


[Oxidative metabolism of funicular tissue. II. Determination of the activity of certain Krebs cycle enzymes]. Previous experiments have suggested that a partial metabolic block might restrain the oxidative metabolism of the cord tissue between the decarboxylation of pyruvate and the oxidation of succinate. Some of the dehydrogenases of the Kreb's cycle were assayed on acetone powders prepared from human cords. Isocitrate dehydrogenases (both NAD and NADP-specific) have much lower activities than the alpha-ketoglutarate- and malate dehydrogenases; a partial block might be located at this level. Moreover, the malic enzyme has a rather high activity, and might play a significant role by regenerating NADPH in a tissue where other sources of this coenzyme are practically absent.


[The effect of amino acids on the excitability of identified autoactive giant neurons of Achatina fulica Férussac]. Effects of the following amino acids were examined on the electrical activity of the two giant neurones (PON and TAN) identified in the subesophageal ganglia of Achatina fulica Férussac : L-Asp, L-Thr, L-Ser, L-Glu, L-Pro, Gly, L-alpha-Ala, beta-Ala, L-cysteine, L-cystine, L-Val, L-Met, L-Ileu, L-Leu, L-Tyr, L-Phe, L-Lys, L-His, L-Arg, L-Cit, L-Try, GABA and GABOB. Among these substances, we observed an inhibitory effect of GABA and GABOB on the TAN excitability. GABA showed stronger effect on the TAN than GABOB. This effect of GABA was due to producing hyperpolarization on the TAN membrane. GABA showed a slight excitatory effect on the PON. The effect of GABOB on the PON was very weak and unstable.


[Rapid production of a biotin deficiency in mice]. This work describes a rapid method for the production of a biotin-deficiency in mice. Besides classical morphological symptoms, a decrease of activities of some biotin-dependent enzymes was also observed. The biotin-enzymes are not inhibited at the same extent in a same organ and the metabolic changes do not always follow the enzymatic modifications.


[Ciguatera: neurophysiologic and histoenzymologic studies of several fractions of ciguatoxic extracts]. Two fractions of ciguatoxic extracts were isolated by chromatography. The first showed anticholinesterasic properties, while the other acted directly on the muscular fibre. These findings were histoenzymologically confirmed. Controls with non-toxic extracts of fishes from the coral benthos reinforce the theory of the ecological origins of ciguatoxins.


Lorazepam as a premedication.

Lorazepam as a premedication. A double-blind random study compared the effects of lorazepam and pantopon an intra-muscular premedication in healthy women for uterine curettage (D & C). Anxiety, as assessed by a self-rating test by the patient and by a trained observer, showed a significant reduction at one and one-half hours after lorazepam and a smaller reduction after pantopon, which was not significant. Sedation was satisfactory with no significant difference between the two drugs in the change before and after the premedication. Lorazepam showed much more amnesia than pantopon (p less than 0.001). The patients who had lorazepam required higher doses of thiopentone for the operation, and this, in part, led to longer intervals in recovery times after lorazepam. However, it is suggested that lorazepam itself was partly responsible for the longer recovery. Pantopon was followed by more nausea, vomiting and headaches, than lorazepam. The intra-muscular injection of lorazepam hurt more patients than did pantopon, but other local complications were negligible and comparable in both groups. The results of this study show that lorazepam produces better reduction of anxiety and much more amnesia than pantopon, with comparable sedation and much less nausea and vomiting. The only disadvantage of lorazepam is the lack of analgesia and, therefore, the need for more anaesthesia during the operation. The conclusion is that lorazepam is a very satisfactory premedication and warrants more use as such.


Studies on fazadinium bromide (ah 8165): a new non-depolarizing neuromuscular blocking agent. Intravenous dose-response relationships were used to correlate neuromuscular paralysis with the effects of fazadinium (AH 8165) on autonomic mechanisms in anaesthetized cats and rhesus monkeys and with cardiovascular effects in man. In cats and monkeys neuromuscular paralysis of the twitch responses of the gastrocnemius muscle by fazadinium was accompanied by impairment of the vagally induced bradycardia, but cardiovascular disturbances were small. Blockade of sympathetic mechanisms and hypotension were only evident with supra-maximal doses. In man tachycardia was a common occurrence and in some patients hypertension occurred with doses of the drug needed for complete neuromuscular paralysis. Fazadinium was three to four times more potent in rhesus monkeys than in cats and its course of action was considerably longer. The potency of the drug in man corresponded more closely to that in cats than in rhesus monkeys but its course of action in patients was similar to that in monkeys. In man, dose-response curves were constructed for the contractions of the adductor pollicis muscles elicited by tetanic and single twitch stimuli applied to the corresponding ulnar nerves. The onset of paralysis of the tetanic contractions after the intravenous injection of fazadinium (0.4 mg/kg) occurred within two minutes, but recovery was slow and about 50 minutes were needed for its completion. Depression of the simultaneously recorded twitch responses was less marked, slower in onset and recovery was slightly more rapid. These effects were similar to those obtained with tubocurarine (0.2 mg/kg) but the action of fazadinium was slightly shorter. Tetanic-tension ratios were computed after 30 and 50 per cent recovery from neruomuscular blockade in man. These ratios were lower with fazadinium than with tubocurarine and indicated taht tetanic fade was greater and more persistent after fazadinium than after tubocurarine.


Biology of large bowel cancer. Present status and research frontiers. Man and laboratory rodents exposed to chemical carcinogens both show changes in growth characteristics of colonic epithelial cells during neoplastic transformation. Progressive phases of abnormal cell development appear in colonic epithelial cells which gain an increased ability to proliferate and accumulate in the mucosa. These phases in the expression of neoplastic transformation of colonic cells are best defined in the dominant inherited disease of man as adenomatosis of the colon and rectum. Individuals with inherited adenomatosis and those in lesser risk categories can be classified by cell phenotype based on changes in the proliferation and maturation of colonic and other cells. These classifications are leading to new predictive indices which identify heightened degrees of susceptibility of individuals who are at increased risk for colon cancer, and the stage of development of their disease. The indices also are being used to study the contribution of specific elements in the enviroment that modify or accelerate the progression of disease.


Laboratory aids to diagnosis--enzymes.

Laboratory aids to diagnosis--enzymes. A summary is presented of those organ specific enzyme assays traditionally used in evaluation of the patient with cancer. In addition, the use of certain serum enzymes such as gamma-glutamyl transpeptidase, phosphohexose isomerase or 5'-nucleotidase as aids in following the course of the disease, particularly in patients with metastatic spread to the liver is outlined. Also considered is the utility of enzyme analysis in biopsy tissue, biologic fluids, and washings of body cavities. Newer enzymes are considered which might, in the future, be developed as diagnostic tools or as probes for the understanding of the etiology of cancer.


Increase in alkaline phosphatase activity in the liver of mice bearing Ehrlich ascites tumor. In mice bearing Ehrilich ascites tumors, alkaline phosphatase activity was increased fivefold in the liver and by 50% in the kidney. In mice bearing solid tumors caused by inoculation of tumor cells into the axillary region, the activity of this enzyme in the liver was increased 11-fold, whereas the activity in the kidney did not change. Alkaline phosphatase activities in the liver and kidney were not altered by administration of adrenal steroids. Adrenalectomy, fasting, and pregnancy did not affect the activity of alkaline phosphatase in the liver and kidney. Treatment with tumor extracts or ascites fluid of normal mice increased liver alkaline phosphatase activity. These findings suggested that the elevation of liver alkaline phosphatase activity was cuased primarily by the tumor itself, and not by hormonal imbalance provoked secondarily by the presence of the tumor.


Monoamine storage sites in the rat superior cervical ganglion following synthesis inhibition. Monoamine storage sites in paraganglionic (PG-) cells of the rat superior cervical ganglion were investigated by electron and fluorescence microscopy following treatment with p-chlorophenylalanine (pCPA), disulfiram or guanethidine respectively. Dense core vesicles in PG-cells are significantly decreased (p less than 0.001) in number following pCPA, and in the majority of these cells following disulfiram and guanethidine. However in a minor portion of PG-cells the latter agents cause an increase in number and in size of dense core vesicles, in parallel with structural alterations. In agreement with these electron microscopic findings fluorescence microscopic and cytophotometric evaluations reveal a general decrease in catecholamine content with few cells showing an increase. The findings provide a morphological basis for the assumption, that monoamine storage sites in PG-cells can be decreased by inhibition of monoamine synthesis, following administration of pCPA, disulfiram and guanethidine. However the two types of responses of PG-cells which occur after disulfiram and guanethidine demonstrate a functional heterogeneity of this cell system in the rat superior cervical ganglion which is discussed.


Ultracytochemistry of the synaptic ribbons in the rat pineal organ. The synaptic complexes of the rat pinealocytes are neither cholinergic nor adrenergic. In the synaptic vesicles, a neurotransmitter carrier substance of lipid nature reacting with OsO4-Zn I2 mixture (similar to that present in both cholinergic and adrenergic vesicles) was not found. In addition, there were no indications of glucose-6-phosphatase or thiamine-pyrophosphatase activity in the synaptic vesicles. Thus, it appears that the synaptic vesicles do not originate from the rough or smooth endoplasmic reticulum. The synaptic ribbons do not contain carbohydrates, are of protein nature and possess some chemical resemblance to microtubules and microtubular bouquets. Appropriate ultracytochemical reactions have not shown detectable quantities of sodium and calcium ions in pinealocyte synaptic complexes.


Afferent neural pathway in the regulation of cardiopulmonary responses to tissue hypermetabolism. We studied the role of neural transmission from hypermetabolic peripheral tissues in the regulation of cardiac output and pulmonary ventilation in chloralose-anesthetized dogs. Cross-circulation techniques with femoral-femoral or femoral-aortic anastomoses were used to produce a vascularly isolated, but normally innervated, hindlimb or lower half-body, 2,4-Dinitrophenol (DNP) was infused into the arterial side of the perfusion circuit to triple oxygen consumption and to increase lactate production by the cross-perfused area. After infusion of DNP, cardiac output and mean systemic arterial blood pressure increased, but neither heart rate nor pulmonary artery wedge pressure changed significantly. Pulmonary minute ventilation and arterial pH also increased, while arterial PCO2 fell. These changes were abolished when the nerve connections between the perfused limb and its parent body were severed. Normal saline, when administered in a similar manner, did not increase either ventilation or cardiac output, and simple denervation without previous infusions of DNP also had no effect. These results indicate that there are receptors sensitive to metabolic changes in the tissue, and that neural transmission is an important afferent link in regulating the cardiopulmonary responses to increased tissue metabolism.


Determination of the molar absorptivity of NADH. The molar absorptivity of NADH at 340 nm has been determined by an indirect procedure in which high-purity glucose is phosphorylated by ATP in the presence of hexokinase, coupled to oxidation of the glucose-6-phosphate by NAD+ in the presence of glucose-6-phosphate dehydrogenase. The average value from 85 independent determinations is 6317 liter mol-1 cm-1 at 25 degrees C and pH 7.8. The overall uncertainty is -4.0 to +5.5 ppt (6292 to 6352 liter mol-1 cm-1), based on a standard error of the mean of 0.48 ppt and an estimate of systematic error of -2.6 to +4.1 ppt. Effects of pH, buffer, and temperature on the molar absorptivity are also reported.


Molar absorptivities of beta-NADH and beta-NADPH. Re-investigating the accuracy of the commonly used values for molar absorptivities (epsilon) of beta-NADH and beta-NADPH at Hg 334, Hg 365, or 340 nm, we obtained the following results: The maximum of absorbance of NADH is shifted from about 340 nm at 0 degrees C to about 338.5 nm at 38 degrees C; the corresponding maxima of NADPH are located at about 0.5-nm longer wavelengths. In addition, the absorption curves of both coenzymes broaden with increasing temperature. For these reasons, the epsilon-values of NADH and NADPH are generally different from each other, and are temperature-dependent. Only at 334 nm are they almost identical and nearly independent of temperature. Therefore this wavelength is recommended for precise measurements. The epsilon-values of these coenzymes are influenced by ionic strength and pH. To determine the absolute values of the molar absorptivities, we performed the glutamate dehydrogenase or lactate dehydrogenase assay with carefully purified 2-oxoglutaric acid or pyruvic acid in the presence of excess coenzyme. The purity of the substrates was checked through differential scanning calorimetry, moisture analysis, gas-liquid chromatography, gas chromatography in combination with mass spectrometry, and nuclear magnetic resonance spectroscopy. The epsilon-values observed under the various conditions are about 1-7% higher than those currently used.


Analysis for indole compounds in urine by high-performance liquid chromatography with fluorometric detection. We describe a chromatographic system involving a high-performance chemically-bonded reverse-phase column and fluorescence detection for measurement of indoles in urine. We controlled retention and selectivity by optimizing the methanol content and pH of the mobile phase. Six reference indoles were separated in less than 20 min; three 5-hydroxyindoles were eluted in less than 7 min. About 5-15 ng of aqueous solutions of these compounds can be detected. The combination of selectivity (from use of the chromatographic column) and fluorescence detection permitted analysis for five of the six indoles after a single urine-deproteinization step.


Effect of halothane anesthesia on muscle, liver, thyroid, and adrenal-function tests in man. Seven healthy men volunteers received 6.6 +/- 1.3 (SD) percent-hours of halothane oxygen anesthesia without surgery. Serum bilirubin, alanine aminotransferase, and aspartate aminotransferase significantly increased after anesthesia, which may indicate subclinical liver-cell damage. Creatine kinase of skeletal muscle origin increased above 90 U/liter in six subjects, indicating subclinical muscle-cell damage. Cortisol, triiodothyronine uptake, thyroxine, and free thyroxine index increased significantly immediately after anesthesia. Serum bromide concentrations had increased by fivefold on the second day after anesthesia, and on the ninth day was still elevated fourfold. Oral temperatures increased 0.7 degrees C 6 h post-anesthesia, possibly because of increased thyroxine activity. Lactate dehydrogenase, hydroxybutyrate dehydrogenase and gamma-glutamyltransferase activities did not change significantly. No drugs administered during the course of this study chemically interfered with any of the test methods used.


Improved assay for urinary 17-hydroxycorticosteroids. We describe a simple, rapid, reliable method for determining urinary 17-hydroxycorticosteroids. A neutral resin (Amberlite XAD-2), which is a non-ionic cross-linked polystyrene, is used to extract and concentrate the steroids, which are then quantitatively determined with the Porter-Silber reaction. Use of the resin eliminates the need for enzymatic hydrolysis and n-butanol extractions, thereby decreasing analysis time considerably; results can be obtained within 3 h of receipt of specimens. Most of the nonsteroidal Porter-Silber chromogens are removed, resulting in a method that is highly specific and sensitive, even at low concentrations (0.6 mg/liter), and so more accurate and reproducible than currently used methods.


Radioimmunoassay of plasma renin activity. We describe a sensitive, simplified radioimmunoassay method for determination of plasma renin activity. Plasma was acidified to the optimal pH (6.0) of angiotensin l generation with the least possible dilution, by using a single addition of hydrochloric acid and the enzyme inhibitor hydroxyquinoline. Recovery of unlabeled angiotensin l added to plasma was 92-97%; that of monoiodinated angiotensin l exceeded 90%, indicating satisfactory protection from proteolytic enzymes. Plasma constituents interfered little with the radioimmunoassay. Bland values for plasma kept at 0 degrees C were 10.7 +/- 2.3 (mean +/- SD) percent of the activity values for samples kept at +37 degrees C (n equals 63). In the routine setting, 6.25 pg of angiotensin l or 10(-6) Goldblatt units of Standard Human Renin was detected. We report results of plasma renin activity measurements and a comparison with seven renin kits, and with bioassay for plasma renin activity.


P-Hydroxybenzoic acid hydrazide procedure for serum glucose adapted to the Technicon "SMA 12/60," and compared with other glucose methods. We adapted the p-hydroxybenzoic acid hydrazide procedure for serum glucose for use with the Technicon SMA 12/60 AutoAnalyzer. Like the o-toluidine method, this method is based on a general carbohydrate reaction except that it occurs in a mildly alkaline medium and the intense yellow color formed is measured at 400 nm. Advantages of this reagent over o-toluidine include lower cost, less toxicity, and higher purity. Aside from those carbohydrates that are present in serum in insignificant quantities, there are no interferences from various physiological compounds or drugs (hypoglycemic agents) found either in normal persons or diabetics. Within-run and day-to-day values had coefficients of variation of 1.39% and 3.44%, respectively; recoveries ranged from 100 to 102% (mean, 101%). Comparative data showed excellent agreement with the hexokinase (r equals 0.998; y equals 0.950x + 5.91) and glucose oxidase (r equals 0.996; y equals 0.986x + 5.34) enzymatic ("true") glucose methods, and with the o-toluidine procedure (r equals 0.998; y equals 0.979x + 3.14).


Lipoamide dehydrogenase in serum: a preliminary report. Lipoamide dehydrogenase was identified in serum and the optimal conditions for its assay at 30 degrees C were defined. The pH optimum in tris(hydroxymethyl)aminomethane buffer is 7.8, and activity is inhibited if buffer concentration exceeds 100 mmol/liter. Saturating concentrations of the substrates NAD+ and lipoamide are 3 mmol/liter and 5 mmol/liter, respectively. Activity is decreased eightfold when lipoic acid is substituted for lipoamide. Activity is linearly related to enzyme concentration up to limiting absorbance change of 0.300 at 340 nm, and both within-day and day-to-day precision are satisfactory. Data suggest a normal range (2 SD) of 3-19 kU/liter. The highest value measured in serum was 473 kU/liter. A correlation with direct bilirubin concentrations (r equals 0.435, P less than 0.01) was found.


Direct radioimmunoassay of plasma cortisol. The simplification of the measurement of circulating cortisol by direct radioimmunoassay of plasma samples sets the problem of inhibiting the carrier proteins competing with antibodies. This was accomplished by exploiting the much higher effectiveness of pH and temperature variations on steroid binding to carrier proteins than to antibody sites. A solid-phase system was set up, using antisera to cortisol-21-BSA conjucates coupled to CNBr-activated cellulose. The standardized procedure consisted of an incubation at pH 3.5 and room temperature, directly assaying 10 mul of plasma. A methodological and clinical validation of the measurement was carried out through a series of tests aimed at assessing the reliability of results (assay of steroid-deprived plasma, recovery test and serial dilution of samples, comparison between different antisera and with different methods including extraction, responsiveness to well-established physiological situations). The results obtained are reported and the validity of the method discussed in terms of more general applicability to steroid assay.