The synthesis and degradation of presumptive messenger RNA in cultured mouse leukemia cells during the inhibition of protein synthesis. RNA synthesis in mouse leukemia L5178Y cells was inhibited depending upon the time of treatment by blasticidin S or by ricin, which inhibits specifically protein synthesis. When blasticidin S or ricin blocked protein synthesis by more than 90% of the control, marked accumulation of monosome was accompanied by the decrease of pulse-labeled RNA (20% of that in the control) in the polysomes and monosome fraction. The size distribution of pulse-labeled RNA among polysomal fractions including monosome obtained from the cells treated with either blasticidin S, ricin of L-asparaginase showed that the size of presumptive mRNA was shifted from 18 S to 9--10 S. TReatment of a blasticidin S-resistant (Bla-R) subline derived from L5178Y cells (Kuwano, M., Matsui, K., Takenaka, K., Akiyama, S. and Endo, H. (1977) Int. J. Cancer 20, 296--302) with L-asparaginase or ricin induced smaller size (9--10 S) RNA, but treatment of Bla-R cells with blasticidin S did not. Such shorter RNA fragments could not be observed even when cellular protein synthesis was inhibited by treatment for short time with blasticidin S (40--80% of the control activity). Smaller RNA fragments accumulated after drastic inhibition of protein synthesis were composed of 74% of polyadenylate sequence lacking poly(A)(-)RNA with peak of approx. 10 S and 26% of polyadenylate sequence containing poly(A)(+)RNA with a peak of 18 S, whereas cytoplasmic polysomal RNA of the control contained 46% poly(A)(+) with a peak of 18 S and 54% poly(A)(-)RNA with a 10--18 S peak. Cytoplasmic poly(A)(+)RNA degraded biphasically with half-lives of approx. 2 h and 8--10 h in exponentially growing mouse cells. However, in degradation of poly(A)(+)RNA molecules being formed in the cells pretreated with blasticidin S for 3 h, the rapid phase of decay with a half-life of approx. 2 h was interrupted by successively appearing poly(A)(+)RNA with a longer half-life of 8--10 h in cytoplasm. However, when the cells were pretreated with blasticidin S for 6 h, there appeared no poly(A)(+)RNA population with the rapid-decay in cytoplasm.


Transcription and circular dichroism of chromatin in lymphoblastoid cell lines during proliferation and quiescence. RNA polymerase activity of nuclei and positive circular dichroism ellipticity in the 250--300 nm region of chromatin were studied in three lymphoblastoid cell lines: HRI, an Epstein-Barr Virus (EBV) positive virus-producing line; Raji, and EBV positive virus-producing line; Raji, an EBV positive non-producer line; and SU-AMB-1, and EBV negative line. Raji consistently exhibited the highest and SU-AMB-1 the lowest polymerase activity and ellipticity. All lines showed higher RNA polymerase activity and positive ellipticity when exponentially growing than when stationary. In Raji it was shown that changes in RNA polymerase activity and chromatin ellipiticity were parallel. The changes in RNA polymerase activity of nuclei and circular dichroism ellipticity correlated with those of the proliferative state but not with the number of viral genomes or the production of virus or viral antigens.


Estradiol-induced synthesis of vitellogenin. IV. The isolation of non-degraded polysomes from avian liver using an endogenous ribonuclease inhibitor. A procedure allowing the isolation of intact polysomes from rooster liver is described. Good recovery of polysomes is achieved by the presence of Triton X-100 in the homogenization and centrifugation steps since the detergent prevents the sedimentation of microsomes with the nuclear fraction. This sedimentation of microsomes leads to considerable losses of polysomes, especially the larger ones. In the detergent-treated homogenate the integrity of the polysomes is threatened by various ribonucleases, some of which can be effectively inhibited by the addition of both heparin and yeast RNA. The remaining nuclease activity is counteracted by the endogenous ribonuclease inhibitor of the liver. In estradiol-treated roosters, sufficient endogenous inhibitor is present to inhibit its specific ribonuclease, but in control roosters there is not. This difference is due to a hormone-mediated increase in inhibitor level and decrease in nuclease level. Consequently, for an estrogenized rooster, the addition of both heparin and yeast RNA to the homogenate suffices to stabilize the polysomes, whereas control rooster liver homogenate needs supplementation with endogenous ribonuclease inhibitor. The cytosol of estrogenized rooster liver can be used as a crude inhibitor preparation. Rat liver cytosol is only partially effective; this may indicate a certain degree of species specificity of the inhibitor. The isolation procedure described also yields large polysomes from the livers of duck and Xenopus.


Nuclear chromatin changes during post-natal myocardial development. The proliferative capacity of rat myocardium declines rapidly during the first few weeks of post-natal life. In order to gain insight into the mechanisms involved in this decline, we studied the structure and function of nuclear chromatin from isolated rat myocardial cells during post-natal growth. Chromatin template activity decreased progressively (7.5 +/- 0.3 pmol [3H]UTP/microgram DNA per min at age 5 days compared to 2.2 +/- 0.1 pmol [3H]UTP/microgram DNA per min at age 6 months) and was associated with a decrease in the number of transcription initiation sites. This decline was accompanied by changes in chromatin structure as evidenced by: (a) decreased susceptibility to DNAase I digestion with advancing age, (b) decreased poly-L-lysine binding (60% decrease between day 5 and six months of age), (c) progressive decline in positive ellipticity of circular dichroism spectra between 250--300 nm, and (d) derivative melting profiles showing a decrease in DNA regions bound by non-histone proteins and concomitant increase in histone-bound regions. The protein composition of myocardial chromatin also changed during post-natal development, chiefly due to a progressive increase in the histone/DNA ratio. These results indicate substantial changes in the organization and functional capacity of myocardial chromatin during early post-natal growth. These changes accompany, and may contribute to, the restriction in the proliferative capacity of myocardial cells.


RNA from the insect trypanosome Crithidia luciliae contains transcripts of the maxi-circle and not of the mini-circle component of kinetoplast DNA. We have hybridized total cellular RNA of Crithidia luciliae with the kinetoplast DNA of this organism. To allow the discrimination of DNA from mini-circles (2300 base pairs) and maxi-circles (33 000 base pairs), kinetoplast DNA was digested with restriction endonucleases and the fragments were separated by electrophoresis through an agarose gel and transferred to nitrocellulose filters by blotting. No mini-cricle transcripts were found under conditions where maxi-circle fragments showed extensive and specific hybridization. Since maxi-circle sequences are present at less than 1% of the concentration of mini-circle sequences, we conclude that mini-circles may not be transcribed at all. Predominant hybridization with the maxi-circle fragments is obtained with a segment of only 2300--2500 base pairs. The possibility that this segment codes for unusually small mitochondrial ribosomal RNAs is discussed.


Metabolic fate of pyrimidines and purines in dietary nucleic acids ingested by mice. 1. In order to study the metabolism and tissue utilization of pyrimidines or purines ingested as dietary nucleic acid components, [14C]uracil, [14C]cytosine-labeled RNA, [14C]thymine-labeled DNA, or [14C]adenine-labeled RNA was fed to mice. 2. Absorption and catabolism of each ingested radioactive material were rapid; 80% or more of the ingested radioactivity was excreted as catabolic products over an 8-h period. 3. Utilization of the ingested radioactive materials for tissue synthesis of nucleic acids was limited under the usual conditions, the extent being 2--5%, 4 h after feeding. Such acid-insoluble radioactivity was localized principally in gastrointestinal tissue, and much lesser amounts, albeit significant, were found in the liver. 4. With increase in the dose of dietary nucleic acids, the amounts of utilized (nucleic acids and nucleotides) and utilizable (nucleosides and free bases) forms of uracil and cytosine and of adenine were increased in all tissues examined. Relationship between the dose and utilization together with additional findings support the view that gastrointestinal tissue and the liver utilize and degrade a greater part of the exogenous nucleic acid bases before their entry into the systemic circulation. 5. The metabolism of DNA thymine was unique in that it was significantly utilized for DNA synthesis in tissues other than the gastrointestinal tissue and liver to a comparative extent. The spleen was particularly active in this respect, and the hyperplastic, hematopoietic spleen was three times more active than the normal spleen. 6. Principal components of partially digested products in the intestinal lumen 1 h after the ingestion were uridine (33%) and cytidine (22%) in the case of [14C]uracil, [14C]cytosine-labeled RNA and inosine (53%) in the case of [14C]adenine-labeled RNA, in accordance with the view that purines and pyrimidines in nucleic acids are absorbed mainly in the form of nucleosides.


10-S DNA polymerase from calf thymus which copies both poly(rA) . oligo(dT) and activated DNA. A novel DNA polymerase, which could use both poly(rA) . oligo(dT) and activated calf thymus DNA efficiently as template-primers, was purified 20 000-fold from calf thymus extract. These activities were co-purified throughout successive column chromatographies and banded at the same position in either electrofocussing (pI = 6.5--7.0) or sucrose rate-zonal centrifugation (10--10.5 S). The most purified fraction (DNA-cellulose fraction) possessed specific activities of 3900 units/mg of protein with poly(rA) . oligo(dT) and 32 000 units/mg of protein with activated DNA. The poly(rA) . oligo(dT)-dependent activity differed from the previously described DNA polymerase gamma from other sources in the following ways: 1. The activity was inhibited by 100--300 mM KCl and and 80 mM potassium phosphate buffer. 2. The activity was 4-fold higher at 26 degrees C than at 37 degrees C. 3. The Km value for dTTP was 2.6--3.0 . 10(-4) M, which is several hundred-fold greater than that of DNA polymerase gamma. 4. Mn2+ was essential for the reaction and could not be replaced by Mg2+. The activated DNA-dependent activity shared many properties with DNA polymerase alpha, except that it was less sensitive to N-ethylmaleimide and anti-alpha polymerase immunoglobulin G. The 10-S DNA polymerase was dissociated into 8.5-S and 3.3-S by treatment with Triton X-100.


A fast evaluation of diffusion effects on bound enzyme activity. As the kinetic behavior of bound enzymes is frequently affected by substrate diffusion between the bulk solution and the catalytic sites, a fast and simple method is proposed to detect and, subsequently, to remove diffusion effects on measured enzymic activities. The procedure makes use of the effectiveness factor concept and essentially involves the direct determination on two diagrams of the magnitude of both external and internal diffusion limitations. It requires a prior estimation of the volume and external surface area of the matrix, of the substrate external transport coefficient and internal diffusivity, and of the intrinsic Michaelis constant of the bound enzyme. However, it does not necessitate the knowledge of the quantity of bound enzyme. The two basic graphs have been calculated for Michaelis-Menten kinetics. They can also be used to evaluate diffusional effects on two-substrate reactions, as illustrated with previously published data.


Bovine milk xanthine oxidase: purification by ultrafiltration and conventional methods which omit addition of proteases: some criteria for homogeneity of native xanthine oxidase. Methodological difficulties have been encountered when proteases were omitted from the conventional isolation of bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2). The use of these conventional methods has been studied and modified to reduce the problems encountered. Some of the difficulties may be due to the presence of high concentrations of caseins, which exhibit a wide range of charges and sizes, thereby making separations based on charge and size more complicated. In addition, non-covalent interactions may occur between the caseins and xanthine oxidase leading to the formation of casein-xanthine oxidase micellar aggregates. The difficulties encountered in this conventional isolation have been circumvented by purifying the enzyme directly from milk fat globule membranes that first have been washed free of most casein and other milk proteins. The xanthine oxidase is isolated by ultrafiltration through an Amicon XM-100A membrane at 5 degrees C in 0.25 M sucrose/5 mM sodium salicylate. The largest molecular size of globular proteins which can penetrate this ultrafiltration membrane has been previously estimated to be around 100 000 daltons. Xanthine oxidase thus appears to be smaller than 100 000 daltons in its native state. The size observed for active xanthine oxidase previously isolated by other methods has been around 275 000--300 000 daltons. Xanthine oxidase isolated by ultrafiltration appears similar to xanthine oxidase from conventional isolation methods according to empirical criteria of homogeneity based on size and also on the absorbances at 280 and 450 nm. Criteria based on charge were found to be less reliable.


Drug metabolism in the Novikoff hepatoma: evidence for a mixed function oxidase system and partial purification of cytochrome P-450 reductase. Novikoff kepatoma microsomes catalyze the hydroxylation of benzphetamine and ethylmorphine at rates less than 1% of those of liver microsomes but catalyze the hydroxylation of p-nitroanisole and p-nitrophenetole at rates about 40% of those of liver microsomes. Benzo[a]pyrene hydroxylation is also catalyzed by Novikoff hepatoma microsomes at about 2% of the rate of liver microsomes. Like the hepatic microsomal system the rates of substrate hydroxylation by Novikoff hepatoma microsomes can be increased by pretreatment with phenobarbital/hydrocortisone or beta-naphthoflavone and inhibited by carbon monoxide, SKF-525A, and 7,8-benzoflavone. In addition, NADPH-cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) has been partially purified from Novikoff hepatoma ascites cells and some properties are described. The induction and inhibition characteristics of the Novikoff hepatoma microsomal hydroxylation activities and the isolation of a cytochrome P-450 reductase from the hepatoma are consistent with the presence of a functional mixed function oxidase system in the Novikoff hepatoma, analogous to that present in liver endoplasmic reticulum.


The characterization of n-butanol-pseudosolubilized and trypsin-solubilized porcine thyroid iodide peroxidase. Porcine thyroid peroxidase (Iodide: hydrogen-peroxide oxidoreductase, EC 1.11.1.8) was solubilized by proteolytic and non-proteolytic procedures. A kinetic and physical study was undertaken to ascertain the catalytic properties of the peroxidase prepared by the two purported solubilization procedures. Where possible, the properties of the two enzyme preparations were compared with the original microsomal preparation. The n-butanol-solubilized thyroid iodide peroxidase is not truly soluble, but exists as a large molecular weight lipoprotein aggregate. The trypsin-solubilized thyroid iodide peroxidase is truly soluble, active, and contains lipids. The microsomes, butanol-pseudosolubilized enzyme, and trypsin-solubilized enzyme have similar kinetic properties such as pH optima, Km for iodide and H2O2, sigmoid character of the saturation curves, substrate inhibition, and inhibition by 3,5-diiodotyrosine. Since the proteolytic solubilization procedure produced a soluble peroxidase with catalytic properties similar to the microsomal preparation, trypsin-solubilized peroxidase can be studied with reasonable assurance that its properties are essentially unaltered and are not artifacts of the solubilization procedure.


Uridine diphosphate glucose:fatty acid glucosyl transferase activity of rape (Brassica napus L.) anther tissue. Particulate fractions from mature rape anther tissue catalysed the incorporation of glucose from UDPglucose into endogenous lipids. The UDPglucose:fatty acid glucosyl transferase was tightly bound to particulates that could be suspended in 0.1% Triton X-100. The apparent Km for UDPglucose was 3.5 . 10(-6) M. In Tris buffer the enzyme has a pH optimum of 8.0 and a temperature optimum of 30 degrees C. In the presence of NaF, the transferase activity was proportional to protein concentration, time, and other variables of the reaction mixture. ADPglucose, GDPglucose, and CDPglucose did not compete with the activity of the UDP-glucose-dependent glucosyl transferase. Gas chromatographic and high performance liquid chromatographic analyses indicated that the endogenous fatty acids glucosylated in vitro had carbon chain lengths of C16 to C20. This is the first report on the biosynthesis of non-phosphorylated glucosyl esters of fatty acids in higher plants.


Progress curve analysis in enzyme kinetics: model discrimination and parameter estimation. The method of progress curve analysis for enzyme-catalyzed reactions (Duggleby, R.G. and Morrison, J.F. (1977) Biochim. Biophys. acta 481, 297--312) has been extended to a two substrate, reversible reaction through the use of enzyme-catalyzed recycling of one of the products. The reaction investigated was that catalyzed by aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) and the product, alpha-ketoglutarate was recycled to glutamate using NADH and NH4Cl in the presence of glutamate dehydrogenase. The values determined for the kinetic parameters of the aminotransferase were found to agree well with those obtained from steady-state velocity measurements. The standard errors of the parameters, as calculated by the procedure originally described, were found to underestimate the observed variation between different experiments. Therefore, a procedure of data compression was devised which leads to more realistic values for standard errors. The compressed data obtained with aspartate aminotransferase have been fitted to the integrated rate equations that describe a variety of kinetic mechanisms. The best fit was obtained with the Ping-Pong model which is applicable to the aspartate aminotransferase reaction. Thus, progress curve analysis may be used to determine the kinetic mechanism of, and values of the kinetic parameters associated with, an enyzme-catalyzed reaction.


The structural requirements of glucose for phosphorylation by phosphoglucomutase. During catalysis, the phosphoryl group of phosphoglucomutase (alpha-D-glucose-1,6-bisphosphate:alpha-D-glucose-1-phosphate phosphotransferase, EC 2.7.5.1) is transferred through a nucleophilic displacement reaction to the monophosphate substrates to form the diphosphate. Some non-phosphorylated analogs of glucose have been shown to serve as effective acceptors of the active phosphate albeit at a much reduced rate. Several other analogs exhibit little or no reactivity. The relative reaction rates of the reactive analogs follow the order: thioglucose greater than alpha- or beta-D-glucose greater than D-xylose, greater than L-arabinose greater than myo-inositol. The rate of transfer increased with the increased concentration of glucose or its analogs. The products of the reaction may be acid stable ester phosphate or acid labile glycosyl phosphate as well as inorganic phosphate. S-phosphoryl (hemiacetal) thioglucose was identified as a product of the 1-thioglucose reaction. It was possible to define certain steric requirements for the orientation of the hydroxyl groups in all the reacting sugars. These requirements are limited to 3 hydroxyl groups and pertain to loci or receptors on the active site of the enzyme. These would correspond in topography to carbons 2, 3 and 4 of the glucose molecule in the enzyme substrate complex. These hydroxyl groups should be oriented equatorially and project below, above and below the plane of the pyranose ring for C-2, C-3 and C-4, respectively.


A novel method for determining equilibrium constants. CTP:phosphorylcholine cytidyltransferase. A novel method for the determination of equilibrium constants for reversible reactions is described. The method is based on the measurement of initial velocities of isotope transfer for a given substrate-product pair of both the forward and reverse reactions as a function of the mass action of reactants. The reciprocal values of these initial velocities are plotted against the mass action ratios of reactants. The observed Keq is the abscissa of the intersection point of these reciprocal plots, i.e. the mass action ratio at which the initial velocities of isotope transfer for both the forward and reverse reaction are identical, that is, when isotope exchange is occurring. In this manner, an observed Keq of 0.2 was obtained from CTP:phosphorylcholine cytidyltransferase (CTP:cholinephosphate cytidyltransferase, EC 2.7.7.15) at 37 degrees C and pH 7.5 under physiological conditions 1.0 mM free Mg2+ and 0.15 M salt concentration. A comparison of this value with the in vivo mass action of reactants calculated from published data indicates that this reaction is rate-limiting in the rat liver (Infante, J.P. (1977) Biochem. J. 167, 847--849).


Alkaline phosphatase from Bacillus licheniformis. Solubility dependent on magnesium, purification and characterization. The membrane-associated alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from Bacillus licheniformis MC14, a facultative thermophile, was purified to homogeneity in buffer containing 0.2 M Mg2+. The alkaline phosphatase purified in this manner is insoluble upon removal of the magnesium by dialysis. This insoluble alkaline phosphatase has been characterized and compared to the previously purified heat-solubilized enzyme (Hulett-Cowling, F.M. and Campbell, L.L. (1971) Biochemistry 10, 1364--1371).


Chromatography of dipeptidyl aminopeptidase I on inhibitor-Sepharose columns. A number of affinity materials for the purification of dipeptidyl amino-peptidase (dipeptidylpeptide hydrolase, EC 3.4.14.1) have been prepared and tested. These material include peptide and amino acid inhibitors bound to agarose and reversible sulfhydryl adsorbents. Several of these materials are effective affinity adsorbents. The most useful material to be employed in combination with earlier purification methods is acetoxy-anilinomercuri-Sepharose. This removes proteins which are contaminants of some preparations and yields consistently high specific activities. Results with affinity and hydrophobic columns indicate that the primary interactions of the enzyme with amino acid and peptide derivative inhibitors are ionic in nature. This results is in agreement with the conclusions reached in studies of the interactions of these inhibitors with dipeptidyl aminopeptidase in solution.


Rat renin: purification and characterization. In order to clarify the molecular basis of the unique features of rat renin (EC 3.4.99.19) and to provide materials and basic information for high blood pressure studies in rats, renin was purified from rat kidney. The final step of purification on CM-cellulose separated renin into three major isoenzyme peaks, R-I, R-II, R-III, and an additional minor peak. These preparations were judged homogeneous by multiple criteria, and the isoenzymes were found to have similar amino acid compositions. The amino acid composition is also closely analogous to hog renin, except that rat renin has a higher cysteine content. In contrast to hog renin, the rat enzymes do not contain amino sugars, yet are apparently glycoproteins as judged by their affinity for concanavalin A. The molecular weights of R-I, R-II, and R-III were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 37 000, 36 000 and 35 000, respectively. The isoelectric points were 5.05, 5.15 and 5.22, respectively. The specific activities of the purified enzymes (determined using rat plasma as substrate) were 615, 626 and 452 Goldblatt units/mg, respectively. Comparison of activities with the hog- and rat-derived substrates indicated a preference for that from the rat. The reaction of the rat enzymes with a synthetic peptide substrate had a similar catalytic rate constant to the hog enzyme, indicating close similarity in the active site region of the two enzymes.


Inhibition of bovine adrenocortical adenylate cyclase activity by adenosine. Incubation of bovine adrenocortical membranes with corticotropin and 5-guanylylimidodiphosphate produced a state of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) with maximal catalytic activity and an increased sensitivity to inhibition by adenosine. Due to metabolism of adenine nucleotides during adenylate cyclase assays a quantitative assessment of the nature of this inhibition was not possible. However, when determined at 0.2--1.0 mM MgATP2-, half-maximal inhibition of the basal and maximally active states of the enzyme was observed at adenosine concentrations of 210--330 and 70--90 micrometer, respectively. The inhibition appeared to be partially competitive, suggesting that the nucleoside may act as an allosteric negative effector which reduces the affinity of the active site for substrate. Adenosine was 5--6 times more potent as an inhibitor of adrenal adenylate cyclase than 2-chloroadenosine. Adenosine deaminase abolished the inhibitory effect of the nucleoside, whilst theophylline had no effect on activity either in the absence or presence of adenosine.


Properties of particulate and detergent-solubilized adenylate cyclase of rat testis. Effects of follitropin stimulation. Basal, fluoride and follitropin stimulated activities of adenylate cyclase have been studied in the testes of immature rats. The enzyme was maximally activated (about twice the basal activity) by low concentrations of follitropin, with an apparent Km of about 9 . 10(-10) M. Both Mg2+ and Mn2+ activate the enzyme but the apparent Ka for Mg2+ is about 10 times that for Mn2+. However, the apparent Km values for MgATP2- and MnATP2- are nearly the same (1.4 . 10(-4) M) and the cation activation of the enzyme is mainly through an increase in V. Ca2+ inhibited all expressions of testicular adenylate cyclase activity. Follitropin and fluoride stimulated adenylate cyclase activity at all Mg2+ concentrations but did not significantly affect the apparent Ka for Mg2+ or the Km for the substrate (MgATP2-). The stimulatory effect of the hormone or fluoride is mainly through an increase in V. Testicular adenylate cyclase could be solubilized with Triton X-100 or Lubrol-PX. The detergent-solubilized enzyme exhibited Km for substrate and Ka values for divalent cations similar to those of the membrane-bound enzyme and remained responsive to stimulation by fluoride. The stimulatory effect of follitropin, however, was lost. Responsiveness to follitropin was also lost by membrane-bound adenylate cyclase after treatment with phospholipase, although the fluoride effect was unchanged. These results reflect the essential role of lipids in the regulation of the follitropin-specific response.


Inhibition of chicken erythrocyte AMP deaminase by tetraiodofluorescein compounds. A kinetic study has been performed on the inhibition of the chicken erythrocyte AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) reaction by tetraiodofluorescein and Rose Bengal. These dyes inhibited the enzyme by decreasing its affinity for the substrate without affecting the maximum velocity. Kinetic analysis has shown the inhibition constants for tetraiodofluorescein and Rose Bengal to be 350 and 55 micrometer, respectively, and the presence of 4 binding sites of the enzyme for the inhibitors per enzyme molecule. These results suggest that the fluorescein dyes mimic the AMP binding at the catalytic center of the enzyme, which can be formed by the "dinucleotide fold".


A study of the alkaline mesentericopeptidase active site by means of peptide chloromethyl ketones. The kinetics of inactivation of alkaline mesentericopeptidase was studied using chloromethyl ketone derivatives of amino acids and peptides. It was shown that Tos-LysCH2Cl and Tos-PheCH2Cl did not influence the enzyme activity, while the inhibitory effect of Cbz-Ala-Gly-PheCH2Cl was 35 times that of Cbz-Ala-PheCH2Cl. The dependence of the pseudo-first order rate constant of the enzyme inactivation by Cbz-Ala-Gly-PheCH2Cl on pH and temperature indicated that a group with a pK of 6.59 and deltaHi of 7.7 kcal/mol was the site of the inhibitor's attack. Amino acid analysis of the modified totally inactive enzyme revealed a definite loss of histidine and after performic acid oxidation a recovery of 3-carboxymethyl histidine. The whole set of experimental data is convincing evidence on behalf of a selective alkylation of the N-3 of the active site histidine after treatment with Cbz-Ala-PheCH2Cl and Cbz-Ala-Gly-PheCH2Cl. Alkaline mesenteriocopeptidase possesses an extended active site and only a peptide chloromethyl ketone, covering a determined sequence of theenzyme molecule (S3, S2, S1, S'1, S'2, S'3 ...) is able to provide effective inhibition. The values of the inactivation constant (kinact) for Cbz-Ala-PheCH2Cl and Cbz-Ala-Gly-PheCH2Cl are close to the corresponding values reported for subtilisin Amylosacchariticus.


Catalysis and leaving group binding in anilide hydrolysis by chymotrypsin. The influence of the leaving group on the reactivity of specific anilides in alpha-chymotrypsin-catalyzed hydrolysis (chymotrypsin, EC 3.4.21.2) involves both its binding to the enzyme (steric effect) and electronic nature (electronic effect). These effects are considered in terms of the stereoelectronic theory for the formation and cleavage of the tetrahedral intermediate in acyltransfer reactions. The application of this theory to the enzyme hydrolysis leads to the conclusion that the nature of the reaction products and the effectiveness of the catalysis are controlled by the orientation of the leaving group nitrogen lone pair orbital. The leaving group binding affects the formation of a reactive conformation of the enzyme tetrahedral intermediate that is presumed to intervene between the Michaelis complex and the acylenzyme. The steric and electronic effects could be separated in a straightforward fashion only in the case of equal binding of the leaving groups to the leaving-group-binding site of alpha-chymotrypsin.


Purification and characterization of a carboxyl ester hydrolase from human pancreatic juice. A carboxyl ester hydrolase has been purified 20-fold from human pancreatic juice. It is a glycoprotein with a molecular weight of 100 000. It contains 9% neutral and amino carbohydrates and the amino acid composition is characterized by a high content of proline residue (12.7%). The enzyme catalyzes the hydrolysis of p-nitrophenylacetate and the activity increases in the presence of biliary salts; V is not modified but Km is decreased 10 times by addition of biliary salts. The enzyme migrates on Sephadex G-200 as a protein with a molecular weight of 300 000. This behaviour does not seem to be due to a polymerization but to a peculiar configuration of the enzyme.


Some physicochemical and structural properties of two beta-fucosidases from Achatina balteata. Some properties of two beta-fucosidases from the digestive juice of a giant snail Achatina balteata were studied. Their amino acid composition was similar and both enzymes were shown to be rich in acidic and aromatic residues. Alanine was the single N-terminal residue in the two molecules. The enzymes appeared to be devoid of sialic acid but to contain galactose, glucose, mannose, fucose and hexosamines. The total sugar content was higher in beta-fucosidase I (21.6% w/w) than in beta-fucosidase II (12.7% w/w). Gel filtration and ultracentrifugation assays were consistent with an apparent molecular weight higher than 300 000 for beta-fucosidase I and of about 110 000 for beta-fucosidase II. On SDS polyacrylamide gel electrophoresis, beta-fucosidase I dissociated into two subunits (mol. wt. 190 000 and 170 000), whereas beta-fucosidase II showed a single band with a mol. w. of about 110 000. On the basis of these studies, it is concluded that the two forms are not structurally related proteins.


Studies on brain cytosol neuraminidase. II. Extractability, solubility and intraneuronal distribution of the enzyme in pig brain. The origin and properties of cytosolic neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) from pig brain were studied. 1. The brain extracts containing the cytosol derived from neuronal bodies and glial cells carry 0.69 munits neuraminidase/g fresh tissue. The behaviour of neuraminidase during extraction closely paralleled that of authentic cytosolic enzyme, lactate dehydrogenase; whereas, it differed from that of the lysosomal enzymes, beta-hexosaminidase and beta-galactosidase, also found in the extracts. 2. Nerve endings from either crude or purified preparations, when treated by hypoosmotic shock, released neuraminidase activity up to a maximum of 1.25 munits/g fresh tissue. The behaviour of releasable neuraminidase was always identical to that of lactate dehydrogenase and very similar to that of ATPase and acetylcholinesterase. Typical lysosomal enzymes, however, such as beta-galactosidase and beta-hexosaminidase, behaved differently under the same conditions. This neuraminidase activity is thought to be derived from the cytosol of nerve endings. 3. The specific activity of neuraminidase in nerve-ending cytosol is 15--20 times that in neuronal body and glial cell cytosol. Some properties (pH, Km value, V/t relationship) of the cytosolic enzymes of different origin are similar; others (stability on standing at 4 degrees C; resistance to freezing and thawing) are different. Hypoionic solutions caused both cytosolic neuraminidases to slowly precipitate and to assume a stable insoluble form which was still active.


Use of glyceraldehyde-3-phosphate dehydrogenase-depleted human erythrocyte ghosts as specific high affinity adsorbents for the purification of glyceraldehyde-3-phosphate dehydrogenase from various tissues. Human erythrocyte ghosts depleted of glyceraldehyde-3-phosphate dehydrogenase are used as specific high-affinity adsorbents for the purification of glyceraldehyde-3-phosphate dehydrogenase from mouse muscle, liver, kidney and brain. On incubation with the crude tissue homogenates, the depleted ghosts bind glyceraldehyde-3-phosphate dehydrogenase, aldolase, and a few other proteins. Washing the incubated ghosts several times with 5 mM phosphate buffer(pH 8.0) removed several of the non specifically bound proteins. Aldolase can be eliminated from the membrane by incubating the ghosts for 30 min in 5 mM phosphate buffer (pH 8.0)/2mM fructose 1,6-biphosphate, and then washing with the same solution. Glyceraldehyde-3-phosphate dehydrogenase can then be specifically eluted from the ghosts by incubating them with 2 mM NADH in 5mM phosphate buffer (pH 8.0). Although the enzyme from brain appears to bind less strongly to the ghosts it was possible, using this procedure, to purify glyceraldehyde-3-phosphate dehydrogenase from all the tissues investigated. The purified enzyme exhibits high specific activity and migrates as a single band (during SDS polyacrylamide gel electrophoresis) which corresponds to a protomer molecular weight of 37 000.


The interaction of fatty acids with rabbit liver and muscle glycerol-3-phosphate dehydrogenase. Various fatty acids containing 10--22 carbons and including unsaturated derivatives were found to be inhibitors of rabbit liver and skeletal muscle sn-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8). For the liver enzyme, the logarithm of the inhibition constant was linearly related to the number of carbon atoms in the saturated fatty acids whereas the muscle enzyme, which was generally more strongly inhibited, showed a nonlinear dependence. The liver and muscle enzymes also interacted differently with a series of unsaturated fatty acids for which a high degree of specificity was exhibited which was related to the position, configuration, and number of double bonds in the compound. A steady-state kinetic analysis shows that under some conditions, the kinetics of the NADH reduction of dihydroxyacetone phosphate by NADH in the presence of stearic acid do not follow simple Michaelis-Menten behavior but rather the velocity shows a sigmoidal dependence on fatty acid concentration and strong substrate inhibition. Stearic acid is a much poorer inhibitor of the NAD-dependent oxidation of glycerol-3-phosphate. At low substrate concentrations stearic acid is competitive with respect to NAD with an inhibition constant of 24 micrometer for stearic acid. In addition to the effect of fatty acids on the initial velocities of the enzyme-catalyzed reactions, preincubation of the enzyme with fatty acid leads to a slow, time-dependent irreversible inactivation of the enzyme which is prevented by the presence of NADH. The results are discussed in terms of the differences in the conformations of the hydrophobic binding sites on the two enzymes.


Purification and properties of the major isozymic form of cytoplasmic glycerol-3-phosphate dehydrogenase from rabbit liver. The major isozymic form of sn-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) has been purified from rabbit liver using a simplified three-step chromatographic procedure involving an ion exchange and two affinity chromatography steps. The 1200-fold purified enzyme is electrophoretically homogeneous, nucleotide-free, and possesses a specific activity of 295 units/mg and an isoelectric point of 6.5. A steady-state kinetic analysis was applied to both the forward and reverse reactions. The NADH oxidation reaction was found to adhere to Michaelis-Menten behavior with Km values of 22 micrometer and 75 micrometer for NADH and dihydroxyacetone phosphate, respectively. In the NAD reduction reaction, sigmoidal kinetic patterns were observed when NAD was the variable substrate whereas with sn-glycerol-3-phosphate as the variable substrate, strictly hyperbolic kinetics were observed. The apparent Km values for NAD and glycerol-3-phosphate were 83 and 909 micrometer, respectively. By comparison with published reports, these results demonstrate that the rabbit muscle and liver isozymes of sn-glycerol-3-phosphate dehydrogenase have different kinetic properties and suggest that the liver isozyme is better adapted to participation in glyconeogenesis in vivo.


Purification of thymidine phosphorylase from human amniochorion. Thymidine phosphorylase (thymidine : orthophosphate deoxyribosyltransferase, EC 2.4.2.4) has been purified 1500-fold from extracts of human amniochorion. The purified enzyme catalyzes the phosphorolysis of deoxythymidine and to a lesser extent deoxyuridine but not deoxycytidine nor uridine. Discontinuous gel electrophoresis of the freshly purified enzyme shows a band containing 95% of the stainable protein. Gradient gel electrophoresis resolves the preparations into an active fraction with an apparent molecular weight of about 120 000 and a heavier less active or inactive fraction of about 180 000. Storage of the enzyme results in a decrease of the 120 000 dalton component, a loss in activity, and an apparent increase in the high molecular weight component. Sodium dodecyl sulfate gel electrophoresis shows only a single subunit of about 58 000 daltons which does not change on storage. These data are consistent with an active enzyme dimeric in structure which is capable of being converted to a less active form larger in molecular weight and possibly trimeric or tetrameric in structure.


Purification and properties of the ATP sulphurylase of rat liver. ATP sulphurylase (ATP:sulphate adenylyltransferase, EC 2.7.7.4) as been purified about 2500-fold from rat liver. It was free of ATPase, inorganic pyrophosphatase, adenosine phosphosulphate kinase and ADP sulphurylase activities. The enzyme was homogeneous to chromatography on Sepharose 4B and to density-gradient sedimentation; it was not homogeneous to acrylamide gel electrophoresis nor to sedimentation in the ultracentrifuge. Possible reasons for this heterogeneity are considered. The molecular weight of the enzyme is 410 000 as measured by chromatography on Sepharose 4B. The v is 0.80, suggesting that ATP sulphurylase is a lipoprotein. The enzyme activity is associated with a pigment having a lambdamax of 410 nm. Studies of the forward, reverse and ATP-PPi exchange reactions catalysed by ATP sulphurylase have shown that these are sequential bi-bi reactions, with ATP being the first substrate bound and adenosine phosphosulphate the last product released. The results are incompatible with previous suggestions that the ATP sulphurylase of rat liver catalysed a bi-bi ping-pong reaction.


Purification of indolyl-3-alkane alpha-hydroxylase by affinity chromatography on indolyl-agarose columns. Indolyl-3-alkane alpha-hydroxylase was isolated from soil isolate organism, Pseudomonas XA, by affinity chromatography on indolyl-agarose, using different indole derivatives (L-tryptophan, N-acetyl-L-tryptophan, indole-3-carboxaldehyde and 3-indole-acrylic acid). With the exception of N-acetyl-L-tryptophan-agarose, excellent yields were obtained. The affinity chromatography step caused a 15-fold increase in the specific activity of the enzyme. The purity of indolyl-3-alkane alpha-hydroxylase was comparable to the preparations obtained by conventional isolation techniques; however, it showed a 7- to 10-times higher overall yield. Affinity purified indolyl-3-alkane alpha-hydroxylase exhibited essentially one band in polyacrylamide gel electrophoresis and on isoelectric focusing.


Presence of a single enzyme catalyzing the pyrophosphorolysis of UDP-glucose and UDP-galactose in Bifidobacterium bifidum. The enzyme preparation catalyzing the pyrophosphorolyses of UDP-glucose and UDPgalactose almost at the same rate was purified about 900-fold from Bifidobacterium bifidum grown on glucose medium. The two activities were always associated with each other, and their activity ratio was always constant throughout the purification steps. The final preparations was revealed homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. There was no significant difference in thermal stabilities of the two activities. From these results it was concluded that the UDPglucose and UDPgalactose pyrophosphorylase activities in B. bifidum are catalyzed by a single enzyme protein.


Enhancement of alpha-chymotrypsin-catalyzed hydrolysis of specific p-nitroanilide substrates by 4-phenylbutylamine derivative of hen egg-white lysozyme. Modification of hen egg-white lysozyme by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide in presence of 4-phenylbutylamine yielded derivatives, which contained 0.6--0.7 modified residues and retained about 60% of the original activity. Kinetic studies revealed that the modified-lysozyme increases approx. 20-fold the kcat of hydrolysis of SucGly2Phe-4-nitroanilide by alphachymotrypsin, without changing the Km. The apparent dissociation constant of phenylbutylamine-modified lysozyme . chymotrypsin complex was found to be 0.03 mM and independent of substrate concentration. The accelerating effect of the modified lysozyme was also observed with other p-nitroanilide substrates of alpha-chymotrypsin. However, the hydrolysis of other substrates, acylation by active site titrant or inhibition by irreversible or competitive inhibitors were uneffected. The enhancing effect of the modified lysozyme seems to be very specific since other chymotrypsin-like enzymes, or serine proteinases except delta-chymotrypsin, were not influenced and phenylbutylamine derivatives of alpha-lactalbumin or ribonuclease were lacking any enhancing effect. Smaller, but significant enhancing effect was found also in lysozyme substituted by benzylamine, beta-phenylethylamine and tryptamine and in inactive derivatives of lysozyme substituted by phenylbutylamine. Competitive inhibitors of lysozyme such as N-acetyl-D-glucose amine oligomers, (GlcNAc)2 and (GlcNAc)3 abolished partially the accelerating effect of phenylbutylamine-modified lysozyme, indicating that the substituted group is located in the vicinity of the binding site.


Inhibition of lysozyme by polyvalent metal ions. The rate of hen egg-white lysozyme (mucopeptide N-acetylmuramoylhydrolase, EC 3.2.1.17), catalysis was determined in the presence of various metal ions (Co2+, Zn2+ and eight of the trivalent lanthanide ions). In the assay system employed, the lanthanides were found to inhibit more strongly than either Zn2+ or Co2+. The inhibition data was fitted to several models of the interactions of the metal ion with the enzyme. These models ranged in complexity from a single inhibitory metal binding site on the enzyme (two-parameter fit) to the presence of two non-independent and non-equivalent inhibitory metal binding sites (five-parameter fit). The more complicated models did not fit the data more precisely than the simplest one-site model, suggesting that the adoption of the simpler model is warranted. The fact that the association constants obtained from the simplest analysis for Co2+ (1.3 +/- 1.9 . 10(2) M-1) and Gd3+ (7.0 +/- 2.6 . 10(3) M-1) are consistent with literature values determined from spectroscopic measurements further supports the validity of the simplest model.


Comparison of C18-, C20- and C22-unsaturated fatty acids in reducing fatty acid synthesis in isolated rat hepatocytes. C18-, C20- and C22-unsaturated acids were tested for inhibition of fatty acid synthesis in hepatocytes isolated from essential fatty acid-deficient rats. Fatty acid synthesis was measured by incorporation of radioactivity from [1-14C-A1-acetate or 3H2O into fatty acids. C20-polyunsaturated fatty acids included arachidonic acid (20 : 4 (n-6)) and 4 other fatty acids formed from linoleic acid (18 : 2 (n-6)) or linolenic acid (18 : 3 (n-3)). These were (11,14)-icosadienoic acid (20 : 2 (n-6)), (8,11,14)-icosatrienoic acid (20 : 3 (n-6)), (11,14,17)-icosatrienoic acid (20 : 3 (n-3)) and (5,8,11,14,17)-icosapentaenoic acid (20 : 5 (n-3). All of these have essential fatty acid activity. The fatty acid (5,8,11)-icosatrienoic acid (20 : 3 (n-9)) was also tested. This fatty acid is formed from oleic acid (18 : 1 (n-9)) and is not an essential fatty acid or a prostaglandin precursor. C20-unsaturated fatty acids and (22 : 6 (n-3)) were as effective as stearic acid in inhibiting fatty acid synthesis and were more inhibitory than their precursor C18-unsaturated fatty acids. These results are evidence that C20-unsaturated fatty acids of the linoleic and linolenic acid series can act in short-term inhibition as well as in adaptive inhibition of fatty acid synthesis (Bloch, K. and Vance, D. (1977) Annu. Rev. Biochem. 46, 263--298). The effectiveness of (5,8,11)-icosatrienoic acid indicates that short-term inhibition by C20-unsaturated fatty acids is not limited to those fatty acids which have essential fatty acid activity.


Fatty acyl-CoA inhibition of beta-hydroxy-beta-methylglutaryl-CoA reductase activity. The influence of the fatty acyl-CoA thioesters on rat liver microsomal hydroxymethylglutaryl-CoA reductase activity was tested in vitro to determine if the previously demonstrated inhibition of [14C]acetate incorporation into cholesterol is due to inhibition of this rate limiting step in cholesterol synthesis. The polyunsaturated fatty acyl-CoA thioesters caused the greatest inhibition of enzyme activity, 50 micron arachidonoyl-CoA inhibiting 67% and 5 micron inhibiting 22%. 50 micron linoleoyl-CoA inhibited 56% with the more saturated thioesters causing less inhibition. 50--100 micron free fatty acids, free CoA, cholesterol esters, phospholipids, carnitine derivatives, prostaglandins and non-specific detergents caused little or no inhibition of enzyme activity. Kinetic studies revealed the inhibition to be noncompetitive with respect to hydroxymethylglutaryl-CoA with a Ki for arachidonoyl CoA of 3.10 micron. Fatty acyl-CoA inhibition of in vitro cholesterol synthesis is due to inhibition of hydroxymethylglutaryl-CoA reductase activity. Variation in intracellular concentrations of fatty acyl-CoA thioesters may signficantly alter cholesterol synthesis.


The positional specificity of a desaturase in the psychrophilic bacterium Micrococcus cryophilus (ATCC 15174). The positional specificity of the desaturase activity in the psychrophilic bacterium Micrococcus cryophilus (ATCC 15174) is shown to be delta9. The desaturase is inhibited by sterculic acid. Small amounts of delta8, delta10 and delta11 isomers are present. The implications of these findings for fatty acid metabolism in M. cryophilus are discussed. It is suggested that the temperature-dependent chain length change, known to occur in the phospholipid fatty acids of this bacterium, is not mediated by either a temperature-dependent change in desaturase substrate specificity or the induction of new desaturase enzymes with novel positional specificity. It is concluded that the control by temperature of fatty acid chain length is mediated by either a temperature-dependent change in the products of fatty acid synthetase or a temperature-sensitive palmitate elongase.


Malonyl-CoA decarboxylase from the mammary gland of lactating rat. Purification, properties and subcellular localization. Malonyl-CoA decarboxylase (EC 4.1.1.9) was purified 500--600-fold from the mammary gland extracts by (NH4)2SO4 precipitation, gel filtration with Sepharose 4B, anion-exchange chromatography with QAE-Sephadex, and chromatography with NADP-Agarose. This enzyme (spec. act. 200--300 nmol/min per mg protein) had a molecular weight of approx. 170 000. It did not cross-react with rabbit antiserum prepared against either fatty acid synthetase from the mammary gland or malonyl-CoA decarboxylase from the uropygial gland of goose. The decarboxylase showed a pH optimum near 8.5--9.0 and a Km of 0.33 mM, decarboxylated neither malonic acid nor methylmalonyl-CoA and was inhibited by thiol directed reagents but not by avidin. Sucrose density gradient centrifugation of the gland homogenate showed that the major peak of decarboxylase activity coincided with that of cytochrome oxidase. Breakage of mitochondria released greater than 80% of the decarboxylase activity into the 105,000 X g supernatant, suggesting that malonyl-CoA decarboxylase may be located in the mitochondrial matrix.


Abnormal liver cholesteryl ester storage in a strain of golden hamsters. Possible model for studies on cholesterol metabolism. Very high values of hepatic cholesteryl esters were found in a strain of golden hamsters isolated in our laboratory: 2 g/100 g in animals kept at 22 degrees C and more than 10 g/100 g in starved cold-adapted animals. The rate of incorporation of labelled mevalonate was not increased, but the esterification of the newly-synthetized cholesterol was markedly stimulated.


Lipoprotein lipase of cultured mesenchymal rat heart cells. III. Effect of glucocorticoids and insulin on enzyme formation. Lipoprotein lipase activity was studied in mesenchymal cells isolated from rat hearts and cultured for up to 8 days. The enzyme activity increased markedly between day 3 and 5 while the subsequent increase was less pronounced. Addition of hydrocortisone to complete culture medium resulted in an increase in lipoprotein lipase activity at all stages of culture. Lipoprotein lipase activity did not increase after addition of insulin to the complete culture medium. In the presence of serum-poor medium between day 3 and 6, the increase in lipoprotein lipase activity was much lower than in the presence of complete culture medium. Addition of hydrocortisone and insulin to the serum-poor medium resulted in a significant rise in lipoprotein lipase activity while less consistent effects were obtained after addition of each hormone alone. Transfer of cells to serum-poor medium between day 6 and 7 of culture caused a fall in enzyme activity. Addition of hydrocortisone alone and with insulin restored enzyme activity to control values. No effect on lipoprotein lipase was seen with estradiol, growth hormone, or glucagon when added to serum-containing medium, or serum-poor medium. These results indicate that the lipoprotein lipase of heart is controlled by glucocorticoids and that this control might require the presence of insulin for optimal expression.


Synthesis of trinitrophenylaminolauric acid and the use of its glyceryl esters for assaying lipase by a spectrophotometric procedure. Trinitrophenylaminolauric acid was synthesized from omega-aminolauric acid and trinitrobenzenesulfonic acid and then condensed with glycerol to yield mono-, di- and triacylglyceryl esters of this acid. Hydrolysis of these glycerides was followed by isolation of the yellow fatty acid with the aid of one solvent extraction step and estimating its content by spectrophotometry. This procedure was used to assay the activities of lipases from hog pancreas, rat bile, microsomes of rat brain and Rhizopus arrhizus delamar.


Purification, some properties and the primary structures of three reduced and S-carboxymethylated toxins (CM-5, CM-6 and CM-10a) from Naje haje haje (Egyptian cobra) venom. Three reduced and S-carboxymethylated toxins (CM-5, CM-6 and CM-10a) were purified from Naja haje haje (Egyptian cobra) venom. Whereas toxin CM-5 comprises 71 amino acid residues and five intrachain disulphide bridges, toxins CM-6 and CM-10a contain each 61 residues and four disulphide bridges. The complete primary structures of the three toxins have been established. The toxicity, the immunochemical properties, the sequence and the invariant amino acid residues of toxin CM-5 resemble the properties of the long neurotoxin group, while those of toxin CM-6 and CM-10a are related to the short neurotoxin group. Further, the sequences of the three toxins from Naja haje haje venom reveal a high degree of homology with those of the corresponding neurotoxins isolated from Naja haje annulifera or Naja nivea venoms.


Sulfhydryl reactivity of human erythrocyte superoxide dismutase. On the origin of the unusual spectral properties of the protein when prepared by a procedure utilizing chloroform and ethanol for the precipitation of hemoglobin. 1. During purification of human superoxide dismutase by the McCord-Fridovich procedure (McCord, J.M. and Fridovich, I. (1969) J. Biol. Chem. 244, 6049--6055) the "extra" sulfhydryl groups react with a variety of sulfur containing compounds including zero-valent sulfur to yield several dismutase fractions containing excess sulfur atoms and having a unique absorption band in the region of 325 nm. This is shown to be artefact of the purification procedure. 2. Cysteine trisulfide and glutathione polysulfide were found to react with native human superoxide dismutase to yield derivatives having no reactive sulfhydryl groups and possessing spectral properties similar to the various fractions obtainable from the above purification procedure. A structure of the type protein-CH2-S-(S)n R is proposed to account for the results. The value of n is variable, and the additional sulfur reactive toward thiol reagents is thought to be due to persulfides (R = H). The 325 nm band is probably due to a n leads to sigma ss transition associated with a strained S-S bound.


Hydrodynamic studies on the self association of vertebrate skeletal muscle myosin. Sedimentation velocity studies on myosin A solutions at high ionic strength combined with computer-simulation of the concentration dependence of the sedimentation coefficient for a rapidly reversible monomer-dimer equilibrium have confirmed that if such an equilibrium does exist it has an equilibrium constant of less than 10 ml/g. A new hydrodynamic treatment has been used to calculate the molecular weight of myosin from s0 and ks alone and has yielded a value of 470 000. Combination of viscosity and sedimentation velocity results has shown that the myosin molecule displays little swelling (Vs/v = 1.1 +/- 0.1). A new picture of the myosin molecule is presented in which a conformational change in the head region is suggested to account for the variation in published s 0 values.


Radioelectrophoresis: a specific microassay for parvalbumins. Application to muscle biopsies from man and other vertebrates. A two-dimensional radioelectrophoretic method is described, by which parvalbumins from minute biopsy samples (approx. 50 mg) can be detected and quantitated by their 45Ca2+-binding properties. In the first dimension, parvalbumins are purified by sieving through a gradient polyacrylamide gel and collected at the bottom of the electrophoresis tubes. The second dimension is a disc electrophoresis in the presence of 45Ca2+. Parvalbumins can thus be identified and quantitated by three criteria: low molecular weight, acidic character and calcium-binding properties, since they are never exposed to denaturing conditions. Validity of the technique was demonstrated on carp myogen, and on extracts from rabbit psoas and heart muscles. Application of this method to the shrew fast beating myocardium shows that it does contain parvalbumin, in agreement with the proposed role of soluble relaxing factor (Pechère et al. (1977) FEBS Lett. 75, 111--1141. When applied to human muscle biopsies, radioelectrophoresis points to an uneven distribution of parvalbumin among different skeletal muscles. For the human limb muscles tested in this study, the parvalbumin content is similar to that of rabbit psoas muscle.


Heterogeneity of avenin, the oat prolamin. Fractionation, molecular weight and amino acid composition. The prolamin, avenin, was extracted from oat seeds and shown to be maximally extractable in 45% (w/w) ethanol. A purification procedure is described and some properties of avenin are examined. As with wheat and barley prolamins, salt soluble and glutelin fractions were simultaneously extracted. Starch gel electrophoresis revealed two novel fractions, which were isolated by ion-exchange chromatography. These fractions have similar amino acid composition, threonine as the N-terminal amino acid and both have a 22 500 molecular weight. It is suggested that the avenin constituents have a common ancestral gene.


Purification and characterization of two different agarose-degrading enzymes. Agarase was concentrated and purified from culture filtrates of an agar-degrading Pseudomonas-like bacteria by affinity chromatography on divinyl sulphone cross-linked Sepharose 4B. By fractionation on Sephadex G-200 three fractions were obtained two of which, agarases I and II, had agarase activity. Agarase II was further purified by isoelectric focusing, and the main peak, agarase IIb, was isoelectric at pH 5.1. Molecular weight determinations indicated agarase I to be a dimer with Mr approximately 210 000. The Mr of agarase IIb was 63 000 as determined by analytical ultra-centrifugation, polyacrylamide gel electrophoresis in sodium dodecyl sulphate and molecular sieve chromatography on Sepharose 4B in 6M Gdn-HCl. The amino acid compositions of the two proteins were very similar and both were found to be glycoproteins. The pH optimum of the enzymes was 6.7 and the optimal temperature was 38 degree C for agarase I and 43 degree C for agarase IIb. Melted agarose and agarose gel were used as substrates for the enzymes. The ratio of the activities towards the different substrates was 4.3 for agarase I and 1.0 for agarase IIb. Agarase I hydrolyzed the beta-linkages in neoagarooctaose so as to produce two moles of neoagarotetraose or one mole of neoagarohexaose and one mole of neoagarobiose. Agarase IIb hydrolyzed only the central beta-linkage to form two moles of neoagarotetraose.


Purification and properties of porcine platelet aggregating factor. Porcine platelet aggregating factor was purified from porcine plasma by a rapid batch procedure which included polyethylene glycol precipitation and adsorption on calcium citrate. The aggregating factor was separated from antihemophilic factor by gel chromatography in the presence of 1 M MgCl2. It appeared homogenous when examined by immuno- or SDS-gel electrophoresis. The molecular weight was estimated to be 10 million by exclusion chromatography. After reduction, subunit molecular weight, by sodium dodecyl sulfate (SDS)-gel electrophoresis, was 225 000. Amino acid and carbohydrate composition were similar to those reported for the bovine material. The porcine platelet aggregating factor was found to have no free sulfhydryl groups or exposed disulfide bonds. Binding of formalin-fixed washed human platelets to the aggregating factor linked to Sepharose was inhibited in 0.5 M NaCl or 2.7 M urea and reversed by the presence of free aggregating factor in a concentration-dependent manner. Ristocetin had little or no discernible effect on binding.


Demonstration of human pancreatic anionic trypsinogen in normal serum by radioimmunoassay. A specific radioimmunoassay for human pancreatic anionic trypsin has been developed. The trypsin employed as radioiodinated tracer in the assay was inactivated with tosyl-L-lysine chloromethyl ketone in order to prevent binding of the tracer to the serum inhibitors alpha1-antitrypsin and alpha2-macroglobulin. A normal serum level of immunoreactive anionic trypsin of 5.45 ng/ml was determined. The results of experiments in which serum was fractionated by Sephadex G-200 gel filtration suggest that essentially all of the immunoreactive material in normal human serum is trypsinogen. This finding implies that a small fraction of the zymogens synthesized in the pancreas are released directly into the circulation.


Mucin degradation in the intestine.

Mucin degradation in the intestine. Rat intestinal mucin was labelled biologically by intraperitoneal injection of radioactive amino acids and monosaccharides 3--6 h prior to killing, followed by isolation and purification of the mucin from mucosal scrapings. The labelled product was then introduced into intestinal segments of rats under ether anesthesia for periods up to 3 h, removed by washing and assessed for evidence of degradation. In segments containing the pancreatic ducts the total mucin precipitable by cetyltrimethylammonium bromide fell from 80% to 5% in 3 h. At 3 h, chromatography on Sephadex G-100 and Sepharose 4B revealed multiple products, including very small molecular weight fragments deficient in carbohydrate label. With the introduction of neomycin sulfate into the segments to reduce bacterial growth, only two products were found, one corresponding in size to the original mucin and one somewhat smaller, although still in excess of 200,000 daltons. These products occurred independently of the presence of the pancreatic ducts in the segments, and in chronically pancreatectomized rats. The smaller product could not be produced by incubation with trypsin or elastase. Both products were altered antigenically as compared with the original mucin. Both products also retained the same ratio of carbohydrate and protein label as the original. It is concluded that mucins undergo early degradative changes in the intestine which do not involve deglycosylation but which involve partial loss of antigenicity and a fall in molecular weight. The pancreas is not responsible for these changes.


Biosynthesis of formic acid by the poison glands of formicine ants. The biosynthesis of formic acid in the poison glands of formicine ants is closely related to the C-1 metabolism of the glandular cells. Experiments utilizing radiolabeled amino acids revealed that serine is a major precursor, contributing both its alpha and beta carbons to formic acids. 5,10[14C]methylene H4folate and 5,10[14C]methenyl H4folate also serve as precursors of formic acid in the poison gland, suggesting that they are intermediates in the pathway. Furthermore, these H4folate derivates were isolated from poison glands following incubation with [3-14C]serine and proved radioactive. The glandular cells are also exceptionally rich in the enzymes responsible for these reactions, supporting the proposed pathway. Although this pathway has been established in various organisms, the uniqueness of the poison gland system is that it accumulates formic acid to large extent, yet avoids its cytotoxicity. This is made possible by a combination of the biochemical characteristics of the pathway and the special morphological features of the poison gland.


Sources of the proteins of rat bile.

Sources of the proteins of rat bile. The protein composition of rat bile has been studied systematically using two-dimensional agarose-polyacrylamide gel electrophoresis, with or without prior absorption by immobilised antisera, and by crossed immunoelectrophoresis. Sixteen bile proteins were distinguished. Of these, thirteen are immunologically identical to proteins present in rat serum and only one is identical to a protein present in rat liver plasma membrane but not in rat serum. Of the remaining two proteins, one is bile lipoprotein and the other has many of the properties of immunoglobulin A secretory component. The serum-related proteins in rat bile fall into two distinct groups. In the first group are immunoglobulin A and an alpha2-globulin. These proteins are major constituents of bile but only minor constituents of serum. In the second group are albumin and some other major serum proteins which are found in bile at concentrations less than 1% of their concentrations in serum. The relative proportions of these proteins in bile appear to differ from their proportions in serum. It therefore appears that, although the majority of bile proteins are derived from serum, there cannot be direct leakage of serum into bile. Examination of the proteins contained within liver lysosomes indicates that, although discharge of lysosomal contents at the bile canalicular face of the hepatocyte may contribute to the bile proteins, an additional mechanism, with a considerable degree of selectivity, must also be involved in the transport of proteins from serum to bile.


Effects of bile salts on the structural integrity of liposomes. The effects of the bile salts, sodium taurocholate and sodium glycocholate on the structural integrity of liposomes of different lipid compositions were studied. The bile salt concentrations were in the range encountered in the human intestine and the incubations were conducted at 37 degrees C. Two measures of liposome integrity were used: (a) changes in their light-scattering properties; and (b) release of entrapped potassium chromate. The results show that "fluid" liposomes, i.e., those prepared from phospholipids whose phase transition temperatures are below 37 degrees C, were totally disrupted in the presence of 10 mM bile salts at pH 7.4, and their entire contents were released. In the case of liposomes prepared from phospholipids with phase transition temperatures above 37 degrees C, the effect of the bile salts became less pronounced as the phase transition temperature increased. Nevertheless, a considerable proportion of the entrapped marker compound was released from liposomes with phase transition temperatures as high as 60 degrees C. The relevance of these results to the reported liposome-mediated gastrointestinal uptake of insulin is discussed.


A comparison of purine metabolism and nucleotide pools in normal and hypoxanthine-guanine phosphoribosyltransferase-deficient neuroblastoma cells. Purine nucleotide synthesis and interconversion were examined over a range of purine base and nucleoside concentrations in intact N4 and N4TG (hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient) neuroblastoma cells. Adenosine was a better nucleotide precursor than adenine, hypoxanthine or guanine at concentrations greater than 100 micron. With hypoxanthine or guanine, N4TG cells had less than 2% the rate of nucleotide synthesis of N4 cells. At substrate concentrations greater than 100 micron the rates for deamination of adenosine and phosphorolysis of guanosine exceeded those for any reaction of nucleotide synthesis. Labelled inosine and guanosine accumulated from hypoxanthine and guanine, respectively, in HGPRT-deficient cells and the nucleosides accumulated to a greater extent in N4 cells indicating dephosphorylation of newly synthesized IMP and GMP to be quantitatively significant. A deficiency of xanthine oxidase, guanine deaminase and guanosine kinase activities was found in neuroblastoma cells. Hypoxanthine was a source for both adenine and guanine nucleotides, whereas adenine or guanine were principally sources for adenine (greater than 85%) or guanine (greater than 90%) nucleotides, respectively. The rate of [14C]formate incorporation into ATP, GTP and nucleic acid purines was essentially equivalent for both N4 and N4TG cells. Purine nucleotide pools were also comparable in both cell lines, but the concentration of UDP-sugars was 1.5 times greater in N4TG than N4 cells.


Comparison of glucocorticoid-receptor complex binding to nuclei and DNA cellulose. Evidence for different forms of interaction. Binding of dexamethasone . receptors with isolated nuclei, DNA-cellulose and cellulose has been compared with respect to dependence on salt concentration and resistance to KCl extraction and DNAse I digestion. A solution of cytoplasmic dexamethasone . receptor complexes was prepared by the incubation of rat thymus cells with steroid at 3 degrees C and breaking the cells by hypotonic lysis. Activation of the complexes was accomplished by warming the solution at 25 degrees C for 15 min. Activation significantly increased the ability of dexamethasone . receptors to bind to nuclei and DNA-cellulose but not to cellulose. Dexamethasone-receptor complexes bound to nuclei at 3 degrees C are completely resistant to extraction with 0.1 M KCl, 76% resistant to 0.2 M KCl and 20% resistant to 0.4 M KCl. Dexamethasone . receptors bound to DNA-cellulose are 45% resistant to extraction with 0.1 M and 0.2 M KCl and 29% resistant to 0.4 M KCl extraction. Cellulose-bound dexamethasone . receptors are not resistant to any of these extractions. DNAase I treatment releases 60% of the dexamethasone . receptors bound to DNA-cellulose but only 13% of those bound to nuclei, though at least 60% of the nuclear DNA is solubilized. The presence of 0.15 M KCl decreases binding of activated dexamethasone . receptors to nuclei by 73% but to DNA-cellulose by only 17%. Pretreatment of nuclei with 0.1--0.4 M KCl reduces their capacity to bind activated dexamethasone . receptors by 90% whereas similar treatment reduces the capacity of DNA-cellulose to bind dexamethasone . receptors by only 29%. Nuclei extracted with 0.1 M KCl appear to have a limited capacity to accept dexamethasone . receptors. These studies demonstrate that binding of dexamethasone . receptors to nuclei and DNA-cellulose differs by (a) the higher resistance of nuclear complexes to KCl and DNAase I treatment; (b) the much greater sensitivity of nuclei to KCl treatment.


Effect of acriflavine on ultraviolet inactivation of Acholeplasma laidlawii. An increased sensitivity to inactivation was observed when ultraviolet light-irradiated Acholeplasma laidlawii cells were plated on medium containing either acriflavine or chloramphenicol. Chloramphenicol reduced liquid holding recovery (dark repair) to about 10% of that in untreated irradiated cells. In acriflavine treated cells no dark repair could be observed and there was a progressive degradation of cell DNA during holding. While the primary effect of acriflavine may be to inhibit excision repair, since ultraviolet-irradiated Mycoplasma gallisepticum (cells which lack an excision repair mechanism) show a slight increase in inactivation when plated on medium containing acriflavine the dye must also have some other effects on ultraviolet repair processes. Acriflavine treatment of A. laidlawii cells before ultraviolet irradiation has a protective effect, as seen by an increased cell survival.


Separation of retinoid receptors from cultured retinoblastoma cells. Receptor proteins for [3H]retinol and [3H]retinoic acid in cultured human retinoblastoma cells have been separated rapidly and reproducibility by two different methods. By isoelectric focusing, the isoelectric point of the retinol receptor is at pH 4.0; the retinoic acid receptor has a higher isoelectric point of 4.3. Polyacrylamide slab gel electrophoresis revealed a slower migration rate for the [3H]retinoic acid receptor compared to the [3H]retinol receptor. The separate nature of the two proteins has thus been established in this unique human cell line.


Impairment of glycoprotein secretion by phenobarbital in rat liver slices. The effects of phenobarbital on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Phenobarbital (2 mM) decreased [14C]glucosamine and [14C]leucine incorporation into liver proteins and markedly inhibited their incorporation into medium (secretory) proteins. The inhibitory effect of phenobarbital was dose dependent and not reversible under the conditions of this study. In the presence of cycloheximide, an inhibitor of peptide synthesis, phenobarbital still inhibited the release of glycoproteins into the medium; however, the specific activity of liver glycoproteins was increased. The effects of phenobarbital on hepatic macromolecular secretion, independent of its effects on synthesis, were determined by prelabeling proteins in a liver slice system with either [14C]leucine or [14C]glucosamine. When phenobarbital was present, the secretion of these prelabeled proteins into the medium was inpaired. 12 h after intraperitoneal injections of phenobarbital, glycoprotein secretion was inhibited from liver slices prepared from the pretreated rats. This inhibition of secretion occurred even though protein synthesis was stimulated and intracellular glycosylations unaffected. The results of this study indicate that phenobarbital impairs the secretion of glycoproteins by the liver.


Evidence for the incorporation of molecular oxygen, a pathway in biosynthesis of N-glycolylmuramic acid in Mycobacterium phlei. The hypothesis that the biosynthesis of the glycolyl group of muramic acid in the peptidoglycan of Mycobacterium phlei is catalyzed by a N-acetyl hydroxylase is strongly supported by the experiments reported in this paper. 18O is incorporated into the N-substituent of muramic acid isolated from the peptidoglycan of M. phlei grown under pure oxygen enriched with the 18O isotope.


Studies on the cytochrome P-450 product complexes formed during the metabolism of N,N-dimethylaniline. The oxidative metabolism of N,N-dimethylaniline by partially solubilized cytochrome P-450 from rabbit liver was found to be associated with the formation of a 424- and 448-nm product adduct of the hemoprotein. From the effects of temperature, hydrogen ion concentration, n-octylamine, extraction of the enzyme preparations with organic solvents and pretreatment of the animals with inducers of drug metabolism on both the formation of the spectral species and the enzymic C- and N-oxidation of N,N-dimethylaniline it is concluded that the 424-nm spectral change is generated from an intermediate in the C-oxidation reaction, whereas formation of the 448-nm spectral perturbation is the result of binding to cytochrome P-450 of a metabolite arising from N-oxidation of the arylamine; N-dealkylation of the parent amine is not a obligatory intermediary step in 448-nm complex formation. The 448-nm ferrohemochrome is supposed to be formed through coordination of the N-oxidized intermediate via the oxygen atom. This type of interaction appears to require considerably stronger thermal activation as compared with the 424-nm complex. The 448-nm product adduct of cytochrome P-450 is unstable in the ferric state or in the presence of sodium dithionite.


Theoretical investigations on the conformation of 1,5,N(4),N(4)-tetramethylcytosine. Theoretical investigations (Perturbative Configuration Interaction over Localized Orbitals (PCILO) and Intermediate Neglect of Differential Overlap (INDO) methods) of the conformation of tetramethylcystosine, an overcrowded molecule with planar structure, have been carried out. The physical features of rotational bending potentials of the dimethylamino group are discussed. Particularly, the controversial problem concerning the planarity of the molecule is investigated. The obtained results show that the planarity of tetramethylcytosine is an intrinsic property of the molecule. Nevertheless, because of the repulsion between the methyl groups, the planar structure of tetramethylcytosine is slightly destabilized. Further, the functional dependence of the dipole moment of tetramethylcytosine on rotation and bending of the dimethylamino group has been analyzed.


In vitro studies of the interaction between ascorbic acid and rat tail tendon. Ascorbic acid (in its normal and oxidised forms) enhances the mechanical and thermal stability of rat tail tendon. Its effectiveness increases with the concentration but levels off at a value approximately 5 times normal physiological concentration (1--2 mg/100 ml). An analogue, D-isoascorbic acid is also effective, but to a lesser extent. There is some evidence that it reduces reducible aldimine links, especially in young tissues. However, for the most part, its effects are reversible.


Increased uptake and desulphation of heparin by mouse macrophages in the presence of polycations. Heparin uptake and desulphation by cultured macrophages were investigated. Histones, polyamino-acids, protamine and eosinophil-basic protein stimulated both heparin uptake and desulphation, processes found to be non-related. Poly-L-ornithine and poly-DL-lysine increased the heparin uptake by about 33-fold, and histone produced up to 7.5-fold increase in the desulphation. The same polycations inhibited heparin desulphation by macrophage extracts.


Glucosamine metabolism in Drosophila salivary glands. Separation of metabolites and some characteristics of three enzymes involved. The conversion of fructose 6-phosphate to mucopolysaccharide precursors was studied in extracts of Drosophila virilis salivary glands. 1. Methods for chromatography of sugar phosphates were adapted and modified to allow routine separation and quantitation of radioactivity of the metabolites from milligram amounts of tissue. Anion exchange chromatography was performed on Dowex 1-X8 employing steps of increasing ammonium formate. Final isolation of each compound was achieved by various thin-layer chromatographic systems. 2. Data obtained by isotope incorporation into glucosamine 6-phosphate compare well with results of the Morgan-Elson colorimetric assay for amino-sugars. 3. Glucosaminephosphate isomerase (glutamine-forming) (EC 5.3.1.19) in gland extracts has a Km of 0.35 mM for fructose 6-phosphate, and of 0.25 mM for glutamine. The enzyme is inhibited at glutamine concentrations exceeding 1 mM and by UDP-N-acetylglucosamine (50% at 0.6 mM). Feedback inhibition by UDP-N-acetylglucosamine is enhanced by AMP and by glucose 6-phosphate. 4. Glucosaminephosphate isomerase (EC 5.3.1.10) has a twenty fold lower affinity towards fructose 6-phosphate (Km = 6.0 mM) compared to the glutamine-forming isomerase. Km (NH+4) is 7.4 mM. In the presence of 20 mM glucose 6-phosphate, the pH optimum is shifted from 6.6 to 7.4, and V increased by a factor of 2.5. Furthermore, the affinity is approximately doubled for both substrates. 5. Glucosamine acetyltransferase (EC 2.3.1.3) has a Km of 2 mM for glucoseamine 6-phosphate. Its activity is not rate-limiting in salivary glands. Since N-acetylglucosamine 6-phosphate and 1-phosphate were found near equilibrium concentrations, acetylglucosamine phosphomutase (EC 2.7.5.2) must also be present in the extracts.


The effects of insulin on the turnover of glucose-6-phosphate dehydrogenase in epididymal adipose tissue of the rat. The effects of insulin on the turnover of glucose-6-phosphate dehydrogenase in rat epididymal adipose tissue were studied by immunochemical technique in in vitro incubations. Insulin increased the relative rate of synthesis of glucose-6-phosphate dehydrogenase by two-fold in tissue obtained from normal rats. Insulin also had an effect on the rate of degradation of this enzyme. In the absence of insulin in the incubation medium the rate constant of degradation was 0.11 h-1 (half-life, 6.3 h). When insulin was added to the medium degradation of this enzyme was slowed. The new rate constant of degradation was 0.04 h-1 (half-life, 17 h). In the presence of insulin, the rate constant of degradation of total protein in adipose tissue was unchanged; therefore the effects of insulin on the degradation of glucose-6-phosphate dehydrogenase are specific to that protein and perhaps to a few other specific proteins.


alpha-Bungarotoxin binding properties of a central nervous system nicotinic acetylcholine receptor. High-affinity, specific binding of radiolabeled alpha-bungarotoxin to particulate fractions derived from rat brain shows saturability (Bmax approximately 37fmol/mg, KDapp = 1.7 nM) and insenstivity to ionic strength, and is essentially irreversibel (Kon = 5 . 10(6) min-1 . mol-1; Kdisplacement = 1.9 . 10(-4) min-1, tau1/2 = 62 h). Subcellular distribution of specific sites is consistent with their location on synaptic junctional complex and post-synaptic membranes. These membrane-bound binding sites exhibit unique sensitivity to cholinergic ligands; pretreatment of membranes with cholinerin binding sites to a high affinity form toward agonist. The effect is most marked for the natural agonist, acetylcholine. These results strongly support the notion that the entity under study is an authentic nicotinic acetylcholine receptor.


The formation of oxalate from hydroxypyruvate, serine, glycolate and glyoxylate in the rat. [14C]Oxalate was found in the urine of fasted male Wistar rats fed L-[14C3]-serine, DL-[14C1]serine, [14C1]ethanolamine, [14C1]hydroxypyruvate, [14C3]-hydroxypyruvate, [14C1]glycolate or [14C2]glyoxylate. [14C1]Hydroxypyruvate and [14C1]ethanolamine were the least effective precursors of [14C]oxalate. 20% of the [14C1]serine and [14C3]serine administered was recovered as 14CO2, while less than 4% was metabolized by pathways known to contribute to oxalate synthesis. Oxalate synthesis from serine involved both the transamination to hydroxypyruvate and the conversion to glycine, but not decarboxylation to ethanolamine. The oxidation of [14C2]glyoxylate to [14C]oxalate in the rat was inhibited by hydroxypyruvate, but the oxidation of [14C1]glycolate to [14C]-oxalate was not significantly altered. [14C]Glycolaldehyde, [14C]glycolate, [14C]glyoxylate and [14C]oxalate were recovered in the urine of rats administered [14C3]hydroxypyruvate. This is consistent with the oxidation of hydroxypyruvate via glycolaldehyde leads to glycolate leads to glyoxylate leads to oxalate and is identical to the metabolic pathway for the oxidation of ethylene glycol to oxalate. However, the major metabolic intermediate recovered from [14C3]hydroxypyruvate was [14C]glyoxylate rather than [14C]glycolate, suggesting that an alternate pathway is contributing to the oxidation of hydroxypyruvate to oxalate in the rat.


Role of calcium in exocrine pancreatic secretion. IV. Calcium movements in isolated acinar cells of rabbit pancreas. 1. A study has been made of the calcium movements in isolated acinar cells of rabbit pancreas in relation to the process of enzyme secretion. 2. After 90 min, the 45Ca2+ uptake level of the acinar cells reaches a steady state level which depends on the extracellular calcium concentration: it increases with increasing calcium concentration. 3. Carbachol, in addition to stimulating enzyme secretion, causes a decrease in the 45Ca2+ content of pre-loaded acinar cells. This decrease is virtually independent of the extracellular calcium concentration. It is followed by an uptake of 45Ca2+ which only in a medium with 2.5 mM Ca2+ leads to a significantly higher 45Ca2+ level than the steady state level. 4. Carbachol, added to acinar cells not preloaded with 45Ca2+, increases the 45Ca2+ uptake in medium with 2.5 mM and in that with 0.1 mM Ca2+. 5. The amount of enzyme released by carbachol depends on the extracellular calcium concentration: it is larger in media with higher calcium concentration. 6. It is concluded that the increase in cytoplasmic calcium concentration which takes place immediately upon addition of a stimulus and which is necessary for the stimulation of the enzyme secretion, is caused by a release of calcium from an intracellular pool and not by an influx of calcium from the extracellular medium. 7. The results suggest that there are at least three different calcium pools in the pancreatic acinar cells.


Purine metabolism in Leishmania donovani and Leishmania braziliensis. We have studied purine metabolism in the culture forms of Leishmania donovani and Leishmania braziliensis. These organisms are incapable of synthesizing purines de novo from glycine, serine, or formate and require an exogenous purine for growth. This requirement is better satisfied by adenosine or hypoxanthine than by guanosine. Both adenine and inosine are converted to a common intermediate, hypoxanthine, before transformation to nucleotides. This is due to the activity of an adenine aminohydrolase ((EC 3.5.4.2), a rather unusual finding in a eukaryotic cell. There is a preferential synthesis of adenine nucleotides, even when guanine or xanthine are used as precursors. The pathways of purine nucleotide interconversions in these Leishmania resemble those found in mammalian cells except for the absence of de novo purine biosynthesis and the presence of an adenine-deaminating activiting.


Bovine adrenocortical microsomal hydroxylase and thermotropic transition. Substrate-cytochrome P-450 binding reaction versus substrate hydroxylation. The effect of temperature on steroid C-21 hydroxylation and substrate-cytochrome P-450 binding reaction under turnover conditions (NADPH + O2 are investigated. The Arrhenius activity plot exhibited a single break, while the van 't Hoff plot of the substrate dissociation constant (Ks) exhibited four breaks between 10 and 40 degrees C which corresponded to the characteristic temperatures of the lipids' phase transitions. Unlike the case of the Ks value, the detergent Triton X-114 was without effect on the Arrhenius activity plot. This indicates that the single break in the case of the enzyme activity is distinct from but not necessarily independent of the multiple breaks inthe case of the Ks. At physiologic temperature and concentration of the substrate, the free energy (--9.5 kcal/mol) of the substrate-cytochrome binding reaction is more than sufficient to account for the apparent activation energy (6.6 kcal/mol) of the overall hydroxylation. This suggests that the substrate-cytochrome P-450 binding reaction has the potential of being a source of energy for the overall reaction.


Laser Raman investigation of intact single muscle fibers. On the state of water in muscle tissue. Laser Raman spectroscopy has been used to investigate the state of water in intact single muscle fibers of the giant barnacle (Balanus nubilus). The spectra in the region of the O-H (or O-2H) stretching modes of water in unfrozen fibers show that there is no appreciable difference between the shape and relative intensity of the Raman bands due to the water molecules located inside a muscle fiber and those of the corresponding bands in the spectrum of pure water. The presence of significant amounts of "structured" intracellular water, greater than approx. 5% of the total water content, in these fibers is thus excluded. The Raman spectra of frozen fibers have also been recorded in order to evaluate the amount of intracellular water which remains unfrozen at temperatures below the normal freezing point of water. We have been able to reproduce these spectra by assuming that the spectrum of a frozen fiber is the sum of the individual spectra of water and ice. To calculate the amount of unfrozen water from these curve fittings, it was also necessary to determine the intensities of the water and ice Raman bands relative to one another. We have found the I(ice)/I(water) ratio is 1.07 +/- 0.01 for H2O and 1.05 +/- 0.03 for 2H2O With these figures, we have calculated that for a fiber with a normal water content of 80%, 20% of the water molecules remain in the supercooled state of -5 degrees C, which corresponds to 1 g of water per g of fiber dry weight. This amount of bound water was also found to be independent of the water content of the fibers.


Preparation and properties of biologically active fluorescent heparins. Hog mucosal heparin (N-sulfate, 0.84 mol; O-sulfate, 1.55 mol; N-acetyl, 0.12 mol; anticoagulant activity assayed by the method of U.S. Pharmacopeia, 161 USP units/mg) or its partially N-desulfated heparin (N-sulfate, 0.71 mol; O-sulfate, 1.47 mol; N-acetyl, 0.12 mol; anticoagulant activity, 117 USP units/mg/ was reacted with 5-isothiocyanatofluorescein in 0.5 M carbonate buffer (pH 8.5) at 35 degrees C for 6 h to yield the corresponding N-fluoresceinylthiocarbamoyl heparins (lambdaem 516 nm, lambdaex 491 nm; degree of substitution 0.006 and 0.013, respectively, anticoagulant activity, 174 and 140 USP units/mg, respectively). The fluorescent heparin (degree of substitution, 0.006; 174 USP units/mg) was injected into rabbits intravenously. The half-life of the fluorescent heparin determined by fluorometry was 24 min, that determined by the clotting time assay was 39 min. The time-course of concentration and the half-life of the fluorescent heparin and of the starting heparin obtained by the clotting time assay were virtually identical.


3-O-Methyl galactose found in polysaccharides of Lampteromyces japonicus. A polysaccharide fraction from Lampteromyces japonicus contained a sugar (4%), which was identified as 3-O-methyl galactose by demethylation with boron trichloride, periodate oxidation of the methyl glycoside derivative and gas chromatography-mass spectrometry of the alditol acetate derivative.


The uptake of liposome-entrapped 125I-labelled poly(vinylpyrrolidone) by rat jejunum in vitro. The uptake of free and liposome-entrapped 125I-labelled poly(vinylpyrrolidone) was measured in an intestinal sac preparation from adult rats. At an equal concentration of 125I-labelled poly(vinylpyrrolidone), the rate of uptake of the liposome-entrapped material was four times that of the free macromolecule.


[DNA packing in the DNP fibrils of polytene chromosomes]. Isolated polytene chromosomes stained with acridine orange were stretched with micromanipulator needles. Changes of chromosome fluorescence polarization led to the conclusions: 1) almost complete orientation of DNP fibers is attained at very low levels of chromosome stretching (3--8 times) without irreversible disruption of chromomeres; 2) internucleosomal DNA in native DNP fibers is supercoiled and makes 30 degrees angle to the fiber axis.


[Equilibrium surface charge distribution in phospholipid vesicles. III. Effect of nonelectrostatic factors]. The different curvature of the two vesicle layers causes a difference in the standard free energies mu01 and mu02 of the lipid molecules in the inside and outside layers. In the present paper the influence of this difference on the equilibrium surface charge distribution is studied. The values of mu02--mu01 in the numerical calculations were in the range 0--3 kT. The results show the relative influence of electrostatic and non electrostatic forces on the equilibrium distribution of the charged lipid molecules. In the range of low surface charge density the charged molecules localize predominantly in the outside layer at low electrolyte concentrations, and in the inside layer at high electrolyte concentrations. A good agreement between the theoretical data and the experimental results of Berden et al. [3] about the phosphatidylserine distribution in lecithin-phosphatidylserine vesicles is demonstrated.


[Free radical oxidation of biological membrane lipids. VIII. Effect of linoleic acid and its oxidation products on the functional parameters of mitochondria]. Effects of linoleic acid and its oxidation products (with 75--80% content of hydroperoxide) on rat liver mitochondria were studied by polarographic and spectrophotometric methods. Both agents according to their concentration are able to uncouple oxidation and phosphorylation and to inhibit the electron transport in the initial region of the respiration chain as well. Unlike the unoxidized linoleic acid, its oxidation products increase the activity of some enzymatic systems of mitochondria, and at high concentrations they produce a more pronounced effect on the electron transport.


[Spectral transformations in purple membranes of Halobacterium halobium: effect of 560-570 transition and blue light on photochemical processes]. Effect of reversible photodependent spectral transition 560 in equilibrium 570 of the basic absorption band in purpur membranes (PM) in water on the accumulation of short-living forms 412 and 650 of bacteriorhodopsin (BR) photochemical cycle was studied. It has been shown that photochemical transformations in BR proceed as a minimum by two independent cycles, whose initial spectral forms were represented by BR with retinene in the form 13 cis and BR with retinene in the form of trans. In the course of the transition 560-- greater than 570 a long-living spectral form 370--410--430 sensitive to the blue light is formed. On the basis of our own and literature data a new scheme of photochemical processes in PM was suggested and connection between some stages of these processes and ejection and absorption of PM protones proposed.


[Quantitative model of human erythrocyte glycolysis. II. Effect of arsenate on glycolysis. Experimental study of the relationship between the rate of glycolysis and the ATP concentration]. The effect of arsenate on human erythrocyte glycolysis was studied. The stationary rate of glucose consumption rises and then drops with a gradual increase of arsenate concentration while ATP and glucose-6-phosphate concentrations drop monotonically. A plot of glucose consumption rate against ATP concentration gives a bell-like curve. This curve is the same for different donors if it is plotted in relative units with values at zero arsenate concentration taking for 100%, while the absolute values obtained for separate donors are very different. The normal (physiological) point is situated on the steeply descending part of the curve.


[Application of autocorrelation analysis to the study of conformational changes in chloroplasts]. The process of conformational oscilations of chloroplasts has been studied using autocorrelation analysis technique. The process is shown to differ from the non-correlated random process of "white noise" type. Four types of autocorrelogram have been obtained which are characteristic of the phone oscilations of chloroplast cross-section area in time. The authors suggest that the different types of autocorrelograms are due to the different possible states of chloroplasts. The periodical components with the frequency of 16; 10--11; 8; 4; 2; 0.5 and 0.3 hertz have been obtained in the kinetics of chloroplast cross-section changes. Two magnitude ranges have been found for the moment of first zero volume in autocorrelation function (ACF): 5--10 and 15--20 shifts. ACF slow attenuation towards zero may be due to quite a good organization of the process of chloroplast conformational oscilations and availability of the well-developed control system of adaptive type.


[Stability of spatially nonuniform states of diffuse systems]. The problem of stability of inhomogeneous stationary states in kinetic systems with nonlinear local dynamics and diffusion is studied. Stability criterion applicable in the case of one rate equation or two coupled rate equations with the diffusion term presented at one of them is formulated in the integral form. It is made the conclusion on the absence of stable inhomogeneous stationary states in the unicomponent system with zero fluxes at the boundaries and the case of periodic boundary conditions. In the case of non-zero fluxes at the boundaries the condition of stability of exfoliated stationary structures in the systems with bistable local dynamics is given.


[Theory of kinetic patterns. Chains and networks]. The kinetic principles of the migration of population in the homogeneous net schemes of the different dimensions are investigated. One-sided and two-sided connections between the states are compared. The general solutions are given in the operator form. The concrete form of the transfer processes is defined for the exponential own functions of states. The distribution of the average life time of the population among the states is analogous to the stationary distribution of the potential in the syncitial tissue.


[Problem of sphere formation in mathematical models of morphogenesis]. The model of a process of morphogenesis is described, being a new link in the series of mathematical models of morphogenesis based on the local interactions of cells (see [6]) and the first space model of the series. A corresponding mathematical problem is formulated. The results of the proper computer calculations are published, showing that the chosen rule of motion solves the problem of sphere formation for a very large class of initial states of all investigated types of nets. The processes taking place in solving the problem of sphere formation are considered.