Showing papers on "Enzyme assay published in 1983"
621 citations
TL;DR: In intact liver, the enzyme was found to be localized exclusively within a very small population of the parenchymal cells surrounding the terminal hepatic venules, demonstrating an interesting aspect of liver zonation and might have important implications for liver glutamine and, more generally, nitrogen metabolism.
Abstract: The distribution of glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.1)] among rat liver parenchymal cells in situ and in primary culture was investigated by indirect immunofluorescence using a specific antiserum. In intact liver, the enzyme was found to be localized exclusively within a very small population of the parenchymal cells surrounding the terminal hepatic venules. Other parts of the parenchyma including non-parenchymal cell types did not stain for this enzyme. Heterogeneity was preserved during isolation of liver parenchymal cells and persisted in cultured cells for at least 3 days. Despite alterations in enzyme activity due to the adaptation of the cells to the culture conditions or due to the hormonal stimulation of the enzyme activity, no change in the relative number of cells expressing this enzyme could be detected. This rather peculiar localization of glutamine synthetase demonstrates an interesting aspect of liver zonation and might have important implications for liver glutamine and, more generally, nitrogen metabolism. Furthermore, it raises the question of whether there might be a phenotypic difference among liver parenchymal cells.
350 citations
TL;DR: NadP-dependent formate dehydrogenase (NADP+) from Clostridium thermoaceticum has been purified to a specific activity of about 1100 mumol min-1 mg-1 when assayed at 55 degrees C and pH 7.5.
274 citations
TL;DR: A type IV collagen-degrading enzyme activity secreted by a highly metastatic mouse tumor was purified by concanavalin A- and typeIV collagen-agarose affinity chromatographies followed by gel filtration on Bio-Gel A-0.5 m, indicating that the enzyme is a hydrophobic protein.
247 citations
TL;DR: Carbon monoxide dehydrogenase from Clostridium thermoaceticum has been purified to homogeneity using a strict anaerobic procedure, indicating that the enzyme dissociates to form a dimer with an alpha beta structure.
213 citations
TL;DR: The results suggest that relatively high activities of tyrosine protein kinase exist in a normal tissue (rat spleen) and major endogenous substrates of the enzyme(s) appear to be represented by two proteins of Mr = 53,000 and 56,000; one or both of these substrates may be the tyrosines itself.
160 citations
TL;DR: The correlation between increased collagenolytic activity and the early osteoarthritic changes in cartilage suggests a role of this enzyme activity in the disease process.
Abstract: Recent reports on the Pond-Nuki model of osteoarthritis in the dog have provided evidence for partial disruption of the collagen network. The possibility of collagenase involvement in these localized changes was studied. Animals were killed 2, 4, 8, and 12 weeks after surgery. The left knee served as a sham-operated control. Cartilages from femoral condyles were processed for light and electron microscopy and assayed for collagenolytic activity by a direct tissue assay, based on the measurement of digestion of endogenous cartilage collagen. Animals killed at 2 and 4 weeks showed fibrillation and mild erosion of femoral condyles, which usually progressed to ulceration by 8 and 12 weeks. Electron microscopy demonstrated fiber disruption of the mid-zone perilacunar collagen as early as 2 weeks after the operation. Total collagenolytic activity, measured after activation by aminophenylmercuric acetate, was significantly higher in decreased cartilage than in controls of 2, 4, and 8 weeks; the peak value was at 4 weeks. Collagenase was shown, by its specific action on type I collagen, to be present at 2 and 4 weeks; however, other metalloproteases may also contribute to the digestion. The correlation between increased collagenolytic activity and the early osteoarthritic changes in cartilage suggests a role of this enzyme activity in the disease process.
144 citations
TL;DR: The observation that these transition state analog inhibitors bind poorly to thiol Subtilisin while other compounds bind nearly equally well to both enzymes suggests that thiolsubtilisin may not be able to stabilize the transition state during acylation by specific substrates.
Abstract: Subtilisin is a bacterial serine protease with a broad specificity in the S1 subsite. It has been very extensively studied using a variety of kinetic and physical techniques. A chemical derivative, thiolsubtilisin, has been subjected to similar studies in order to analyze the effects of the OH to SH conversion on enzyme activity.
135 citations
TL;DR: The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol synthase was purified 1,000-fold from the microsomal fraction of Saccharomyces cerevisiae and was reasonably stable at temperatures of up to 60 degrees C.
Abstract: The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol synthase (cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified 1,000-fold from the microsomal fraction of Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of the microsomal membranes, CDPdiacylglycerol-Sepharose (Larson et al., Biochemistry 15:974-979, 1976) affinity chromatography, and chromatofocusing. The procedure resulted in the isolation of a nearly homogeneous protein preparation with an apparent minimum subunit molecular weight of 34,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphatidylinositol synthase was dependent on manganese and Triton X-100 for maximum activity. The pH optimum was 8.0. Thioreactive agents inhibited enzyme activity. The energy of activation was found to be 35 kcal/mol (146,540 J/mol). The enzyme was reasonably stable at temperatures of up to 60 degrees C.
133 citations
TL;DR: Results indicate that fluctuations of the enzyme activity in this organ are mediated via changes in the amount of enzyme protein rather than by post-translational modifications or interaction with inhibitors or activators.
132 citations
TL;DR: Ouabain binding studies show that the inhibition of cholesterol content is not due to a loss of enzyme from the membrane, and the most likely mechanism of inhibition is suggested to comprise direct cholesterol-protein interactions which lead to a low activity conformation.
TL;DR: Results provide the first strong evidence that the induced ribonucleotide reductase activity is at least partially virus-coded.
Abstract: Ribonucleotide reductase, an enzyme found in all prokaryotic and eukaryotic cells that synthesize DNA, is induced by herpes simplex virus (HSV). In this study the effect of anti-HSV antiserum on the induced ribonucleotide reductase has been examined and the ability of different temperature-sensitive (ts) mutants of HSV-1 to induce the enzyme has been investigated. The HSV-1-induced ribonucleotide reductase was inhibited by antiserum raised against infected cell lysates but not by preimmune serum. The wild-type (ts+) virus induced similar levels of ribonucleotide reductase at 31 degrees C and 38.5 degrees C (the permissive and non-permissive temperatures respectively for the ts mutants). All ts mutants induced approximately wild-type levels of the enzyme at 31 degrees C. At 38.5 degrees C, two of the four ts mutants studied also induced wild-type levels of enzyme but ts G failed to induce any activity while ts K induced variable but low levels. The enzyme activity induced by ts G at 31 degrees C was thermolabile both in vivo and in vitro. These results provide the first strong evidence that the induced ribonucleotide reductase activity is at least partially virus-coded.
TL;DR: Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration.
Abstract: Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. Optimum enzyme activity was recorded at pH 5.0 to 6.0 and at 30 to 40°C. The enzyme was stable up to 30°C and within the pH range of 4.0 to 6.5. The Km value was found to be 0.48 × 10−4 M when tannic acid was used as the substrate. Metal salts at 20 mM inhibited the enzyme to different levels.
TL;DR: D-Amino acid oxidase activity in the kidney homogenates of mice of seven strains was measured to search for a mutant for this enzyme, suggesting that Dao-1c is a null allele and that there is a gene dosage effect on the enzyme activity.
Abstract: D-Amino acid oxidase activity in the kidney homogenates of mice of seven strains was measured to search for a mutant for this enzyme. There was a consistent sex difference in the enzyme activity in these strains: male mice showed higher levels of the enzyme activity than females. In contrast to other strains, some mice of the ddY strain did not possess enzyme activity. This trait was inheritable, and a mouse stock without enzyme activity (DAO-) was established. The allele (Dao-1c) carried by the DAO- mice was recessive and behaved as a single autosomal gene in inheritance. Heterozygous mice for this gene (Dao-1+/Dao-1c) showed nearly half the enzyme activity of the wild-type homozygotes (Dao-1+/Dao-1+), suggesting that Dao-1c is a null allele and that there is a gene dosage effect on the enzyme activity.
TL;DR: A rationale for the clinical application of human blood platelet monoamine oxidase is developed and some thoughts for further experimentation are considered.
TL;DR: It is demonstrated that in the chloroplasts ribulose-5-phosphate kinase can not only be very efficiently switched off in the dark, but also be subjected to fine control during the illuminated state through the action of stromal metabolites.
TL;DR: Measurements of the metabolic flux in the conversion of ethanol to the two products, CO2 and lipids, for six different active genotypes, containing the predominant naturally recurring alleles and covering a threefold range of in vitro activity are reported.
Abstract: Although there are many in vitro studies of enzyme activity of genetic variants at the Adh locus in D. melanogaster, little is known about the corresponding metabolic activity in living flies. We report here such measurements of the metabolic flux in the conversion of ethanol to the two products, CO2 and lipids, for six different active genotypes, containing the predominant naturally recurring alleles and covering a threefold range of in vitro activity. In adult flies we have found nonsignificant differences between genotypes in metabolic flux when estimates for individual genotypes had standard errors of approximately 10% of the mean value. In vitro activities are, therefore, poor predictors of the physiological consequences of enzyme variation since such determinations ignore the interactions inherent in multienzyme systems. We have no evidence that heterozygote show overdominance either at the enzyme or the flux level. Since fitness differences between genotypes must be generated by physiological differences, investigations of polymorphisms should be based on in vivo studies.
TL;DR: A new flavoprotein enzyme, l-glutamate oxidase, was purified to homogeneity from an aqueous extract of a wheat bran culture of Streptomyces sp.
Abstract: A new flavoprotein enzyme, l-glutamate oxidase, was purified to homogeneity from an aqueous extract of a wheat bran culture of Streptomyces sp. X-l 19–6. It showed absorption maxima at 273, 385 and 465 nm and a shoulder around 490 nm, and contained 2 mol of FAD per mol of enzyme. The enzyme had a molecular weight of approximately 140,000 and consisted of three sizes of subunits with molecular weights of 44,000, 16,000 and 9,000. Balance studies showed that 1 mol of l-glutamate was converted to 1 mol of α-ketoglutarate, ammonia and hydrogen peroxide with the consumption of 1 mol of oxygen. In addition to l-glutamate, l-aspartate was oxidized by the enzyme but only to an extent of 0.6% at pH 7.4; the Michaelis constants were as follows: 0.21 mM for l-glutamate and 29 mM for l-aspartate. The isoelectric point was pH 6.2, and the enzyme activity was optimal between pH 7.0 and 8.0. When the enzyme was heated at pH 5.5 for 15 min, the remaining activity was 100% of the original activity level at 65°C, 87% at 75...
TL;DR: Comparison of the developmental patterns of glutamine synthetase and S- 100 protein showed that the maximum acquisition of both these astrocytic proteins occurs at a comparable age; however, the former continues to increase for a much longer time than the S-100 protein.
TL;DR: The results indicate that the recently identified phospholipid-sensitive species of Ca2+-dependent protein kinase was inhibited by a variety of agents, probably via their abilities to interfere with a hydrophobic interaction between phospholIPid and the enzyme, an interaction presumably required to confer upon the enzyme a Ca2+.
TL;DR: 2-methyl-branched chain acyl-CoA dehydrogenase was purified to homogeneity from rat liver mitochondria and showed a single protein band with Mr = 41,500, suggesting that this enzyme is composed of four subunits of equal size.
TL;DR: Fucokinase (EC 2.7.52) activity was estimated in supernatants of homogenate from day‐old chick forebrain, finding the enzyme is considerably more active than was reported for other species and tissues.
Abstract: Fucokinase (EC 2.7.1.52) activity was estimated in supernatants of homogenate from day-old chick forebrain. Enzyme kinetic studies gave a Km of 4.5 X 10(-6) M and Vmax of 3.72 nmol fucose converted into fucose-1-phosphate/mg prot/h. The pH optimum was 7.5. The enzyme is thus considerably more active than was reported for other species and tissues. There were no differences in enzyme activity between the four forebrain regions studied. One hour after chicks were trained on a one-trial passive avoidance learning paradigm, enzyme activity in the right forebrain base increased 14% over control values (p less than 0.02). The 11.3% increase in activity in the left forebrain base and 10.3% increase in the left roof were not statistically significant. The relationship of this change to the increased fucose incorporation into glycoproteins known to occur over a similar time period and the significance of the lateralization of the increase are discussed.
TL;DR: The alpha-amylase produced by Bacillus licheniformis CUMC305 was purified with a 42% yield through a series of four steps as discussed by the authors, and the enzyme showed maximal activity at 90 degrees C and pH 9.0, and 91% of this activity remained at 100 degrees C. The activation energy for this enzyme was calculated as 5.1 x 10 J/mol.
Abstract: alpha-Amylase produced by Bacillus licheniformis CUMC305 was purified 212-fold with a 42% yield through a series of four steps. The purified enzyme was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme showed maximal activity at 90 degrees C and pH 9.0, and 91% of this activity remained at 100 degrees C. The enzyme retained 91, 79, and 71% maximal activity after 3 h of treatment at 60 degrees C, 3 h at 70 degrees C, and 90 min at 80 degrees C, respectively, in the absence of substrate. On the contrary, in the presence of substrate (soluble starch), the alpha-amylase enzyme was fully stable after a 4-h incubation at 100 degrees C. The enzyme showed 100% stability in the pH range 7 to 9; 95% stability at pH 10; and 84, 74, 68, and 50% stability at pH values of 6, 5, 4, and 3, respectively, after 18 h of treatment. The activation energy for this enzyme was calculated as 5.1 x 10 J/mol. The molecular weight was estimated to be 28,000 by sodium dodecyl sulfate-gel electrophoresis. The relative rates of hydrolysis of soluble starch, amylose, amylopectin, and glycogen were 1.27, 1.8, 1.94, and 2.28 mg/ml, respectively. V(max) values for hydrolysis of these substrates were calculated as 0.738, 1.08, 0.8, and 0.5 mg of maltose/ml per min, respectively. Of the cations, Na, Ca, and Mg, showed stimulatory effect, whereas Hg, Cu, Ni, Zn, Ag, Fe, Co, Cd, Al, and Mn were inhibitory. Of the anions, azide, F, SO(3), SO(4), S(2)O(3), MoO(4), and Wo(4) showed an excitant effect. p-Chloromercuribenzoic acid and sodium iodoacetate were inhibitory, whereas cysteine, reduced glutathione, thiourea, beta-mercaptoethanol, and sodium glycerophosphate afforded protection to enzyme activity. alpha-Amylase was fairly resistant to EDTA treatment at 30 degrees C, but heating at 90 degrees C in presence of EDTA resulted in the complete loss of enzyme activity, which could be recovered partially by the addition of Cu and Fe but not by the addition of Ca or any other divalent ions.
TL;DR: In this article, a modified continuous spectrophotometric assay for threonine aldolase overcame interference resulting from ThDehydrogenase activity and revealed that thDehydratase activity was very low in rat liver, irrespective of the metabolic state of the animal.
Abstract: Threonine dehydratase, threonine aldolase and threonine dehydrogenase activities were assayed in livers of rats that had been normally-fed, starved for 72 h, fed a high-protein diet or normally-fed and injected with glucagon or cortisone. A modified continuous spectrophotometric assay for threonine aldolase overcame interference resulting from threonine dehydratase activity and revealed that threonine aldolase activity was very low in rat liver, irrespective of the metabolic state of the animal. The concentration of free threonine was determined in livers of animals subjected to the same treatments as described above. Using Michaelis-Menten kinetics to estimate enzyme activities in vivo at intracellular threonine concentrations it was calculated that in the normally-fed state, 87% of the threonine degraded was catabolized by threonine dehydrogenase. In other metabolic states (except in glucagon-treated animals) threonine dehydratase was the major enzyme catalysing threonine catabolism. It was concluded that threonine dehydrogenase activity plays a hitherto unrecognized role in the metabolic homoeostasis of threonine in the normally-fed rat and that this enzyme activity, in association with 2-amino-3-oxobutyrate CoA-ligase, accounts for the known rate of glycine formation from threonine in the rat.
TL;DR: A 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography.
Abstract: A 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography. A single protein band in polyacrylamide-gel electrophoresis and one arc in immunoelectrophoresis corresponded to the enzyme activity. The Mr was 65 000. The pI was 4.2-3.6. The purified enzyme contained about 10% hexose. The enzyme differs from previously described cellobiohydrolases in being more effective in the hydrolysis of cellulose.
TL;DR: Anions were found to inhibit the enzyme activity in an almost chaotropic order and the enzyme demonstrated a wide substrate specificity for both ribo- and deoxyribonucleoside triphosphate and hydrolyzed these nucleotides at almost the same rate.
TL;DR: Two alternate screening methods have enabled the detection of monoclonal antibodies with different specificities toward the lysosomal enzyme alpha-mannosidase of Dictyostelium discoideum using an indirect enzyme-linked immunoadsorbent assay and a direct assay based on the antibody binding to enzyme adsorbed on plastic.
TL;DR: A phospholipid-sensitive Ca2+-dependent protein kinase was purified to homogeneity, for the first time, from extracts of pig spleen, employing the steps of DEAE-cellulose, octyl-agarose, Sephacryl S-200 and phosphatidylserine-Affigel 10 affinity chromatographies.
Abstract: A phospholipid-sensitive Ca2+-dependent protein kinase was purified to homogeneity, for the first time, from extracts of pig spleen, employing the steps of DEAE-cellulose, octyl-agarose, Sephacryl S-200 and phosphatidylserine-Affigel 10 affinity chromatographies. The purified enzyme appeared as a single protein band on both analytical (non-denaturing) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, having a minimum mol.wt. of 68 000 +/- 200. The molecular weight of the enzyme was also determined to be 74 500 +/- 4600 by gel filtration and 80 000 based on its sedimentation coefficient (5.52 S) and Stokes radius (3.52 +/- 0.09 nm), indicating that the enzyme was a monomeric protein. The frictional ratio (f/f0) of the enzyme was 1.24, indicating it was non-globular in shape. The enzyme had a pI of 5.3, and a pH optimum of 6.5 for its reaction. Amino acid analysis indicated that the enzyme apparently was not similar to myosin light-chain kinase (a calmodulin-sensitive species of Ca2+-dependent protein kinase) or cyclic AMP-dependent and cyclic GMP-dependent protein kinases. The enzyme had an apparent Km for ATP of 7.5 microns. Histone H1 and myelin basic protein were effective substrates for the enzyme, with apparent Km values of 0.3 and 0.2 microns, and Vmax, values of 0.06 and 0.09 mumol/min per mg of enzyme respectively. The enzyme activity was dependent on both phosphatidylserine (apparent Ka = 6.25 micrograms/ml) and Ca2+ (apparent Ka = 160 microns). Calmodulin was unable to substitute for the phospholipid as a cofactor, nor was it a subunit of the enzyme. Sr2+ and Ba2+ could partially mimic Ca2+ to activate the enzyme in the presence of phosphatidylserine. An endogenous substrate protein (mol.wt. 41 000) for the enzyme was found in the total, solubilized fraction of pig spleen. Monoclonal antibodies against the enzyme interacted similarly with the homogeneous and impure enzyme; the antibodies, however, did not bind to cyclic nucleotide-dependent protein kinases.
TL;DR: Evidence is presented that mRNA purified from chicken myeloblasts codes for two reductase polypeptides of Mr = 94,000 and 102,000, suggesting that 25-hydroxycholesterol must inhibit the rate of reduct enzyme synthesis by translational regulation.
TL;DR: The specific activity of alkaline phosphatase was increased in two human osteogenic sarcoma cell lines after treatment with 1,25 dihydroxy-vitamin D3 (1,25(OH)2D3).