Showing papers on "Enzyme assay published in 1984"
TL;DR: The application of this enzyme assay in a visible spectrophotometer, along with the considerable evidence that a single aromatic hydrocarbon-inducible cytochrome P-450 isozyme is responsible for the catalysis, enhances the utility of this substrate in microsomal monooxygenase assays.
314 citations
TL;DR: Students in which gel electrophoresis was used, in conjunction with an enzyme activity stain and elution and re-electrophoreis of protein bands, showed that the heavy subunit contains all of the structural requirements for enzymatic activity and also for feedback inhibition of the enzyme activity by glutathione.
220 citations
TL;DR: The effects of phytic acid and its interactions with divalent cations (Ca++ and Mg++) on α-amylase activity were investigated in model systems and were found to be of noncompetative type.
Abstract: The effects of phytic acid and its interactions with divalent cations (Ca++ and Mg++) on α-amylase activity were investigated in model systems. Amylase activity was influenced by both preincubation time with phytate as well as phytate concentration. At 6-30 mM concentrations, Ca++ and Mg++ ions lowered the enzyme activity by 9-34% and 24-49%, respectively. When divalent cations were added simultaneously with phytate, a slight increase in enzyme activity with Ca++ and lowered enzyme activity with Mg++ were observed, as compared to when added independently. The enzyme activity was only moderately lowered when phytate was first preincubated with the divalent cations. Amylase inhibition by phytate was found to be of noncompetative type with an apparent inhibitor constant of 1.75 mM under the assay conditions.
207 citations
TL;DR: Damage to the plasma membrane of rabbit epididymal spermatozoa during spontaneous lipid peroxidation was examined by means of trypan blue uptake and expression of activity of the intracellular enzymes, lactate dehydrogenase and pyruvate kinase, and there is a linear correlation between increase in expressed enzyme activities and malondialdehyde production.
Abstract: Damage to the plasma membrane of rabbit epididymal spermatozoa during spontaneous lipid peroxidation was examined by means of trypan blue uptake and expression of activity of the intracellular enzymes, lactate dehydrogenase and pyruvate kinase. Both the dye uptake and the expression of enzyme activity probe cell damage from lipid peroxidation as loss of integrity of the plasma membrane. A linear correlation was obtained between trypan blue staining of the cells and malondialdehyde production, a quantifiable measure of the extent of lipid peroxidation. At the point of trypan blue staining of all cells, 0.5 nmol malondialdehyde/10(8) cells was produced. This is the same amount produced at the point of complete loss of motility and superoxide dismutase activity. We have defined this as the "lipoperoxidative lethal end point." Expression of lactate dehydrogenase and pyruvate kinase activities increased with time of aerobic incubation. In the high Na+ medium, NTP, in which lipid peroxidation is slow, there is a linear correlation between increase in expressed enzyme activities and malondialdehyde production. But in the high K+ medium, KTP, in which lipid peroxidation is rapid, there is an initial rapid rise in expressed enzyme activity over 3 h, followed by a slower increase. Activities of rabbit sperm lactate dehydrogenase, pyruvate kinase, and flagellar ATPase were unaffected by aerobic incubations for up to 48 h, double the incubation period used for the assay of enzymatic activities for the first two. The activity of glyceraldehyde-3-phosphate dehydrogenase decreased during aerobic incubation, the time course matching the loss of motility. The subcellular distribution of lactate dehydrogenase in rabbit spermatozoa was determined: 4% in the mitochondrial matrix, 10% in the plasma membrane and 85% in the cytosolic compartment.
163 citations
TL;DR: A trypsin-like, membrane-bound protease from Bacteroides gingivalis was solubilized by Triton X-100 and partially purified by a combination of DEAE-Sepharose and aminophenylmercuric Sepharose chromatography, by taking advantage of the thiol group on the enzyme.
160 citations
TL;DR: A considerable amount of evidence now makes it clear that aerobic tissues require an elaborate enzyme system to remove the harmful reaction products of oxygen reduction, and a portion of this protective system has been studied in human muscle and rat tissues.
Abstract: A considerable amount of evidence now makes it clear that aerobic tissues require an elaborate enzyme system to remove the harmful reaction products of oxygen reduction. A portion of this protective system has been studied in human muscle and rat tissues. The VO2 max as well as the superoxide dismutase and catalase activity of vastus lateralis muscle of 12 healthy, male subjects was measured. The subjects with a high aerobic capacity (VO2 max greater than 60 ml . kg-1 . min-1) had significantly greater levels of both superoxide dismutase and catalase. There was also a linear relationship between both superoxide dismutase and catalase and VO2 max. The tissue oxygen consumption, and enzyme activity of the liver, heart, lung, and gastrocnemius from 24 rats was also studied. There VO2 and tissue enzyme activity of both superoxide dismutase and catalase.
154 citations
TL;DR: Each species of folate-binding proteins of rat liver cytosol was homogeneous, as judged by NaDodSO4/polyacrylamide gel electrophoresis, and they migrated identically.
Abstract: A comparison of the amino acid compositions of one of the folate-binding proteins of rat liver cytosol, folate-binding protein-cytosol II, and that of glycine N-methyltransferase (S-adenosyl-L-methionine:glycine methyltransferase, EC 2.1.1.20) from the same source indicated a great deal of structural homology between the two proteins. Antiserum prepared against the purified folate-binding protein almost completely inactivated the enzyme activity in crude liver cytosol. Purification of glycine N-methyltransferase resulted in the separation of two enzyme species, one that contained bound folate and one that did not. Each species was homogeneous, as judged by NaDodSO4/polyacrylamide gel electrophoresis, and they migrated identically.
150 citations
TL;DR: Examination of the distribution of microsomal glutathione transferase in different organelles, in different organs, and in different organisms found that Toad was the only species that had a notable (twofold) sex difference in their level of hepatic microsomes from extrahepatic tissues.
148 citations
TL;DR: Pregnant DEX treatment results in an acceleration of the normal developmental increase in lung antioxidant enzyme activity which occurs late in gestation in the rat (as in the rabbit), and the DEX-treated prematurely-born may be in a more favorable biochemical state to avoid toxic lung changes associated with the relatively O2-rich ex utero environment and any supplemental hyperoxic treatment it may require.
Abstract: DEXAMETHASONE STIMULATES FETAL RAT LUNG ANTIOXIDANT ENZYME ACTIVITY IN PARALLEL WITH SURFACTANT STIMULATION
140 citations
TL;DR: Results indicated that the gamma-glutamyl kinase from the 3,4-dehydroproline-resistant strain was 100-fold less sensitive to feedback inhibition by proline, and this enzyme was insensitive to the glutamate analog L-methionine-DL-sulfoximine, but ADP was a potent competitive inhibitor.
Abstract: gamma-Glutamyl kinase, the first enzyme of the proline biosynthetic pathway, was purified to homogeneity from an Escherichia coli strain resistant to the proline analog 3,4-dehydroproline. The enzyme had a native molecular weight of 236,000 and was apparently comprised of six identical 40,000-dalton subunits. Enzymatic activity of the protein was detectable only in assays containing highly purified gamma-glutamyl phosphate reductase, the second enzyme of the proline pathway. Plots of gamma-glutamyl kinase activity as a function of glutamate concentration were sigmoidal, with a half-saturation value for glutamate of 33 mM, whereas plots of enzyme activity as a function of ATP concentration displayed typical Michaelis-Menten kinetics with a Km for ATP of 4 X 10(-4) M. Enzyme activity was insensitive to the glutamate analog L-methionine-DL-sulfoximine, but ADP was a potent competitive inhibitor. Characteristics of the enzyme were compared with those of a gamma-glutamyl kinase partially purified from a 3,4-dehydroproline-sensitive E. coli. These results indicated that the only major difference was that the enzyme from the 3,4-dehydroproline-resistant strain was 100-fold less sensitive to feedback inhibition by proline.
130 citations
TL;DR: Results of isotopic exchange reactions between substrates and products suggest that the enzyme catalyzes a sequential Bi Bi reaction.
TL;DR: A useful modification of the method for isolating this enzyme from kidney was developed and studies on the structural features of cystamine that facilitate its interaction with the enzyme showed that selenocystamines, monodansylcystamine, and N-[2[2-aminoethyl)-dithio)ethyl]-4-azido-2-nitrobenzeneamine are also good inhibitors.
TL;DR: Various detoxifying enzymes, including microsomal oxidases, glutathione S -transferases, esterases, epoxide hydrolase, and DDT-dehydrochlorinase were assayed in adult worker bees using midguts as the enzyme source, suggesting that the high susceptibility of honey bees to insecticides is not due to low detoxication capacity.
TL;DR: The level of protein in the diet had a greater effect on the enzyme total activities in the large shrimp (17–30 g) than in small shrimp ( a p ratio diets displayed lower activities than those fed the 2:1 ratio diet).
TL;DR: The denaturation of creatine kinase in urea solutions of different concentrations has been studied by following the changes in the ultraviolet absorbance and intrinsic fluorescence as well as by the exposure of hidden SH groups to observe conformational changes observed during urea denaturation.
Abstract: The denaturation of creatine kinase in urea solutions of different concentrations has been studied by following the changes in the ultraviolet absorbance and intrinsic fluorescence as well as by the exposure of hidden SH groups. In concentrated urea solutions, the denaturation of the enzyme results in negative peaks at 285 nm with shoulders at 280 and 290 nm and positive peaks at 244 and 302 nm in the denatured minus native enzyme difference spectrum. The fluorescence emission maximum of the enzyme red shifts with increasing intensity in urea solutions of increasing concentrations. At least part of these changes can be attributed to direct effects of urea on the exposed Tyr and Trp residues as shown by experiments with model compounds. The inactivation of this enzyme has been followed and compared with the conformational changes observed during urea denaturation. A marked decrease in enzyme activity is already evident at low urea concentrations before significant conformational changes can be detected by the exposure of hidden SH groups or by ultraviolet absorbance and fluorescence changes. At higher urea concentrations, the enzyme is inactivated at rates 3 orders of magnitude faster than the rates of conformational changes. The above results are in accord with those reported previously for guanidine denaturation of this enzyme [Yao, Q., Hou, L., Zhou, H., & Tsou, C.-L. (1982) Sci. Sin. (Engl. Ed.) 25, 1186-1193] and can best be explained by assuming that the active site of this enzyme is situated near the surface of the enzyme molecule and is sensitive to very slight conformational changes.
TL;DR: The cyclic peptide antibiotic echinocandin was found to inhibit 1,3‐β‐D‐glucan synthase activity present in a mixed membrane fraction from Candida albicans and GTP stimulated enzyme activity ~ 4‐fold, but did not affect the percentage inhibition of the enzyme by echinOCandin.
TL;DR: The enzyme was nearly homogeneous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (ca. 5%), and the spectral properties of the enzyme were similar to those published for CO dehydration from acetogenic anaerobes.
Abstract: Carbon monoxide-dependent production of H2, CO2, and CH4 was detected in crude cell extracts of acetate-grown Methanosarcina barkeri. This metabolic transformation was associated with an active methyl viologen-linked CO dehydrogenase activity (5 to 10 U/mg of protein). Carbon monoxide dehydrogenase activity was inhibited 85% by 10 microM KCN and was rapidly inactivated by O2. The enzyme was nearly homogeneous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (ca. 5%). The native purified enzyme displayed a molecular weight of 232,000 and a two-subunit composition of 92,000 and 18,000 daltons. The enzyme was shown to contain nickel by isolation of radioactive CO dehydrogenase from cells grown in 63Ni. Analysis of enzyme kinetic properties revealed an apparent Km of 5 mM for CO and a Vmax of 1,300 U/mg of protein. The spectral properties of the enzyme were similar to those published for CO dehydrogenase from acetogenic anaerobes. The physiological functions of the enzyme are discussed.
TL;DR: Diurnal variations of in vitro activity of 5 enzymes of nitrogen metabolism were studied and supported the assumption that changes in extractable enzyme activity are due to changes in the amount of (active) enzyme protein.
Abstract: Diurnal variations of in vitro activity of 5 enzymes of nitrogen metabolism were studied. Barley (Hordeum vulgare L. cv. Herta) seedlings were grown in 8 h short days, in daylight or under fluorescent lamps. During, the photoperiod nitrite reductase (EC 1.7.7.1) increased by an average of 18% in daylight and 10% under fluorescent lamps. Glutamine synthetase (EC 6.3.1.2) activity increased by 14 and 10%, respectively. The increase in enzyme activity reflected the overall increase in soluble proteins which was 8% in daylight and 3% under fluorescent lamps. Alanine aminotransferase (EC 2.6.1.2) increased by 82% in daylight and 37% under fluorescent lamps. Desalting of the extracts did not alter the enzyme activity and thus supported the assumption that changes in extractable enzyme activity are due to changes in the amount of (active) enzyme protein. Glutamate synthase (EC 1.4.7.1) activity did not show regular diurnal variations, and aspartate aminotransferase (EC 2.6.1.1) activity was almost constant.
TL;DR: Dissociation in substrate specificity, as well as differences in the inhibition profile, distinguish the enzyme activity in the particulate fraction from rat basophil leukemia cell homogenates from the microsomal glutathione S -transferase which has been described in rat liver homogenate, suggesting that this “leukotriene C synthetase” is a new and unique enzyme.
TL;DR: In a study using bacterial and brain micro-somal enzymes, l-Cycloserine was shown to be 100 times more inhibitory than the d-isomer for the brain microsomal enzyme in vitro as discussed by the authors.
Abstract: d- and l-cycloserine were shown to be irreversible inhibitors of the first enzyme of the sphingolipid pathway, 3-ketodihydrosphingosine synthetase, in a study using bacterial and brain microsomal enzymes, l-Cycloserine was shown to be 100 times more inhibitory than the d-isomer for the brain microsomal enzyme in vitro. In vivo, l-cycloserine caused a 70% inhibition of brain microsomal enzyme. Following one injection, enzyme activity recovered 80% of normal after 16 hours. Daily dosages of l-cycloserine on a regimen of intraper-itoneal injection for 7 days caused a significant reduction in total brain ganglioside and cerebroside plus sulfatide levels.
TL;DR: The level of tyrosinase activity in Cloudman melanoma cells is a direct reflection of the abundance of enzyme protein, directly proportional to the increase in enzyme activity.
TL;DR: A lignin-degrading enzyme has been detected in culture supernatants of Phanerochaete chrysosporium strain INA-12 grown under non-limiting nitrogen conditions.
Abstract: A lignin-degrading enzyme has been detected in culture supernatants of Phanerochaete chrysosporium strain INA-12 grown under non-limiting nitrogen conditions. Highest levels of enzyme activity were observed when glycerol served as carbon source. Veratryl alcohol, a known secondary metabolite of P. chrysosporium, was also produced in high nitrogen/glycerol cultures of strain INA-12 and closely followed the development of the ‘ligninase’ activity. Evolution of 14CO2 from 14C-ring-DHP was readily observed when a hydrogen peroxide-generating system was added to 5-day-old high nitrogen/glycerol cultures which contained high amounts of enzyme.
TL;DR: The oxygen-labile, activating enzyme for iron protein from the photosynthetic bacterium, Rhodospirillum rubrum, was purified 11,800-fold using a combination of chromatophore washing, DE52-cellulose Chromatography, hydroxylapatite chromatography, reactive red-120 cross-linked agarose chromatography; and Sephadex G-75 gel filtration.
TL;DR: Data are consistent with the hypothesis that oxygen-mediated damage is responsible for the time-dependent decrease in 17 alpha-hydroxylase and C17-20 lyase activities of control Leydig cells, and is the mechanism by which these microsomal P-450 activities are further decreased in desensitized Leydenig cells.
TL;DR: It is clear that the medulla develops its glycolytic potential, as indicated by its potential enzyme activity, considerably earlier than the other regions (hypothalamus, striatum and mid-brain), with the cortex and cerebellar activities developing even later.
Abstract: The development of key enzyme activities concerned with glucose metabolism was studied in six regions of the rat brain in animals from just before birth (-2 days) through the neonatal and suckling period until adulthood (60 days old). The brain regions studied were the cerebellum, medulla oblongata and pons, hypothalamus, striatum, mid-brain and cortex. The enzymes whose developmental patterns were investigated were hexokinase (EC 2.7.1.1), aldolase (EC 4.1.2.13), lactate dehydrogenase (EC 1.1.1.27) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Hexokinase, aldolase and lactate dehydrogenase activities develop as a single cluster in all the regions studied, although the timing of this development varies from region to region. Glucose-6-phosphate dehydrogenase activity, however, declines relative to glycolytic enzyme activity as the brain matures. When the different brain regions are compared, it is clear that the medulla develops its glycolytic potential, as indicated by its potential enzyme activity, considerably earlier than the other regions (hypothalamus, striatum and mid-brain), with the cortex and cerebellar activities developing even later. This enzyme developmental sequence correlates well with the neurophylogenetic development of the brain and adds support to the hypothesis that the development of the potential for glycolysis in the brain is a necessary prerequisite for the development of neurological competence.
TL;DR: A proteinase capable of degrading the helical region of native human type V collagen was identified in serum-free culture medium from "in vivo-activated" rabbit alveolar macrophages and found to stain positively using the periodic acid-Schiff technique indicating that it was glycosylated.
Abstract: A proteinase capable of degrading the helical region of native human type V collagen was identified in serum-free culture medium from “in vivo-activated” rabbit alveolar macrophages. This enzyme was purified to homogeneity using a combination of gelfiltration, ion-exchange and affinity chromatography. Analysis of the purified material by gel electrophoresis revealed a single broad band with relative molecular mass of 82,000 daltons Enzyme activity was eluted from the gel in a region corresponding to the stained band. The protein band was also found to stain positively using the periodic acid-Schiff technique indicating that it was glycosylated. Amino acid analysis revealed a composition rich in acidic residues. The enzyme cleaved type V collagen into three large molecular weight doublets consistent with two cleavage sites within the helix but was inactive against native type I collagen. However, when type I or type V collagen was heat-denatured, the enzyme degraded the alpha chains to small molecular weight peptides.
TL;DR: An inducible cephalosporinase was purified from Pseudomonas maltophilia GN12873, and showed a broad substrate profile, hydrolyzing cefazolin, cefsulodin, penicillin G, ceftizoxime, and ampicillin at a high rate.
Abstract: An inducible cephalosporinase was purified from Pseudomonas maltophilia GN12873. The pI was 8.4, and the molecular weight was ca. 56,000 by gel filtration or 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that this enzyme had two subunits. The optimal pH and optimal temperature were 7.5 and 45 degrees C, respectively. Enzyme activity was inhibited by clavulanic acid, sulbactam, cephamycin derivatives, carbapenem antibiotics, iodine, HgCl2, and p-chloromercuribenzoate. The enzyme showed a broad substrate profile, hydrolyzing cephaloridine, cefazolin, cefsulodin, penicillin G, ceftizoxime, and ampicillin at a high rate.
TL;DR: Cellular metabolism of squalene 2,3:22,23-dioxide to compounds with the chromatographic properties of polar sterols led to an inhibition of reductase activity that could be prevented by U18666A, and the drug was unable to prevent the inhibition of enzyme activity by 25-hydroxycholesterol or mevalonolactone, but totally abolished the inhibitory action of low density lipoproteins.
TL;DR: Rat liver microsomes devoid of free thiols were prepared in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer in the presence of 30 microM leupeptin and results are compatible with a model in which HMG-CoA reductase activity is GSH-dependent, allosterically modulated under physiological hepatic conditions.
TL;DR: Cathepsin L was purified from rabbit liver by a method involving whole-tissue homogenization, pH precipitation, ammonium sulphate fractionation and chromatography on CM-Sephadex C-50, phenyl-Sepharose and SephadeX G-75, and the enzyme catalysed the hydrolysis of azocasein, collagen and Z-Phe-Arg-NMec optimally at pH 5.9.
Abstract: Cathepsin L was purified from rabbit liver by a method involving whole-tissue homogenization, pH precipitation, ammonium sulphate fractionation and chromatography on CM-Sephadex C-50, phenyl-Sepharose and Sephadex G-75. Pure enzyme was obtained without the necessity of laborious subcellular fractionation techniques. The Mr of the enzyme was determined to be 29 000 by gel filtration, and affinity for concanavalin A-Sepharose indicated that it was a glycoprotein. A novel technique for detection of enzyme activity in agarose isoelectrofocusing gels showed that the enzyme existed in multiple isoenzymic forms with pI values ranging from 5.0 to 5.9. The enzyme catalysed the hydrolysis of azocasein, collagen and Z-Phe-Arg-NMec (where Z and NMec indicate benzyloxycarbonyl and N-methylcoumarin derivative respectively) optimally at pH 5.2, 3.3 and 6.0 respectively. In addition, cathepsin L was found to degrade benzoyl-Phe-Val-Arg-NMec and 3-carboxypropionyl-Ala-Phe-Lys-NMec. However, cathepsin B also cleaved all of these substrates. One major difference between these two enzymes was in their Michaelis constants for Z-Phe-Arg-NMec; cathepsin B had Km 75 microM whereas that of cathepsin L was 0.7 microM. Cathepsin L was inhibited by all of the usual chemical inhibitors of thiol proteinases as well as the more specific inhibitors Z-Phe-Phe-CHN2, Z-Phe-Ala-CHN2, compound E-64 and compound Ep-475. Active-site titration with compound E-64 showed that the purified sample contained 80% active protein, which had kcat. 20s-1 for the substrate Z-Phe-Arg-NMec. Antibodies were raised to active cathepsin L, and these did not cross-react with cathepsin B, thus demonstrating that these two enzymes are immunologically distinct.