Showing papers on "Enzyme assay published in 1978"

An improved assay of 7-ethoxycoumarin O-deethylase activity: induction of hepatic enzyme activity in C57BL/6J and DBA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Abstract: An improved in vitro fluorometric assay of the O-deethylation of 7-ethoxycoumarin which is at least 10 times more sensitive than existing methods is described. Nearly quantitative recovery of the product which is essentially free of fluorescent impurities is obtained by a simple two-step extraction procedure. In C57BL/6J mice O-deethylating activity was induced 3-fold by 3-methylcholanthrene and 7-fold by phenobarbital. Administration of phenobarbital resulted in a 10-fold stimulation of enzyme activity in DBA/2J mice. Kinetic analysis indicated that at least two and possibly more components are involved in the metabolism of 7-ethoxycoumarin. Differential stimulation of the maximal activities associated with these components was observed after the administration of phenobarbital, 3-methylcholanthrene, or 2,3,7,8-tetrachlorodibenzo-p-dioxin. The ED50 values for the induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon hydroxylase activities in C57BL/6J mice were almost identical and approximately 10-fold less than in DBA/2J mice similarly treated.

Oxygen toxicity in neonatal and adult animals of various species

Abstract: Neonatal and adult animals of five species were exposed to 95+% O2. Survival time and changes in lung antioxidant enzyme activity (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase...

Kinetic studies on the inactivation of Escherichia coli RTEM beta-lactamase by clavulanic acid.

Abstract: Incubation of clavulanic acid with the beta-lactamase from Escherichia coli RTEM leads to enzyme-catalyzed depletion of clavulanic acid, to transient inhibition, and to irreversible inactivation of the enzyme. Both the transiently inhibited and the irreversibly inactivated species show a marked increase in the absorbance at 281 nm that is proportional to the decrease in enzyme activity. Hydroxylamine treatment of irreversibly inactivated enzyme restores about one-third of the catalytic activity, with a concomitant decrease in absorbance at 281 nm. Polyacrylamide isoelectric focusing of the irreversibly inactivated enzyme shows three bands of approximately equal intensity, different from native enzyme. Upon hydroxylamine treatment, one of the three bands disappears and now focuses identically with native enzyme. It is evident that the irreversible inactivation of enzyme by an excess of clavulanic acid generates three products, one of which can be reactivated by hydroxylamine.

Chitin synthesis in Candida albicans: comparison of yeast and hyphal forms.

Abstract: Chitin synthesis was studied in both yeast and hyphae of the dimorphic fungus Candida albicans. Incorporation of N-acetyl-d-[1-(3)H]glucosamine ([(3)H]GluNAc) into an acid-alkali-insoluble fraction was 10 times greater in hyphal-phase cells. A crude preparation of chitin synthetase was obtained from sonically treated protoplasts of both forms of Candida. Enzyme activity, which was determined by using [(14)C]UDP-GLuNAc as a substrate, was exclusively associated with the 80,000 x g pellet from sonically treated protoplasts of both forms. It was determined that enzyme activity (nanomoles of [(14)C]UDP-GluNAc incorporated per milligram of protein) was approximately 2 times greater in hyphae versus yeast cells. Enzyme activity in both yeast and hyphae increased six- to sevenfold when the enzyme preparations were preincubated with trypsin. A vacuolar fraction, obtained from yeast cells but not from hyphae, stimulated enzyme activity when incubated with either yeast or hyphal enzyme preparations. Membrane fractions from protoplasts coated with [(3)H]concanavalin A before disruption were isolated by Renografin density gradient centrifugation. Chitin synthetase activity was preferentially associated with the concanavalin A-labeled fraction, suggesting that the enzyme was located on the plasma membrane. In addition, enzyme activity in protoplasts treated with cold glutaraldehyde before disruption was significantly greater than in protoplasts that were sonically disrupted and then treated with cold glutaraldehyde, indicating that the enzyme resides on the inner side of the plasma membrane.

Induction of pulmonary indoleamine 2,3-dioxygenase by intraperitoneal injection of bacterial lipopolysaccharide.

Abstract: Indoleamine 2,3-dioxygenase [indoleamine: oxygen 2,3-oxidoreductase (decyclizing)] activity in the supernatant fraction (30,000 X g, 30 min) of the mice lung homogenate increased approximately 30- to 50-fold after an intraperitoneal administration of bacterial lipopolysaccharide. In all other tissues tested, no significant increase in enzyme activity was observed. The effect appeared to be specific for the lipopolysaccharide fraction because glycogen and zymosan were almost ineffective under the same experimental conditions. In the lung, the enzyme activity increased almost linearly during the first 24 hr after a single injection of the lipopolysaccharide fraction (20 microgram per mouse). The enzyme activity started to decrease after 48 hr and reached a normal value after about 6 days. The increase in enzyme activity was completely abolished by cycloheximide or actinomycin D. Other enzymes in the lung such as beta-glucuronidase, acid phosphatase, and monoamine oxidase did not change significantly with this treatment.

Thyroxine 5′-Deiodinase Activity of Rat Kidney: Observations on Activation by Thiols and Inhibition by Propylthiouracil

Abstract: Enzymatic 5′-deiodination of T4 in various tissues is known to be stimulated by thiols and inhibited by propylthiouracil (PTU). Dithiothreitol (DTT) stimulation of rat kidney T4 5′-deiodinase followed saturation kinetics. Inhibition of the enzyme by PTU (10-5 M) was overcome in a concentration-dependent manner by DTT, methimazole (MMI), and thiourea. Pretreatment of catalytically active kidney microsomal preparations with PTU caused persistent inhibition of enzyme activity, assayed in the absence of PTU. Similarly, injection of PTU in rats in a dosage of 5 μg/100 g BW or greater significantly impaired T4 5′-deiodination in subsequently isolated kidney microsomal preparations. Reagents that cleave disulfide bonds (DTT and KCN), as well as the reductant, Na2S2O4, and the thioureylene, MMI, partially restored activity to PTU-inactivated enzyme in a time- and concentration-dependent way. These observations suggest that PTU inhibition of T4 5′-deiodinase results from an interaction of PTU with the enzyme, poss...

Thyroid hormones control lysosomal enzyme activities in liver and skeletal muscle

Abstract: Because protein degradation in liver and skeletal muscle is increased by thyroid hormones and decreased by thyroidectomy; we investigated the influence of thyroid hormones on the level of lysosomal enzymes. Hypophysectomized rats received daily injections of L-thyroxine or L-triiodothyronine. After 3 days of this regimen, homogenates of liver and skeletal muscle showed a 2- to 3-fold increase in the activities of cathepsin D, cathepsin B, and other lysosomal enzymes including leucine aminopeptidase, acid phosphatase, β-galactosidase, N-acetylglucosaminidase, and α-mannosidase. In liver, this effect reflected increased enzyme activity in the two subcellular fractions that normally contain lysosomes. Titration of cathepsin D with pepstatin indicated that the increase in this activity resulted from an increase in the number of enzyme molecules. These effects occurred with both pharmacologic (thyrotoxic) and physiologic (growth-promoting) doses of thyroid hormones. Liver and skeletal muscle from thyroidectomized rats had approximately 50% of the normal levels of lysosomal enzyme activities. Under these various conditions, heart and kidney, tissues in which protein degradation does not appear to be influenced by thyroid hormones, showed no significant changes in lysosomal hydrolases. Thus, thyroid hormones regulate proteolytic and other lysosomal enzyme activities in those tissues in which these hormones influence protein degradation. Many characteristic features of hyperthyroidism and hypothyroidism may result from changes in levels of lysosomal enzymes.

Biochemical, immunological, and cell genetic studies in glycogenosis type II.

Abstract: Fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis type II (Pompe disease) were cultured under standardized conditions, and the activity of acid alpha-glucosidase (E.C.3.2.1.20) towards glycogen, maltose, and 4-methylumbelliferyl-alpha-D-glucopyranoside was measured. Glycogen levels in muscle biopsies and in cultured fibroblasts from patients were determined. Residual enzyme activities varying from 7%-22% were detected in fibroblasts from patients with the adult form but not from patients with the infantile form of glycogenosis II. An inverse correlation was found between the severity of the clinical manifestation and the degree of residual enzyme activity in the fibroblasts. The kinetic and electrophoretic properties of acid alpha-glucosidase in fibroblasts from the adult patients and from control individuals were similar. Immunological studies suggested that the decrease of acid alpha-glucosidase activity is caused by a mutation that affects the production or degradation of the enzyme rather than its catalytic activity. Complementation studies were carried out by fusing fibroblasts from patients with the adult, juvenile, and infantile form of glycogenosis II, but neither conventional assays on multikaryons nor enzyme assays on single binuclear heterokaryons gave any evidence for genetic heterogeneity among these forms.

Regulation by Dietary Fats of 3-Hydroxy-3-Methylglutaryl-Coenzyme a Reductase in Rat Liver

Abstract: TAKASHI IDE, HIROSHI OKAMATSU ANDMICHIHIRO SUGANOLaboratory of Nutrition Chemistry, Department of FoodScience and Technology, Kyushu University School ofAgriculture, Fukuoka 812 JapanABSTRACT The effects of various dietary fats on the activity of3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA ) reducA­asein rat livermicrosomes, the rate-limiting enzyme in cholesterogenesis, were examined.A series of experiments demonstrated the dependency of the HMG-CoAreducA­aseactivity on the nature of dietary fats. When saturated fats withchain length of 12 to 18 were the dietary sources and were fed at the 10%level for 19 days, feeding fats with shorter chain fatty acids caused a lowerenzyme activity compared to those with longer chain fatty acids. Theactivity was also regulated by the degree of unsaturation of dietary fats;when safflower oil, camellia oil or tristearin were fed at the 10% level for18 days, the higher the unsaturation, the lower the activity. When tri-myristin or tripalmitin were fed at the 10% level for 14 days, addition ofessential fatty acid, at the level of minimum daily requirement (1% wasreplaced by safflower oil), did not affect the enzyme activity. Though therate of incorporation of mevalonate into cholesterol in the 12,500 X gsupernatant fraction of the liver was also found to be influenced by thetypes of dietary fats, the extent of the response appeared much smallerthan that of HMG-CoA reductase. No consistent correlation between theHMG-CoA reductase activity and the content of microsomal cholesterol orcholesteryl ester and the fatty acid composition of microsomal lipids wasobserved. J. Nutr. 108: 601-612, 1978.INDEXING KEY WORDS hepatic cholesterogenesis •HMG-CoAreductase •dietary fats •essential fatty acid •microsomal cholesterol3-Hydroxy-3-methylglutaryl-Coenzyme A etary corn oil stimulated the enzyme ac-(HMG-CoA) reductase [EC 1.1.1.34] is tivity in proportion to the amount addedknown to be the rate-limiting enzyme of to a fat-free diet. In the hamster dietarycholesterogenesis in animal tissues. The ethyl palmitate stimulated, whereas ethylactivity of hepatic HMG-CoA reductase is linoleate, depressed, hepatic HMG-CoA re-controlled by the nutritional and hormonal ductase (6). Further information on thestate of the animals (1-3). One of the nu- effects of differences in the composition oftntional factors controlling the activity of dÂiet fats Qn th e actiyify of thethis enzyme in the liver is the amount and ,. , ., ,, u, . .A“nature of dietary fat. Craig et al. (4) ob- Ilver arf not available although there areserved in rats a decrease in the activity by some data showing that hepatic choles-changing the diets from a closed formula, terogenesis measured using acetate as anon-purified stock diet to a fat-free diet.

Determining kinetic parameters for thermal inactivation of heatresistant and heat‐ labile isozymes from thermal destruction curves

Abstract: A simple method is proposed to analyze the kinetics of thermal inactivation of enzyme systems which consist of two groups differing in their thermal stability. Using this approach kinetic parameters for thermal destruction for each fraction (the heat labile and heat resistant fraction) can be estimated directly from thermal treatment data. However, to determine the relative concentration of the fractions within the enzyme system, additional information concerning the enzyme assay used to determine enzyme activity is required. Commercially available horseradish peroxidase was used to test the applicability of the method. Analysis of data generated from thermal treatments of the peroxidase using the proposed method were found to be satisfactory. Ea values of 21 kcal/mole and 34 kcal/mole were obtained for the heat resistant and heat labile horseradish peroxidase isozymes, respectively. D82c values were 42 min and 1.2 min, respectively.

Macromolecular Binding and Metabolism of the Carcinogen 1,2-Dibromoethane

Abstract: Biochemical investigations relating to mechanisms of action, activation, and inactivation have been made on the carcinogen, 1,2-dibromoethane. Measurable amounts of radioactivity from [1,2- 14 C]-1,2-dibromoethane became bound to protein, RNA, and DNA of all major tissues of rats. For each of these classes of macromolecules, the largest amounts of bound radioactivity were in the liver and kidneys. Optimum conditions for the rat liver glutathione S -transferase that utilizes 1,2-dibromoethane as a substrate have been established. The pH optimum was 8.2, the K m for 1,2-dibromoethane was 25 mm, and the V max was 2.1 µmol/min/g liver. Considerable enzyme activity was found in rat kidney; detectable activity was present in lung, testis, spleen, and heart. Enzymatic activity leading to the irreversible binding of radioactivity from [ 14 C]-1,2-dibromoethane to proteins in the reaction system was present in rat liver microsomes. The activity was inducible by phenobarbital but not by benz[ a ]anthracene. Reduced nicotinamide adenine dinucleotide phosphate was required for activity of both the noninduced and the induced reactions; MgCl 2 stimulated both reactions. Bromoacetaldehyde, a reactive compound probably involved in the irreversible binding, was identified as a metabolite formed in the reaction system. Radioactivity from [ 14 C]-1,2-dibromoethane also became bound to microsomal proteins by a nonenzymatic chemical reaction. The enzymatic reaction leading to binding to macromolecules and/or the chemical binding reaction may be involved in the biological activity of 1,2-dibromoethane.

Production, Purification and Partial Characterization of 1,4-β-Glucosidase Enzymes from Sporotrichum pulverulentum

Abstract: The production of 1,4-β-glucosidase activity by the rot fungus Sporotrichum pulverulentum has been investigated. It was found that cellobiose or cellulose is necessary to cause induction. With cellobiose as sole carbon source only cell-wall-bound enzymes are produced. For extracellular excretion cellulose seems to be a necessary carbon source. The purification procedures, for purification of enzyme activity in the culture solution, involve the following steps: (a) ultrafiltration, (NH4)2SO4 precipitation and dialyses; (b) preparative slab gel isoelectric focusing I, pH range 3–10; (c) phenyl-Sepharose chromatography; (d) preparative slab gel isoelectric focusing II, pH range 3–5. On the phenyl-Sepharose column the β-glucosidase activity was associated with two separable enzyme peaks, enzyme A and B. When these enzymes were subjected to further purification on preparative isoelectric focusing II, enzyme A was split into two peaks, A1 and A2, and enzyme B was split into three peaks, B1, B2 and B3. The significance of the separations is discussed. The isoelectric points of all the enzymes have been determined and found to vary between 4.52 to 5.15. The molecular weights, determined by dodecylsulphate-polyacrylamide gel electrophoresis, vary between 165000 and 182000. The kinetic constants Km and Ki have been determined for enzyme A and B as well as for the cell-bound β-glucosidase activity. Km for cellobiose was for both enzyme A and B higher than Km for p-nitrophenyl β-d-glucoside. Km/Ki of the free enzymes for gluconolactone is approximately 2500 (enzyme B) to 13000 (enzyme A) with cellobiose as substrate.

Affinity Purification and Properties of Cathepsin-E-Like Acid Proteinase from Rat Spleen

Abstract: A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.

Enzymatic studies on the metabolism of vesicular-arbuscular mycorrhiza II. Soluble alkaline phosphatase specific to mycorrhizal infection in onion roots

Abstract: Soluble alkaline phosphatase specific to vesicular-arbuscular mycorrhizal infection has been demonstrated in enzyme extracts from onion roots inoculated with Glomus mosseae. A close correlation existed between the mycorrhiza-specific phosphatase activity and development of both the infection and the host plant. Maximum activity occurred whilst the infection was still young (100% arbuscular) and coincided with the start of the mycorrhizal growth response, declining afterwards as plant development and infection continued. Enzyme activity was optimal in the presence of 10−5 m orthomosphate but was markedly depressed at concentrations above 10−4m. The properties of mycorrhiza-specific alkaline phosphatase were characteristic of alkaline phosphomonesterase (EC 3.1.3.1.): optimal activity at alkaline pH; inhibition by metal chelating agents, certain cations and orthophosphate; requirement for Mg2+ and Mn2+ ions; hydrolysis of β-glycerol, phenyl and naphthyl phosphates; inability to hydrolyse more complex phosphate esters. The possible implications of these results with respect to mechanisms involved in the assimilation of phosphorus in the VA mycorrhizal system and/or the establishment of VA mycorrhizal infection are discussed.

Purification, characterization and inhibition of human skin collagenase.

Abstract: 1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

Biosynthesis of Δ-aminolevulinate in greening barley leaves: Glutamate 1-semialdehyde aminotransferase

Abstract: L-Glutamate-1-semialdehyde was synthesized by catalytic hydrogenation of N-carbobenzoxy-L-glutamyl-1-chloride-5-benzyl ester. Soluble protein extracts of chloroplasts isolated from greening barley leaves enzymically converted L-glutamate-1-semialdehyde to δ-aminolevulinate. The enzyme was partially purified by gel filtration on a Biogel column excluding proteins larger than 500,000 daltons. The enzyme had a broad pH optimum around 8.0 and required no specific cofactors for activity. Aminooxyacetate (20mM), cycloserine (20mM), ρ-chloromercuribenzoate (0.1mM), glyoxylate (20mM) and pyridoxal phosphate (5mM) inhibited δ-aminolevulinate formation from L-glutamate-1-semialdehyde. However, β- hydroxyglutamate (1mM) a potent inhibitor of L-glutamate-U-14C conversion to δ-aminolevulinate, had no effect on L-glutamate-1-semialdehyde aminotransferase. The aminotransferase activity was eluted from the Biogel column together with the enzyme activity that converted L-glutamate-U-14C into δ-aminolevulinate. Soluble proteins prepared from etiolated plastids and mature chloroplasts of barley had a low specific activity of L-glutamate-1-semialdehyde aminotransferase compared to soluble proteins from greening plastids. It is proposed that L-glutamate-1-semialdehyde aminotransferase catalyses a part reaction in the conversion of L-glutamate to δ-aminolevulinate in greening barley plastids.

N‐Acetylglutamate Synthetase from Rat‐Liver Mitochondria

Abstract: N-Acetyl-l-glutamate synthetase was purified approximately 300-fold from the extract of rat liver mitochondria. The enzyme shows a high substrate specificity for l-glutamate and acetyl-CoA. No or very low activity is observed with other amino acids and acyl compounds. The reaction velocity fits normal Michaelis-Menten kinetics with respect to both l-glutamate (Km, 3.0 mM) and acetyl-CoA (Km, 0.7 mM). Acetylglutamate, the reaction product, and some of its structural analogues, such as N-acetylaminoadipate, N-propionylglutamate, and N-acetylglutamine, significantly inhibited the enzyme reaction. The Ki for acetylglutamate was 0.07 mM. The enzyme activity is sensitive to thiol-blocking agents and is inhibited by divalent cations. The enzyme activity is markedly stimulated by arginine, presumably by an allosteric mechanism. Other intermediates of the urea cycle and various structural analogues of arginine were practically not effective. Arginine increases the maximal velocity with no influence on Km values for the substrates. The activation shows a sigmoidal dependence on arginine concentrations and reaches a half-maximal level at 5–10 μM. The enzyme behaves as a mixture of multiple forms upon gel filtration (the range of molecular weight, 30000 to 300000) and its mean value is considerably increased in the presence of arginine. The specific activity of the enzyme increases with higher enzyme concentrations in the assay. The effect is more marked in the presence of arginine than in its absence. These observations suggest that the enzyme undergoes association and dissociation with concomitant changes in catalytic properties.