Amount Of Enzyme Activity Research Articles

Active site titration of the serine protease cucumisin from Cucumis melo

The amount of active enzyme in cucumisin solution was determined by titration with N-acetyl- l-alanyl- l-alanyl-α-azaalanine p-nitrophenyl ester. When azapeptide was added to cucumisin, p-nitrophenol was rapidly released and deacylation was slow. The cucumisin used was found to be approximately 94% active.

Production of α-Amylase Isozymes in Barley Caryopses in the Absence of Embryos and Exogenous Gibberellic Acid

The isozyme composition and kinetics of production of α-amylase were studied in three samples of barley. Acid-dehusked caryopses and endosperm halves from a sample of Klages and from two samples of Clipper that differed in their ability to produce α-amylase in the absence of added gibberellic acid were incubated on moist sand at two temperatures in the presence or absence of gibberellic acid. The isozyme composition of the extracts was analysed on isoelectric-focusing-polyacrylamide gels and chromatofocusing columns. The endosperm halves of Klages and Clipper (type A) produced very low levels of enzyme in the absence of gibberellic acid while similar amounts of enzyme activity were found in both (+) and (-) gibberellic acid-treated incubates of Clipper (type B) endosperm halves. In these latter incubates, the initial kinetics of production, the proportion of α-amylase 1 to total enzyme activity and the effect of incubation temperature on the kinetics of production in both acid-dehusked caryopses and endosperm halves were similar, and were comparable to those observed in gibberellic acid-treated incubates of Klages and Clipper (type A). Since Clipper (type B) endosperm halves can produce α-amylase in the absence of added gibberellic acid, its role in this process should be reexamined.

Oxygen protection of nitrogenase in the aerobically nitrogen fixing, non-heterocystous cyanobacterium Oscillatoria sp.

Oscillatoria sp. strain 23 is a filamentous, non-heterocystous cyanobacterium that fixes nitrogen aerobically. Although, in this organism nitrogenase is inactivated by oxygen a high tolerance is observed. Up to a pO2 of 0.15 atm, oxygen does not have any measurable effects on acetylene reduction. Higher concentrations of oxygen inhibited the activity to a relatively high degree. Evidence for two mechanisms of oxygen protection of nitrogenase in this cyanobacterium was obtained. A high rate of synthesis of nitrogenase may allow the organism to maintain a certain amount of active enzyme under aerobic conditions. Secondly, a switch off/on mechanism may reversibly convert the active enzyme into a non-active form which is insensitive to oxygen inactivation after a sudden and short-term exposure to high oxygen concentrations. It is conceived that these mechanisms in addition to a temporal separation of nitrogen fixation from oxygenic photosynthesis sufficiently explain the regulation process of aerobic nitrogen fixation in this organism.

Metabolism of 1-naphthol by tyrosinase

1-Naphthol was metabolized by the polyphenol oxidase, tyrosinase, primarily to 1,2-naphthoquinone and to small amounts of 1,4-naphthoquinone as well as to covalently bound products. The inhibition of covalent binding by ethylenediamine, which reacts specifically with 1,2-naphthoquinone but not 1,4-naphthoquinone, suggested that most of the covalent binding was due to 1,2-naphthoquinone or a metabolite of similar structure. The activation by tyrosinase of 1-naphthol to covalently bound products suggested that it may alter the reaction kinetics of the enzyme. This was investigated by studying the effects of 1-naphthol on the tyrosinase-catalysed oxidation of 4-hydroxy anisole. Preincubation of tyrosinase with 1-naphthol increased the lag period of the oxidation of 4-hydroxyanisole, which may be due to a decrease in the amount of active enzyme, as well as to a reaction of 1-naphthol with 3,4-anisylquinone, an oxidation product of 4-hydroxyanisole. The metabolic activation of 1-naphthol by tyrosinase to covalently bound species suggests that 1-naphthol or a structurally related derivative may be of potential therapeutic application in the treatment of cells high in tyrosinase activity, such as certain melanomas.

Variant Cell Lines of Haplopappus gracilis with Disturbed Activities of Nitrate Reductase and Nitrite Reductase

Selected variant cell lines of Haplopappus gracilis (Nutt) Gray that showed disturbed growth after transfer from an alanine medium to NO(3) (-) medium were characterized. The in vivo NO(3) (-) reductase activity (NRA) was lower in these lines than in the wild type. In vitro NRA assays suggest that decreased in vivo NRA was not caused by a lower amount of active enzyme. Cells of the variant lines revealed up to 75% lower extractable activity of NO(2) (-) reductase as compared with the wild type. This coincided with higher accumulation of NO(2) (-) by the variant than by the wild type cells after transfer from alanine medium to NO(3) (-) medium. NO(2) (-) accumulation was transient or continuous, depending on cell line, metabolic state of the cells, and light conditions.

Antibody preference for the catalytically active form of beta-hydroxy-beta-methylglutaryl coenzyme A reductase.

The catalytically inactivating subset within rabbit serum polyclonal antibody to the solubilized, purified 55,000 to 60,000 dalton active fragment of rat liver microsomal beta-hydroxy-beta-methylglutaryl coenzyme A reductase immunoinactivates this enzyme with little or no diminution of effect by enzyme catalytically inactivated by incubation of microsomes with ATP,Mg++. Reactivation of inactive enzyme with ethanol-treated rat liver phosphatase restores antibody affinity showing that the catalytically inactivating subset of antibody exhibits marked or complete affinity for the active enzyme over the ATP,Mg++- inactivated form. This means that immunoinactivation using this antibody is not a valid way of measuring changes in the specific activity of the enzyme via phosphorylation-dephosphorylation. Preference for the active enzyme has not been obvious because when different amounts of enzyme activity are used in immunotitrations of samples of low activity, apparent differences in specific activity are observed when none actually exist. If precautions are not taken, results are obtained supporting phosphorylation by using an antibody that is not capable of distinguishing it.

Anaerobic biosynthesis of the manganese-containing superoxide dismutase in Escherichia coli.

Iron, particularly in the ferrous state plays a role in regulating the biosynthesis of the manganese superoxide dismutase (MnSOD) in Escherichia coli B. Addition of iron has a repressive effect on the synthesis of MnSOD under normal or inducing conditions (i.e. in the presence of paraquat). Addition of manganese to cultures already depressed for MnSOD biosynthesis causes a further increase in the amount of active enzyme, however, this effect is also abolished by the addition of iron. Removal of metals from the growth medium by Chelex 100 also derepresses the synthesis of MnSOD but repletion of the medium with iron abolishes this effect. Chelators specific for Fe2+, 2,2'-dipyridyl, and 1,10-phenanthroline, cause a 5-7-fold increase in MnSOD. Removal of iron also increases the synthesis of MnSOD in the absence of oxygen. A model is presented to account for the observed effects of oxygen, superoxide anion, and chelators on the increased synthesis of MnSOD. In this model, the regulatory repressor for MnSOD biosynthesis is envisioned as being an iron-containing protein.

Mechanism of stimulation of ornithine decarboxylase activity in transformed mouse fibroblasts.

Stimulation of confluent serum-starved SV-3T3 cells by serum produces a transient enhancement of ornithine decarboxylase activity which fluctuates at least 20-fold over a 12-h period. The mechanism by which this fluctuation is produced was investigated. Two techniques for assaying the amount of enzyme protein in these cells were utilized: radioimmunoassay and titration with alpha-(difluoromethyl) [5-3H]ornithine. The radioimmunoassay was carried out by using a specific antiserum prepared in rabbits against homogeneous mouse kidney ornithine decarboxylase and by using alpha-(difluoromethyl) [5-3H]ornithine-labeled kidney ornithine decarboxylase as the tracer ligand. An exact correlation between the amount of enzyme protein and the amount of enzyme activity was seen during the rise and fall of ornithine decarboxylase activity after serum stimulation. Similarly, the amount of protein which was labeled covalently by reaction with alpha-(difluoromethyl) [5-3H]ornithine (a specific enzyme-activated irreversible inhibitor) correlated with the enzyme activity. Investigation of the protein labeled in this way by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that no change in the size of the protein which had a molecular weight of 53 000 occurred during this time period. These results indicate the alteration in ornithine decarboxylase activity can be accounted for entirely by changes in the amount of enzyme protein rather than by posttranslational modifications, activators, or inhibitors.

Catalytic properties of carp brain monoamine oxidase in delipidated and reconstituted mitochondrial membranes

A possible role of membrane phospholipids in catalytic properties of carp brain mitochondrial monoamine oxidase (MAO) in both intact and delipidated membranes were studied before and after addition of some phospholipids. Deaminations of 5-HT and tyramine were greatly decreased by treatment with either phospholipase A2, C or D, but that of PEA was to a lesser extent. The sensitivity of MAO in the delipidated preparation to clorgyline and deprenyl was similar to that in the intact one; however, the amount of 3H-pargyline bound to MAO was found to be about half, and the Km values for the three substrates were extremely increased after delipidation. Among phospholipids tested, phosphatidylinositol(PI) most effectively and dose-dependently activated activity towards 5-HT in both intact and delipidated mitochondria, but the inhibitor sensitivity of MAO and the Km values for these substrates were not changed as compared before and after the PI addition. As a result, delipidation accounts for loss of activity through decreases in both the Km values and amount of active enzyme, but some phospholipids activate MAO activity without altering affinities towards substrates and selective MAO inhibitors.

Characteristics of the binding of RNA polymerase to template in mouse liver nuclei.

In whole nuclei isolated from mouse liver, the reaction of free RNA polymerase with the artificial template, poly (dA-dT), is temperature dependent. At assay temperatures below 37 degrees C, the amount of free RNA polymerase (RNA polymerase not inhibited by actinomycin D) which can be measured is substantially reduced. However, if the nuclei are preincubated at 37 degrees C in the presence of template and then assayed at lower temperatures, substantial amounts of enzyme activity can be measured. The active complex (enzyme bound to template) is resistant to inactivation by heparin. This evidence is consistent with a two-stage model for binding of eukaryotic RNA polymerase to template similar to the binding of prokaryotic RNA polymerase to template.

Heat stability and pH activity data of alpha-L-fucosidase in human serum vary with enzyme concentration.

alpha-L-Fucosidase from serum of humans with either high or low enzyme activity was separately purified. the enzyme from either source had virtually the same heat stability and pH activity profile. It has been widely reported that alpha-L-fucosidase in crude sera from individuals with high and low enzyme activity differed with respect to heat stability and activity at pH 4 relative to activity at pH 5, the pH optimum of the enzyme. We investigated this discrepancy and found that both the heat stability and relative activity at pH 4 of alpha-L-fucosidase from sera with either high or low enzyme activity was dependent upon enzyme concentration. With decreasing enzyme concentration, the enzyme was more heat labile and had less relative activity at pH 4. Consequently, if the data obtained using high and low enzyme activity sera are compared on the basis of equivalent amounts of serum instead of equivalent amounts of enzyme activity, differences between the enzyme from high and low activity serum would be erroneously inferred. Apparently, this is what other investigators have done. Moreover, we found that alpha-L-fucosidase can exist in heat-stable or labile species with sedimentation coefficients of 9.8 S and 4.8 S, respectively. The interconversion and relative proportion of these species is dependent upon enzyme concentration and pH.

Regulatory Properties of Pyruvate Kinase Isoenzymes from a Chlorophyll-free Chlorella Mutant in Darkness or Blue-Light

Summary In analysing the stimulating effect of blue-light on respiration and carbohydrate degradation of green algae, a dual light effect on pyruvate kinase, one of the regulatory enzymes of glycolysis, was demonstrated in the chlorophyll-free, carotenoid-containing mutant no. 20 of Chlorella vulgaris . It consists of an increase in the amount of active enzyme (V max ) and of an increase in substrate affinity, the latter probably resulting from an interconversion of two iso-enzymes with different substrate affinities. Since a thorough comparison of the respective time courses revealed no agreement between the blue-light action on respiration and pyruvate kinase, however, other mechanisms of regulation may be involved. Indeed, kinetic and regulatory properties of the two pyruvate kinase isoenzymes lead to the conclusion that respiration and carbohydrate degradation may be regulated by a light-influenced fine control, i.e. by a blue-light-controlled supply of respirable substrate and/or allosteric effectors and cofactors.

Fluorescence demonstration of a cathepsin H-like protease in cardiac, skeletal and vascular smooth muscles.

A cathepsin H-like enzyme was localized by histochemical techniques in cardiac muscle, extensor digitorum longus and soleus skeletal muscles, and vascular smooth muscle. Using the specific exopeptidase activity of this enzyme against the substrate arg-4-methoxy-beta-naphthylamide, valid histochemical assay conditions were developed. More fluorescent granules were observed in cardiac muscle than in the soleus and about equal amounts in vascular smooth muscle and the extensor digitorum longus. The reaction rate was enhanced by chloride ions and inhibited by 1 mM p-chloromercuribenzoate. The maximal activity was observed between pH 5.5 and 6.0 Chemical fixation with periodate-lysine-paraformaldehyde preserved a small amount of enzymatic activity.

Identification and quantification of leucine aminopeptidase in aged normal and cataractous human lenses and ability of bovine lens LAP to cleave bovine crystallins.

Rabbit antisera to bovine lens leucine aminopeptidase (LAP) have been prepared. These LAP-specific antisera cross-react with a component, LAP, in human lens homogenates. Bovine and human lens LAP are similar but not identical. Immunodiffusion tests show that LAP is present in a vast majority if not all cataractous and normal human lens homogenates. Results from immunoelectrophoresis indicate that LAP is found in these homogenates as several metazymes as well as in an albuminoid--and possibly membrane-associated form. In contrast to many activity-based studies which imply that very little LAP is present in human lenses, micro-complement fixation tests indicate that the concentration of LAP in aged human lenses is similar to that found in calf lenses. Taken together, these data indicate that LAP undergoes age-related changes. These alterations of enzyme (or environment) result in an enzyme of markedly reduced activity, hence, the discrepancy between amount of LAP and the amount of enzymatic activity in aged human lenses. Active LAP is shown to enhance the rate of hydrolysis of alpha-2 and alpha-B crystallins but not alpha-A2, beta H, beta L or gamma crystallins. The ramifications of LAP inactivation with respect to cataractogenesis are discussed.

Evaluation of bovine rennets in terms of absolute concentrations of chymosin and pepsin A

SummaryA method for determining chymosin and bovine pepsin A in commercial extracts of bovine veils, based on the use of the synthetic hexapeptide (Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe) as reference substrate, is reported. Chymosin and bovine pepsin A were separated chromatographically from extracts and assayed for clotting activity on a reconstituted skim-milk standardized with reference chymosin and bovine pepsin A, themselves standardized in relation to the hexapeptide. The effect of pH on the absorbance difference between the hexapeptide and the Leu-Ser-Phe(NO2) tripeptide resulting from its hydrolysis was studied. It was found that the ‘optimal’ pH for determining the activities of the reference enzyme solutions was 4·7.Six chymosin and 3 bovine pepsin A preparations were assayed on the hexapeptide to define the relation between the proteolytic activity and the amount of active enzyme. At pH 4·7 and 30 °C 1 mg chymosin and 1 mg bovine pepsin A hydrolysed 100 and 2700 μm-peptide/s respectively. The clotting activity of these preparations was assayed on a reconstituted skim-milk to standardize it and thus define the relation between the clotting time and the amount of active enzyme. Chymosin had a specific clotting activity twice that of bovine pepsin A. At equal clotting activities, bovine pepsin A was 55 times more active than chymosin on the hexapeptide at pH 4·7.

Studies on avian erythrocyte metabolism: Purification and properties of myo-inositol 1-phosphatase from chick erythrocytes

An enzyme capable of hydrolyzing myo-inositol 1-phosphate was identified and partially purified from the erythrocytes of 7-day chicks. It has an apparent molecular weight of approximately 60,000, is heat stable, and has a pH of optimal activity between 6.5 and 7.3. In most regards the kinetic properties are similar to the myo-inositol 1-phosphatases of rat testis, rat mammary gland, bovine brain, and of yeast. The enzyme has an absolute requirement for a divalent cation; Mg 2+ gave the greatest activity, with an optimal concentration of 2.5 m m in the standard assay employed. Zn 2+, Co 2+, and Mn 2+ supported activity to a lesser degree. Activity was inhibited by NaF, HgCl 2, and p-hydroxymercuribenzoate. myo-Inositol tetrakis (dihydrogen phosphate) and myo-inositol 1,3,4,5,6-pentakis (dihydrogen phosphate) were not substrates for this enzyme and inhibited the hydrolysis of myo-inositol 1-phosphate. Unlike other phosphatases for myo-inositol 1-phosphate, this enzyme cleaved myo-inositol 1-phosphate ( K m = 8.6 × 10 −5 m) and myo-inositol 2-phosphate ( K m = 2.86 × 10 −4 m) at approximately the same rates. It also hydrolyzed 2′-purine and pyrimidine ribonucleotides about as well as myo-inositol 1-phosphate, but was only 20–30% as active against the 3′-ribonucleotides and had scarcely any activity against the 5′-ribonucleotides. The amount of enzyme activity in erythrocytes of embryos, chicks, and mature chickens was the same (~29 μmol/ml rbc/h). The biological function of this enzyme in avian erythrocytes is unclear at this time. Other tissues containing this phosphatase also have an enzyme which synthesizes myo-inositol 1-phosphate from glucose 6-phosphate, but we have been unable to detect the presence of such an enzyme in avian erythrocytes.

Nutritional Regulation of Organelle Biogenesis in Euglena

Exposure of dark grown resting Euglena to ethanol produced a transient increase in the specific activity of the glyoxysomal enzyme malate synthase. Enzyme specific activity increased during the first 24 hours of ethanol treatment and then declined. Light exposure or malate addition failed to increase enzyme specific activity. The increase and decrease in enzyme specific activity represented changes in the amount of active enzyme. In both wild type cells and the plastidless mutant W(3)BUL, enzyme levels were always higher in the dark than in the light.The specific activity of the peroxisomal enzyme glycolate dehydrogenase began to increase 24 hours after dark grown resting Euglena were exposed to light. Ethanol, but not malate, prevented the increase and promoted a decrease in glycolate dehydrogenase levels. Cycloheximide produced a decline in enzyme levels similar to the decline produced by ethanol addition. Glycolate dehydrogenase was present in the plastidless mutant W(3)BUL indicating that it is coded in the nucleus and synthesized on cytoplasmic ribosomes. Streptomycin, a specific inhibitor of chloroplast protein synthesis and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthetic CO(2) fixation, inhibited the photoinduction of glycolate dehydrogenase while having no effect on the photoinduction of NADP dependent glyceraldehyde-3-phosphate dehydrogenase, another light induced, nuclear coded, cytoplasmically synthesized enzyme. Taken together, these results suggest that microbodies are continually synthesized in resting Euglena and their enzyme complement is determined through substrate induction of glyoxysomal and peroxisomal enzymes.

Phospholipase A activity of culture supernatants from Streptococcus mutans strain 6715

1. 1. Phospholipase A activity was found in the culture broth of growing cultures of Streptococcus mutans strain 6715. 2. 2. The amount of enzyme activity was proportional to the cell density of the cultures. 3. 3. The enzyme had a pH optimum of 7.0 and was inactivated at temperatures greater than 45°C. 4. 4. The enzyme was Ca 2+-dependent, since both EDTA and EGTA were inhibitory and Ca 2+ was stimulatory. 5. 5. Analysis of the fatty acid products resulting from the enzyme's action on 1-palmitoyl-2-oleoyl phosphatidylcholine indicated the enzyme to be a phospholipase A 1, (EC 3.1.1.32).

Explanation of anomalous binding kinetics with a high yield immobilized enzyme system

The activities of glucose oxidase (β- d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) and catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) from commercial preparations do not give typical adsorption curves upon immobilization on non-porous polyethylenimine-coated glass microbeads. The cause of this effect with glucose oxidase was investigated. Protein binding exhibited a rectangular hyperbolic adsorption isotherm, approaching saturation at high concentrations, however, enzyme activities did not. The isotherm for activities exhibited a maxima which corresponded to less than 50% saturation with regard to total protein adsorption. The enzyme preparation was found to contain small quantities of several low molecular weight impurities as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These impurities apparently compete with glucose oxidase for binding. When large excesses of protein are added to beads, the binding of impurities becomes significant and the amount of enzyme activity per unit of bead is reduced.

Quantitative measurement by microdensitometry of tyrosinase (dopa oxidase) development in whole small ascidian embryos.

Embryos of the ascidian, Ciona intestinalis, were fixed in either cold (5 degree C) 70% ethanol or cold absolute methanol during their tyrosinase development phase and incubated in buffered (pH 7.2) solutions of the enzyme substrate L-dihydroxyphenylalanine. Optical density of the reaction product (melanin) was measured in the whole small embryos at 450 nm with a Vickers M85 scanning and integrating microdensitometer. The frequency distribution of the reaction density in embryos of a population was Gaussian, and the mean optical density in embryos samples (N = 25) increased linearly with incubation time when a saturation level of substrate was used. Absolute optical density units of dopa oxidase activity in embryos increased linearly in proportion to the development time preceding melanin granulogenesis thereby suggesting that the enzyme activity measured by this procedure is proportional to the amount of tyrosinase present. Since this developmental increase in activity was blocked by treatment of the embryos with puromycin, an inhibitor of protein synthesis, the change is apparently caused by new enzyme synthesis. The microdensitometry assay also confirmed results obtained previously with a radiometric assay: embryos cleavage-inhibited at 7 h development time with cytochalasin B to produce giant melanocytes developed only the same amount of enzyme activity as control embryos.