Showing papers on "Substrate (chemistry) published in 1972"
TL;DR: The soils investigated exhibited optimal protease activity near pH 8.0 and 60°C, and were consistent in their preferential hydrolysis of dipeptide derivatives containing amino acids with hydrophobic side chains, however, soils varied widely in their relative activities towards a given diPEptide derivative and towards benzoyl arginine amide, a cationic substrate used in the assay of ‘trypsin-like’ enzymes.
Abstract: Conditions are described for the rapid and precise assay of soil proteases, using proteins and dipeptide derivatives as substrates in the absence of added bacteriostatic agents. The rate of substrate hydrolysis was proportional to the soil concentration; the release of amino compounds per unit weight of soil was directly related to the incubation time. The soils investigated varied widely in their pH, texture, organic matter content, and total exchangeable cations, but nevertheless exhibited optimal protease activity near pH 8.0 and 60°C, and were consistent in their preferential hydrolysis of dipeptide derivatives containing amino acids with hydrophobic side chains. However, soils varied widely in their relative activities towards a given dipeptide derivative and towards benzoyl arginine amide (BAA), a cationic substrate used in the assay of ‘trypsin-like’ enzymes. Benzyloxycarbonyl (Z) phenylalanyl leucine was hydrolysed most rapidly by all soils investigated. Activities towards Z-phenylalanyl leucine far exceeded those towards protein substrates and were highly correlated with the clay contents of the soils but not well correlated with the organic matter contents.
945 citations
TL;DR: A new assay procedure for cathepsin B1 is described, which is about 35 times more sensitive than that previously used, in which the substrate was the 4-nitroanilide.
377 citations
TL;DR: The broad substrate specificity of the enzymes suggests that, in addition to the possible contribution of the enzyme to the brain renin‐angiotensin system, other naturally occurring peptides might also be substrates for the enzyme.
Abstract: — Angiotensin converting enzyme of rat brain was studied using Hip-His-Leu as substrate. The highest specific activity of the enzyme was associated with the microsomal fraction. The specific activity of the microsomal enzyme in several regions of the rat brain varied significantly. For example, the specific activities of the striatal and pituitary enzymes were about 10-fold greater than that of the cerebral cortical enzyme. The enzyme required chloride ion; moreover, activity was inhibited in the presence of disodium EDTA or O-phenanthroline, effects suggesting that the converting enzyme of brain is a metalloprotein. SQ-20881, a nonapeptide that inhibits converting enzyme in peripheral tissue, was a potent inhibitor of the enzyme of brain. In addition to Hip-His-Leu, the microsomal fraction was capable of liberating C terminal dipeptides from angiotensin I, Hip-Gly-Gly and Z-Gly- Gly-Val. The broad substrate specificity of the enzyme suggests that, in addition to the possible contribution of the enzyme to the brain renin-angiotensin system, other naturally occurring peptides might also be substrates for the enzyme.
293 citations
TL;DR: The principle of Dixon (1965) has been extended to give rapid graphical methods for determining enzyme constants for substrates and inhibitors, valid even for cases of high affinity, where the usual methods fail.
Abstract: The principle of Dixon (1965) has been extended to give rapid graphical methods for determining enzyme constants for substrates (Km) and inhibitors (Ki). It does away with the sometimes questionable assumption that the amounts of substrate or inhibitor bound by the enzyme are negligible in comparison with the total amount added, and is therefore valid even for cases of high affinity, where the usual methods fail. Besides doing away with the need for calculation, it enables the concentrations of the various components of the system to be read off directly for any point of the velocity curve.
272 citations
TL;DR: Glutamate synthase catalyzes the TPNH-dependent conversion of α-ketoglutarate and l-glutamine to glutamate, which results in significantly lower levels of enzyme produced when glutamate replaces NH4Cl as the source of nitrogen.
261 citations
TL;DR: 2-Ethylaminoethanol supports the highest enzyme activity of any of the compounds tested, and Diethanolamine was shown to have many of the favorable characteristics required for a reference enzyme procedure.
Abstract: We have compared 23 compounds, with and without transphosphorylating properties, as buffer systems for human serum alkaline phosphatase activity, with p -nitrophenylphosphate as substrate. Relative enzyme activity in four representative buffers at near-optimal conditions was ethylaminoethanol > diethanolamine > 2-amino-2-methyl-1-propanol > carbonate. Transphosphorylation was demonstrated in the two buffers in which the enzyme was most active, ethylaminoethanol and diethanolamine. The optima for pH, buffer concentration, and substrate for these four systems were studied in detail. 2-Ethylaminoethanol supports the highest enzyme activity of any of the compounds tested. Diethanolamine was shown to have many of the favorable characteristics required for a reference enzyme procedure.
238 citations
TL;DR: It is confirmed that oxygenation of fatty acids by lipoxygenase occurs with a kinetic lag period which can be abolished by adding product hydroperoxides and it is shown that the lag period may be extended as a result of inhibition of the enzyme by polyenoic acid substrates.
220 citations
TL;DR: The purification of a tyrosinase from cell-free extracts of Streptomyces glaucescens is described, and the enzyme obtained is homogeneous according to the criteria of ultracentrifuge analysis and polyacrylamide gel electrophoresis, which shows optimal activity at pH 6.8.
Abstract: 1
The purification of a tyrosinase from cell-free extracts of Streptomyces glaucescens is described. The enzyme obtained is homogeneous according to the criteria of ultracentrifuge analysis and polyacrylamide gel electrophoresis.
2
An average molecular weight of 29100 was determined by dodecylsulfate-gel electrophoresis and by sedimentation equilibrium analysis in the ultracentrifuge. The amino acid composition of the pure enzyme is also reported.
3
The enzyme contains one atom of copper in the cuprous form per molecule and is strongly inhibited by KCN and 4-nitro-catechol.
4
The enzyme shows optimal activity at pH 6.8; it has an isoelectric point at pH 6.95 and is most stable at pH 8.0.
5
Variations of Km values between pH 5.0 and 8.9 when l-tyrosine methylester and 3,4-di-hydroxy-l-phenylalanine were used as substrate was interpreted by assuming that different ionizable groups are involved in the oxidation of these two substrates.
6
The Michaelis-Menten constants and KCat are strongly dependent on the oxygen concentration for 3,4-dihydroxy-l-phenylalanine and almost independent for l-tyrosine methyl ester. Kinetic parameters obtained for a series of mono- and o-diphenol substrates are also reported.
189 citations
TL;DR: In this article, a series of chemically modified lecithins were used to investigate by kinetic analyses their substrate c.q. inhibitor properties for porcine pancreatic phospholipase A.
186 citations
TL;DR: The p-hydroxybenzoate hydroxylase of Pseudomonas fluorescens has been obtained in crystalline form as an electrophoretically homogenous protein which is very stable in the absence of added cofactors as discussed by the authors.
159 citations
TL;DR: GTP did not appear to change the apparent affinity of the substrate to the enzyme, but markedly increased the PGE1-stimulated enzyme activity.
TL;DR: The regulation of adenylate cyclase in skeletal muscle may be classified as a "V" allosteric system since metal ions, F-, and epinephrine all result in increased maximal velocity of the enzyme reaction.
TL;DR: Studies with 2-chloro-2-deoxy- and 2-O-methyl derivatives of d-glucose and d-mannose have revealed that little chemical modification is possible at position 2 without substantial loss in substrate binding, and the variation in the value of K(m) for the d-hexose derivatives was associated with a corresponding change in the values of MgATP(2-).
Abstract: 1. 2-Deoxy-2-fluoro-d-glucose, 2-deoxy-2-fluoro-d-mannose and 2-deoxy-2,2-difluoro-d-arabino-hexose are good substrates for yeast hexokinase. 2. 3-Deoxy-3-fluoro-d-glucose and 4-deoxy-4-fluoro-d-glucose are poor substrates and have very similar K(m) values (8x10(-2)m). 3. Neither alpha- nor beta-d-glucopyranosyl fluoride is a substrate or inhibitor. 4. Studies with 2-chloro-2-deoxy- and 2-O-methyl derivatives of d-glucose and d-mannose have revealed that little chemical modification is possible at position 2 without substantial loss in substrate binding. 5. The variation in the value of K(m) for the d-hexose derivatives was associated with a corresponding change in the value of K(m) for MgATP(2-) showing that the binding of MgATP(2-) is modified by the binding of the sugar.
TL;DR: Cholesterol esters, at proportions similar to those present in high density lipoproteins, reduced enzyme activity to about 20% of that obtained in the presence of only cholesterol and lecithin as lipid components, which was similar to that found using the native lipoprotein.
TL;DR: Crystalline glutaminase-asparaginase with antitumor activity was prepared from an Achromobacteraceae soil isolate organism and catalyzes the hydrolysis of the d isomers of glutamine and asparagine at about one-third the rate of the l isomers.
TL;DR: Compared with the turnover numbers and substrate specificities of the other enzymes acting in β oxidation of fatty acyl-CoA derivatives, this comparison suggests that crotonase, by virtue of its substrate specificity and its sensitivity to feedback inhibition by acetoacetyl- CoA, may play a regulatory role in fatty acid oxidation.
TL;DR: The enzyme-catalysed exchange of the methyl hydrogen atoms of the ;virtual substrate' monohydroxyacetone phosphate with solvent (2)H(2)O or (3)H(-1)O) was shown, and this exchange is about 10(4)-fold slower than the corresponding Exchange of the C-3 hydrogen of dihydroxy acetone phosphate.
Abstract: The isolation of crystalline triose phosphate isomerase from chicken breast muscle is described. The values of kcat. and Km for the reaction in each direction were determined from experiments over wide substrate-concentration ranges, and the reactions were shown to obey simple Michaelis–Menten kinetics. With d-glyceraldehyde 3-phosphate as substrate, kcat. is 2.56×105min−1 and Km is 0.47mm; with dihydroxyacetone phosphate as substrate, kcat. is 2.59×104min−1 and Km is 0.97mm. The enzyme-catalysed exchange of the methyl hydrogen atoms of the `virtual substrate' monohydroxyacetone phosphate with solvent 2H2O or 3H2O was shown. This exchange is about 104-fold slower than the corresponding exchange of the C-3 hydrogen of dihydroxyacetone phosphate. The other deoxy substrate, 3-hydroxypropionaldehyde phosphate, was synthesized, but is too unstable in aqueous solution for analogous proton-exchange reactions to be studied.
TL;DR: The caldo-active strain YT-P was found to produce a variety of extracellular enzymes, including an amylase and a protease, which were further examined, and it is suggested that they belong to the genus Bacillus.
Abstract: The caldo-active strain YT-P was found to produce a variety of extracellular enzymes, including an amylase and a protease, which were further examined. With azo-casein as a substrate, optimum conditions with respect to enzyme and substrate concentration were determined for the protease. The optimum temperature was found to be 70°C, with a sharp decline to both lower and higher temperatures. The enzyme was found to be extremely heat-stabile, with unaltered activity after 8 hours at 80°C. Optimum conditions for the amylase were also examined. This enzyme was shown to be less heat-stabile, though the temperature optimum was again at 70°C. The activity or stability was not influenced by absence or presence of Ca-ions. The main activity of the amylase was found in the 20–40% ammonium sulfate fraction, which also contained the bulk of the proteolytic enzyme. This strain growth optimally on a variety of carbon sources at 72°C. Typical submicroscopical features are the double-layered cell wall, and a cytoplasmic membrane with a varying number of small dots and dot-free patches. Furthermore the nutritional requirements and submicroscopical features of two other strains, YT-G and YT-F, are described and compared to strain YT-P. Based on the fatty acid composition of the three spore forming caldo-active strains we suggest that they belong to the genus Bacillus, and propose the names B. caldolyticus for strain YT-P, B. caldovelox for strain YT-F, and B. caldotenax for strain YT-G.
TL;DR: Specific enzyme activity in enzyme preparations obtained from rats with free access to food and stimulated in vivo by secretin and pancreozymin is twice as high as that in enzymes preparations from starved animals.
Patent•
06 Jun 1972TL;DR: A method for the pre-preparation of BIOCOMPATIBLE and BIO-functional SURFACES is provided by BIOCO and ATI N GRAFTING as mentioned in this paper.
Abstract: A METHOD FOR THE PREPARATION OF BIOCOMPATIBLE AND BIOFUNCTIONAL SURFACES IS PROVIDED BIOCOMPATIBLE AND ATI N GRAFTING A REACTABLE COMPOUND SELECTED FROM THE GROU CONSISTING OF POLYMERS AND COPOLYMERS ONTO AN INERT UP POL MERIC SUBSTRATE AND THEREAFTER CHEMICALLY BONDING A BIOLOGICALLY ACTIBE MOLECULE TO THE REATABLE COMPOUND SPECIFIC EMBODMENTS INCLUDE CHEMICALLY BONDING OF HUMAN SERUM ALBUMIN HEPARIN, STREPTOKINASE, PROSTAGLANDIN E-1 AND MIXTURES THEREOF TO HYDROGELS OF VARYING COMPOSITIONS WITH OR WITHOUT "ARM" SUC AS E-AMINO CAPROIC MOLECULE OR CHEMICAL "ARM" SUCAN INTERMEDIATE SMALLER ACID OR 1,4 DIAMINO BUTANE, THE HYDROGELS HAING BEEN PREVIOUSLY RADIATION-GRAFTED TO TOUGH INERT POLYMERIC SUBSTRATE. A HIGHLY SYNERGISTIC EFFECT IS ACHIEVED FOR HYDROGELS CONTAINING HIGH WATER CONTENT E.G., ABOVE ABOUT 55% WHEN THE CHEMICALLY BONDING OF THE BIOLOGICALLY ACTIVE MOLECULE IS CARRIED OUT VIA AN INTERMEDIATE SMALLER MOLECULE OR CHEMICALL ARM. FOR THIN FILM SUBSTRATES HAVING RAIATION-GRAFTED THERETO HYDROGELS WITH -CO2 PENDANT FUNCTIONAL GROUPS WHICH ARE ACTIVATED WITH A CARBODIIMIDE SOLUTION A QUICK WASH OF THE THIN FILM WITH ICE WATER IS REQUIRED TO EFFECT SUBSEQUENT CHEMICAL BONDING OF THE BIOLOGICALLY ACTIVE MOLECULES TO THE ACTIVATED SURFACE. THE SAME TECHNIQUE IS ALSO REQUIRED WHERE A CHEMICAL ARM IS EMPLOYED WHICH HAS -CO2H PENDANT FUNCTIONAL GROUPS.
TL;DR: A strain of Escherichia coli K-12 bacteria is isolated that is believed to be conditionally defective in peptidyl-tRNA hydrolase, suggesting that the normal substrate is free for this enzyme.
Abstract: WE have isolated a strain of Escherichia coli K-12 bacteria that we believe to be conditionally defective in peptidyl-tRNA hydrolase. This enzyme (which we shall refer to as hydrolase) catalyses the hydrolysis of peptidyl-tRNA (or N-acyl-amino-acyl-tRNA) yielding free peptide (or N-acyl-amino-acid) and a tRNA capable of accepting its conjugate amino-acid1,2. In the current model of protein biosynthesis3 peptidyl-tRNA is the form in which the growing protein is bound to the ribosome. Ribosome-bound peptidyl-tRNA, however, is a poor substrate for the hydrolase2,4, implying that the normal substrate is free. N-acyl-aminoacyl-tRNAs occur in cells5–7 but the only species the function of which is known3,8, N-formyl-methionyl-tRNAf, is a poor substrate for bacterial peptidyl-tRNA hydrolase1,9,10.
TL;DR: Optimal concentrations of ammonium ions, 2-oxoglutarate, and NADH were defined for serum glutamate dehydrogenase (EC 1.1.2) activity at 37°C, and precision was satisfactory.
Abstract: Optimal concentrations of ammonium ions, 2-oxoglutarate, and NADH were defined for serum glutamate dehydrogenase (EC 1.4.1.2) activity at 37°C. Substrate inhibition was not observed with the last two compounds. The pH optimum in triethanolamine buffer is 7.4, and activity is inhibited by increasing the buffer concentration beyond 50 mmol/liter. Activation by ADP exceeds that promoted by L-leucine, and there is little advantage in having both present. Activity is linear with time and with enzyme concentration to a limiting absorbance change of 0.300 at 340 nm, and precision was satisfactory. Data indicate the normal range to lie between 0 and 4 U/liter.
TL;DR: A gel film technique was adopted for kinetic enzyme activity determination in single cells using cryostat sections by means of a microscope photometer, and it was found that the periportal and central areas of the hepatic lobule in rat contain the same level of lactate dehydrogenase activity.
Abstract: A gel film technique was adopted for kinetic enzyme activity determination in single cells using cryostat sections by means of a microscope photometer. The main principle of the method is a comparative activity determination, based on bipositional recording of initial reaction kinetics in two preselected measuring fields in the same tissue section. Systematic model experiments were performed to prove the fulfillment of the following conditions: (a) validity of Beer’s law; (b) optimal con centrations of substrates, cosubstrates and dye within the reaction film, including evidence that the reaction rate is not limited by insufficient diffusion of these compounds; (c) proportionality between local enzyme concentration or thickness of tissue sections and the recorded reaction rate; and (d) specificity of the method as demonstrated by studying reaction rates in the absence of the substrate as well as in the presence of substrate and a specific inhibitor. The validity of the method was also examined by comparing the levels of succinate dehydrogenase activity in various rat tissues, as measured microphotometrically, with the enzyme levels as determined in homogenates. Microphotometric assays for nicotinamide adenine dintscleotide phosphate isocitrate dehydrogenase, lactate dehydrogenase and succinate dehydrogenase are described. Using these techniques it was found that: the penportal and central areas of the hepatic lobule in rat contain the same level of lactate dehydrogenase activity; succinate dehydrogenase activity is 1.6 times higher in the periportal area as compared to the centrilobular area. In experimental thyrotoxicosis, the ratio of enzyme activities in the periportal and central areas was 1.1.
TL;DR: The principle of radial diffusion in substrate containing agar gel has been applied for the quantitative assessment of several enzymes and has been found very helpful in screening large numbers of chromatography fractions.
TL;DR: Beef liver 5-aminolevulinate dehydratase was purified 700-fold by salt fractionation, heat treatment, gel filtration and ion-exchange chromatography and some evidence was obtained for a requirement for zinc as a co-factor.
Abstract: Beef liver 5-aminolevulinate dehydratase was purified 700-fold by salt fractionation, heat treatment, gel filtration and ion-exchange chromatography. The final product was homogeneous by electrophoretic, ultracentrifugal and immunological criteria and had a molecular weight, by the low-speed equilibrium method, of 260000. Treatment with sodium dodecylsulfate and 2-mercaptoethanol dissociated the enzyme into dimeric subunits which could be further dissociated into monomeric subunits by treatment with 7.3 M guanidine hydrochloride and dithioerythreitol. The molecular weight of the monomeric subunit and the results of amino acid analysis suggested the presence of 14 subunits per mole. Carboxymethylation and hydrolysis showed the presence of 56 cysteine residues per mole, approximately half of which were available for titration with p-chloromercuribenzoate in the urea-denatured enzyme.
The enzyme has a pH optimum of 6.3 and a Km of 0.46 mM in the crude state and 0.15 mM when pure. Thiol activation was required for catalytic activity and some evidence was obtained for a requirement for zinc as a co-factor. Heavy metals inhibited the enzyme in a complex manner. Zinc acted as a competitive inhibitor, lead acted as a non-competitive inhibitor, while cadmium acted in a unique manner, causing inhibition at low concentrations of substrate and stimulation at high levels of substrate.
TL;DR: UDP-glucose:d-fructose 2-glUCosyltransferase (EC 2.14), commonly called sucrose synthetase, has been purified from etiolated Phaseolus aureus seedlings to homogeneity by electrophoretic, immunological, and ultracentrifugal criteria andKinetic data indicate a random mechanism for the enzyme.
TL;DR: It is concluded that heparin stabilizes rather than stimulates the enzyme; furthermore, the effect of inhibitors is partly or completely abolished.
TL;DR: A selective medium was used to isolate a bacterium that produced extracellular alkaline amylase in an alkaline medium and the main final product produced from amylose and amylopectin was beta-maltose; the other final products were maltotriose and small quantities of glucose and maltotetraose.
Abstract: A selective medium was used to isolate a bacterium (Bacillus species NRRL B-3881) that produced extracellular alkaline amylase in an alkaline medium (pH 9.5). Maximal enzyme yield was obtained in an aerated medium after 21 hr at 36 C. The enzyme was purified 18-fold by ultrafiltration and ammonium sulfate precipitation. Three active isoenzymes (one major and two minor) of alkaline amylase were detected by disc electrophoresis in polyacrylamide gel. The enzyme was only 12% inactivated by 20 mm ethylenediaminetetraacetic acid after 1 hr at pH 9.2 and 32 C. The optimal temperature was 50 C at pH 9.2, and the optimal pH was 9.2 at 50 C. The enzyme was stable between pH 7.5 and 10. It had an endomechanism of substrate encounter. The products produced from amylose and amylopectin had the beta-configuration. Cyclomaltoheptaose was hydrolyzed to maltotriose, maltose, and glucose. The main final product produced from amylose and amylopectin was beta-maltose; the other final products were maltotriose and small quantities of glucose and maltotetraose. The predominant product at early stages of hydrolysis was maltotetraose; other products were maltose through maltonanaose.
TL;DR: It can be concluded that MgATP is the substrate of this ATPase, and the catalytic centre of the examined Ca 2+ -ATPase contains three different ionizable groups as follows: one ionizable group is active when dissociated, one is involved in the process of substrate binding and another may be identical with an imidazole nitrogen of histidine.