Showing papers on "Substrate (chemistry) published in 1971"
01 Jan 1971
TL;DR: A modified method for the assay of serum phosphatase activity utilising disodium phenyl phosphate as substrate, based upon the reaction of free phenol with 4-amino-antipyrine and alkaline potassium ferricyanide is described.
Abstract: A modified method for the assay of serum
phosphatase activity utilising disodium phenyl phosphate
as substrate is described. This is based upon the reaction of
free phenol with 4-amino-antipyrine and alkaline potassium
ferricyanide. Study of the kinetics of the colour reaction
has led to an alteration in the concentration of the reagents
used; the colour is formed instantaneously and shows a very slow rate of decay.
Sodium arsenate incorporated with the amino-antipyrine reagent effectively abolishes
further enzyme activity and prevents the dilution of the colour inherent in earlier
methods.
471 citations
TL;DR: It is concluded that the previous reports that tyrosine hydroxylase is stimulated by Fe2+ can be explained by the known ability ofFe2+ to decompose H2O2.
381 citations
TL;DR: The ability of the enzymatically inactive monomer forms to bind to the ligand column suggests that this affinity chromatographic method may be used in the purification of catalytically inactive mutant forms which retain the ability to bind substrate analogues.
248 citations
TL;DR: The enzyme activity is absent in dermatosparaxic connective tissues, thus suggesting that dermatosParaxis is caused by the absence of a normal enzyme function rather than by the production of an abnormal collagen.
Abstract: A heritable connective tissue disorder of cattle, dermatosparaxis, is characterized by an extreme fragility of the skin and the presence of additional peptides at the N-terminal extremities of the collagen α chains, p-α1 and p-α2.
The existence of an enzyme activity is demonstrated in normal connective tissues that is capable of cleaving these additional N-terminal peptides from dermatosparaxic collagen. The activity is demonstratable with dermatosparaxic collagen in solution, as well as with reconstituted dermatosparaxic collagen fibrils polymerized in vitro. It has a pH optimum of about 7.0 and is inhibited by EDTA and mercaptoethanol. Differences in Km and Vmax values exist depending on the substrate utilized, i.e., p-α1 or p-α2; and the presence of additional amounts of one substrate, p-α1, alters the concentration requirement for the second substrate, p-α2. The product of the excision reaction with p-α1 as substrate is an equimolar amount of normal α1 monomer; the product when p-α2 is substrate is an equimolar amount of normal α2 monomer.
The enzyme is present in normal calf skin, tendon, aorta, cartilage, and lung; it can be demonstrated in the skin of rats and humans. The enzyme activity is absent in dermatosparaxic connective tissues, thus suggesting that dermatosparaxis is caused by the absence of a normal enzyme function rather than by the production of an abnormal collagen.
208 citations
TL;DR: The Michaelis constant for the hydrolysis of human renin substrate byhuman renin is 0·931 n-mole/ml, which means substrate concentration in normal serum occurs at a very critical level.
Abstract: The Michaelis constant (Km) for the hydrolysis of human renin substrate by human renin is 0·931 n-mole/ml. This concentration of substrate is higher than that found in a normotensive population. Since the Km is numerically equal to the concentration of substrate that will permit the reaction to proceed at half its maximal velocity, substrate concentration in normal serum occurs at a very critical level.
192 citations
TL;DR: A potato lipoxygenase (EC 1.13) was found to have a pH optimum at 5.5-6.0 and was inactive at pH 9.0 as discussed by the authors.
Abstract: A lipoxygenase (EC 1.13.1.13) was partially purified from potato tubers and was shown to differ from previously characterized soya-bean lipoxygenases in the positional specificity and pH characteristics of the oxygenation reaction. The potato enzyme converted linoleic acid almost exclusively (95%) into 9-d-hydroperoxyoctadeca-trans-10,cis-12-dienoic acid. The 13-hydroperoxy isomer was only a minor product (5%). Linolenic acid was an equally effective substrate, which was also oxygenated specifically at the 9-position. The enzyme had a pH optimum at 5.5–6.0 and was inactive at pH9.0. A half-maximal velocity was obtained at a linoleic acid concentration of 0.1mm. No inhibition was observed with EDTA (1mm) and cyanide (1mm) or with p-chloromercuribenzoate (0.2mm). Haemoproteins were not involved in the lipoxygenase activity. The molecular weight of the enzyme was estimated from gel filtration to be approx. 105. Preliminary evidence suggested that the enzyme oxygenated the n–10 position of fatty acids containing a penta(n–3, n–6)diene structure.
187 citations
TL;DR: A slight modification of the Lineweaver—Burk equation, in which the form is retained but the instantaneous velocity is replaced by the negative change in substrate concentration divided by the time, allows this linear reciprocal form to be used with little error even when as much as 50% of the substrate is consumed.
172 citations
167 citations
158 citations
TL;DR: An enzyme preparation was isolated from liquid cultures of Azotobacter vinelandii by precipitation with ammonium sulphate after removal of cells by centrifugation and attempts were made to follow the formation of the two different types of L-guluronic acid-containing blocks during the epimerization reaction.
149 citations
01 Jan 1971
TL;DR: The use, advantages and limitations of the method are discussed as well as the possible significance of effects of drugs on this key enzyme for their pharmacodynamic effects.
Abstract: A highly sensitive method for the determination of cyclic 3′,5′-nucleotide phosphodiesterase (PDE) is described.
TL;DR: The rate of production of the end product at the first stages of the reaction is markedly higher in the immobilized enzyme system than that predicted for a corresponding homogeneous system.
TL;DR: It is suggested that the enhanced catalytic activity of induced alkaline phosphatase is the result of an alteration in zinc ion binding which produces an entatic effect, lowering the energy requirements of the enzyme substrate transition state.
TL;DR: The catalytic activity of lysozyme measured in terms of chemical equivalents of substrate converted per unit time is quite low and an approximate relation has been determined between the amount of chemical reaction and an activity unit defined in terms the rate of absorbance change.
TL;DR: Comparison of the monolayer and bulk methods showed that both techniques are complementary, and the pH dependence and calcium requirements of the enzymatic reaction are similar under monolayers and bulk conditions.
TL;DR: It is suggested that this Ca2-activated ATPase of isolated membranes of guinea pig placenta aids in maintaining the uphill gradient of Ca2+ between maternal and fetal circulation.
TL;DR: The “specific reactivity” of β-galactosidase from Escherichia coli for different substrates has been studied at the optimal pH value and the pH dependence of the enzyme activity has been reinvestigated in highly controlled conditions with respect to Mg2+ and Na+ concentrations and ionic strength.
Abstract: The “specific reactivity” of β-galactosidase from Escherichia coli for different substrates has been studied at the optimal pH value The pH dependence of the enzyme activity has been reinvestigated in highly controlled conditions with respect to Mg2+ and Na+ concentrations and ionic strength The different kinetic parameters kcat, km and the kcat/km ratio have been determined with o-nitrophenyl-β-d-galactoside and o-nitrophenyl-β-d-fucoside, at different pH values between 516 and 10, for both Mg2+-enzyme and Mg2+-free enzyme, since a residual activity has been found in the absence of Mg2+
The activity of both types of enzyme is controlled by a protonated group which ionizes in the alkaline range and by at least one unprotonated group which ionizes in the acidic range This latter group has a pK smaller than 6 in both types of enzyme; it is plausible to assume that this group is a carboxylate
In the alkaline range, the pK of the involved group shifts from about 65 in Mg2+-free enzyme to 84 in the Mg2+ enzyme Either the substrate or the Mg binding induces this shift in the ionization of the group
The β-galactosidase catalyzed reactions proceed via two intermediary complexes For o-nitrophenyl-β-d-galactoside substrate the limiting process is not the same for the Mg2+ enzyme and for the Mg2+-free enzyme
TL;DR: Experimental results of this work confirm Anderson's observations, and show a further reaction rate increase of nearly 20% between sucrose concentrations of 0.177M and 0.285M under the same conditions of temperature, pH, and enzyme‐concentration.
Abstract: The kinetics of the enzymatic hydrolysis of sucrose by invertase have been examined, with particular emphasis on high substrate concentration. Initial rates of reaction were determined by following the production of glucose directly as a function of time over a wide range of substrate concentrations (0.04M to 2.06M). The resulting data reveal a reaction rate that increases gradually until the sucrose concentration reaches about 0.29M, after which the reaction velocity decreases with increasing sucrose concentration. Previous workers (e.g., Nelson and Schubert1) have reported a peak reaction velocity as determined by indirect polarimetric measurements of glucose, at a sucrose concentration of about 0.17M. These measurements, however, neglect the intermediate oligosaccharides formed by the transferase action of invertase,8–10 and assume equal amounts of glucose and fructose. According to Anderson et al.,10 these oligosaccharides interfere by producing an erroneously low reaction rate. Experimental results of this work confirm Anderson's observations, and show a further reaction rate increase of nearly 20% between sucrose concentrations of 0.177M and 0.285M under the same conditions of temperature, pH, and enzyme-concentration.
Effects of substrate diffusion, solution viscisity, water concentration, and substrate inhibition were experimentally studied and the results incorporated into a kinetic model that has proven satisfactory in modeling the experimental results. This model takes into account inhibition by primary substrate, with concentration of the secondary substrate water, as a rate limiting factor at sucrose concentrations greater than 0.285M.
The effects of the mixing, in terms of volumetric power input, on the relation rate have been tested. Approximately 40-fold increase in volumetric power input caused on increase in the reaction rate. These experiments have shown that bulk mass transfer is not a rate limiting factor under the experimental conditions.
TL;DR: The calcium ion pump of fragmented sarcoplasmic reticulum can be coupled to hydrolysis of p-nitrophenyl phosphate, in the absence of added adenosine triphosphate, and comparison of the activities obtained suggests an analogous mechanism of transport.
Abstract: The calcium ion pump of fragmented sarcoplasmic reticulum can be coupled to hydrolysis of p-nitrophenyl phosphate, in the absence of added adenosine triphosphate. Comparison of the activities obtained with the two substrates suggests an analogous mechanism of transport. Independent of the substrate, a 2 : 1 ratio between calcium ion transport and substrate hydrolysis is displayed by the system, and an identical amount of work is required for ion transport against a given gradient. A phosphate ester appears necessary for substrate utilization in the pump mechanism, whereas the structure of the substrate determines the rates of activity and the affinity of the system for calcium ion.
TL;DR: Magnetic resonance studies of the activation of arginase with manganese have been performed with nuclear magnetic resonance, showing that primarily a binary complex between arginases and mangansese is formed.
TL;DR: Oleyl-CoA is the specific substrate for the microsomal oleyl -CoA desaturase from maturing seeds of Carthamus tinctorius L. Since tritium-labeled CoA does not exchange with nonlabeled oleyL- CoA in the presence of themicrosomal system, the possible roles of ACP and enzyme-bound oley l complexes are excluded.
TL;DR: In this article, the reduced and oxidized forms of addition compounds from diphosphopyridine nucleotide and a number of carbonyl compounds are described; these include pyruvate, pyruvic ethylester, oxaloacetate, α-ketoglutarate, acetaldehyde, butyraldehyde, and α -ketobutyrate.
TL;DR: It appears that the properties of thallium-205 are uniquely suitable in this regard for probing macromolecular environment in the presence of paramagnetic ions.
TL;DR: It is concluded that the enzyme chromophore, rather than ferredoxin, serves as a primary electron acceptor in the pyruvate-ferredoxin oxidoreductase reaction, which suggests that hydroxyethyl thiamine pyrophosphate is an intermediate of the reaction.
01 Jan 1971
TL;DR: The results reveal that the specific glycosidic monomer undergoing hydrolysis is probably highly strained and that long chains are hydrolyzed more rapidly than shorter ones primarily because of increased strain.
TL;DR: By the use of electrophoretic and spectrophotometric methods two types of endopeptidases were demonstrated in crude extracts of Escherichia coli: one was active on N-acetyl-dl-phenyl-alanine-2-naphtyl ester and the other onN-benzoyl-l-arginine-p-nitroanilide.
Abstract: By the use of electrophoretic and spectrophotometric methods two types of endopeptidases were demonstrated in crude extracts of Escherichia coli: one was active on N-acetyl-dl-phenyl-alanine-2-naphtyl ester and the other on N-benzoyl-l-arginine-p-nitroanilide. The two activities were separated by gel filtration on Sephadex G-100.
After gel electrophoresis of crude extracts, three bands of activity toward acetyl-phenylalanine-naphthyl ester were visualized. The enzyme corresponding to the band with the strongest esterolytic activity was also responsible for casein-hydrolytic activity in gel between pH 6 and 8; it was isolated and characterized. Starting with 450 g of wet cells, about 3 mg of a purified enzyme were obtained. This represented a recovery of 16% and almost 1000-fold purification.
The isolated enzyme, designated as protease I, was homogeneous by electrophoresis and sucrose-gradient centrifugation. Its molecular weight was estimated by two different methods to be about 43000.
The enzyme hydrolysed N-acetyl-dl-phenylalanine-2-naphthyl ester, a chymotrypsin substrate, but was inactive upon several synthetic substrates for enzymes with carboxypeptidase-A and trypsin-like specificity.
The esterolytic activity was inhibited by DFP, whereas it was resistant to phenyl methyl-sulfonylfluoride even after prolonged treatment. Metal chelating agents, sulfhydryl reagents and several metal ions were without effect.
The proterolytic activity of the purified enzyme was confirmed by its ability to breakdown native E. coli polynucleotide phosphorylase.
TL;DR: It is suggested that the peptide bond of the substrate adjacent to that about to be cleaved, is bound in the cleft of the enzyme between the NH group of glycine-66 and the backbone C=O group of aspartic acid-158, and provides a sensitive amplification mechanism through which the specificity of the enzymes for hydrophobic amino acids is relayed.
Abstract: The specificity of the proteolytic enzyme, papain, for the peptide bond of the substrate adjacent to that about to be cleaved and for the acyl residue of some N-acylglycine derivatives is manifest almost exclusively in the formation of the acyl-enzyme from the enzyme-substrate complex. Models for the enzyme-substrate complex and acyl-enzyme intermediate are suggested that account for these observations. In particular it is suggested that the peptide bond of the substrate adjacent to that about to be cleaved, is bound in the cleft of the enzyme between the NH group of glycine-66 and the backbone C=O group of aspartic acid-158, and provides a sensitive amplification mechanism through which the specificity of the enzyme for hydrophobic amino acids such as l-phenylalanine is relayed. It is also suggested that the distortion in the enzyme-substrate complex and the binding of the peptide bond adjacent to that about to be cleaved are also linked and behave co-operatively, the distortion of the protein facilitating binding and the stronger binding facilitating distortion. The results imply that between the enzyme-substrate complex and the acyl-enzyme a relaxation of the protein conformation must occur.
TL;DR: The specificity of linoleic acid isomerase from Butyrivibrio fibrisolvens for cis-9,cis-12-dienoic fatty acids with an ω chain length varying from 4 to 8 carbons has been examined as discussed by the authors.
TL;DR: This macromolecule, with a high local concentration of binding and catalytic groups, catalyzes the hydrolysis of uncharged nitrophenyl esters in water at pH 7 with rates markedly greater than previously observed with any other synthetic substances under similar conditions.
Abstract: A synthetic polymer has been prepared which contains dodecyl groups (to bind small substrate molecules) and methyleneimidazole side chains (as nucleophilic catalytic sites) linked to a polyethyleneimine framework. This macromolecule, with a high local concentration of binding and catalytic groups, catalyzes the hydrolysis of uncharged nitrophenyl esters in water at pH 7 with rates markedly greater than previously observed with any other synthetic substances under similar conditions.