Showing papers on "Substrate (chemistry) published in 1970"
TL;DR: The FCR provides an effective method for assessing pollen quality and is primarily one for the integrity of the plasmalemma of the vegetative cell, which is likely to be closely correlated with viability.
Abstract: Viable pollen grains immersed in a solution of fluorescein diacetate, made up in a concentration of about 10−6 M in sucrose of suitable tonicity (usually about 0.5 M), rapidly accumulate free fluorescein, which can be detected by its fluorescence. This fluorochromatic reaction (FCR) probably depends upon the entry of the nonpolar substrate into the vegetative cell where it is hydrolyzed by esterase to give the polar product, fluorescein, which is retained by the cell membrane. The test is primarily one for the integrity of the plasmalemma of the vegetative cell. Since this integrity is likely to be closely correlated with viability, the FCR provides an effective method for assessing pollen quality.
874 citations
TL;DR: Comparison of results obtained in continuous culture with results from exponential‐feed culture systems is shown to offer a novel experimental method for evaluating the effect of the cell age distribution on the properties and metabolic activity of a culture.
Abstract: High substrate concentrations inhibit growth and may distort the metabolism of microorganisms. Mechanisms causing substrate inhibition are discussed and used to derive several mathematical models representative of the entire concentration range, including stimulation of growth by low substrate concentrations. These kinetic models are tested with a variety of batch culture measurements of specific growth rate and respiration rate at widely-ranging substrate concentrations. Using one of the kinetic models, equations are developed for batch, continuous, and exponential-feed reactors. Comparison of results obtained in continuous culture with results from exponential-feed culture systems is shown to offer a novel experimental method for evaluating the effect of the cell age distribution on the properties and metabolic activity of a culture.
521 citations
TL;DR: The present series of investigations on proteolytic enzymes is to compare the activity of the enzyme towards peptides of increasing length, diastereoisomeric pairs of peptides in which a particular amino acid residue has been replaced by its antipode, and pairs of substrates inWhich a particular side chain has been replacement by another (say an aromatic group).
Abstract: The active site of an enzyme performs the twofold function of binding a substrate and catalysing a reaction. The efficiency of these actions determines the overall activity of the enzyme towards the particular substrate, i.e. determines the specificity of the enzyme. It is therefore possible to obtain information on the active site by the kinetics of the enzyme’s reactions with different substrates and inhibitors. An important feature of the active site is its size. It should be possible to 'measure’ this by using substrates or inhibitors large enough to show up the interactions of the furthermost parts of the binding site. In the present series of investigations on proteolytic enzymes, our approach is to compare the activity of the enzyme towards ( a ) peptides of increasing length, ( b ) diastereoisomeric pairs of peptides in which a particular amino acid residue has been replaced by its antipode, and ( c ) pairs of substrates in which a particular side chain (say a methyl group) has been replaced by another (say an aromatic group). The influence of these changes on reaction rates as a function of distance from the point of cleavage indicates the extent of the active site (Schechter, Abramowitz & Berger 1965; Abramowitz, Schechter & Berger 1967).
373 citations
TL;DR: In this paper, short-chain lecithins with C6, C7, and C8 fatty acid esters have been used to study kinetically the enzymatic hydrolysis by pancreatic phospholipase A (EC 3.4) in aqueous systems, without the addition of emulsifiers.
280 citations
TL;DR: By kinetic analysis and specific labeling experiments, it has been shown that, at low concentrations of G3 the formation of products is due to hydrolysis, and at high concentrations, the products are due to a condensation reaction in which 2 molecules of G2 condense to give G6 with subsequent hydroleysis and change in product distribution.
277 citations
TL;DR: It was concluded that nucleotides interact with the enzyme at a site other than the active center and hence that GSH peroxidase is an allosteric enzyme.
234 citations
TL;DR: Investigations in which this new substrate was utilized showed that human converting enzyme inactivated by dialysis was reactivated by anions in the order Cl − > NO 3 − > Br − > F − > I − .
224 citations
TL;DR: A rapid assay for lipoprotein lipase activity employing a (14)C-labeled substrate is described, which is very sensitive and suitable for routine use.
217 citations
TL;DR: Berstad as mentioned in this paper used human hemoglobin as a substrate for the estimation of Pepsin in Gastric Juice, using a modified Hemoglobin Substrate Method for the Estimation of PPI.
Abstract: Berstad, A 1970 A Modified Hemoglobin Substrate Method for the Estimation of Pepsin in Gastric Juice Scand J Gastroent 5, 343-348Human hemoglobin was used as substrate for the estimation of
216 citations
TL;DR: The protein methylase II (PMS II) as discussed by the authors has been reported to have a molecular weight of 35,000 by means of Sephadex G-100 and is electrophoretically homogeneous.
169 citations
TL;DR: Kinetic measurements indicated a complex reaction mechanism which is best described as a random binding of substrates with S-adenosylmethionine being kinetically preferred as the first substrate bound.
TL;DR: The presence of a nonspecific NADPH-linked aldehyde reductase (alcohol:NADP oxidoreductase, EC 1.1.2) was observed in various areas of bovine brain in vitro and was found that the enzyme was localized primarily in the soluble supernatant fraction of rat brain homogenates.
TL;DR: The identification of the hydroperoxide isomerase enzyme clarifies the role of lipoxidase in plant tissue and suggests a participation of lipid in the electron transport system.
Abstract: An enzyme has been isolated from flaxseed ( Linum usitatissimum ) which utilizes the product of lipoxidase for its substrate. The enzyme, termed hydroperoxide isomerase, converts the conjugated diene hydroperoxide of linoleic acid to the corresponding monoenoic ketohydroxy fatty acid. The structure of the latter has been determined by ultraviolet, infrared, and nuclear magnetic resonance spectroscopy; periodate and permangate oxidation; gas chromatography; and thin layer chromatography. Hydroperoxide isomerase activity has also been demonstrated in crude extracts from barley ( Hordeum vulgare ), wheat germ ( Triticum aestivum ), mung beans ( Phaseolus aureus ), and corn ( Zea mays ) and from partially purified extracts of soybean ( Glycine max ). The hydroperoxide isomerase enzyme from flaxseed has a pH optimum of 7.0. The enzyme was not inhibited by nordihydroguairetic acid, p-chloromercuribenzoic acid, or cyanide, but it was inhibited by cupric ion. One hundred per cent of the activity was lost by heating the enzyme for 1 minute at 68 C. Both linoleic and linolenic acid hydroperoxides can serve as substrates for the enzyme. The pH optimum for the hydroperoxide isomerase enzyme from barley is 6.2; from wheat germ, 6.1; and from soybean, 6.1. The identification of the hydroperoxide isomerase enzyme clarifies the role of lipoxidase in plant tissue and suggests a participation of lipid in the electron transport system.
TL;DR: Three parameters involved in the binding of substrate to linoleate isomerase of Butyrivibrio fibrisolvens have been identified and two chelators, o-phenanthroline and EDTA, were identified as reversible inhibitors of the enzyme.
TL;DR: Kinetic capability of cytochrome b(T) is evidenced by its rapid electron transfer and energization time of less than 200 msec, its thermodynamic capability-by a 280 mV potential span suitable for providing one of the two electron transfer reactions required in ATP formation.
Abstract: The primary event of coupled electron transfer at phosphorylation site II is identified with a modification in one of the two chemically distinct forms of cytochrome b, designated as the energy-transducing cytochrome bT. This modification is expressed through a change in the redox midpoint potential and by an increase in its reaction half time with cytochrome c1. In pigeon heart mitochondria cytochrome bT exhibits an absorption maximum at 564 nm and on this basis, it can be distinguished from Keilin's cytochrome b which exhibits an absorption maximum at 560 nm and serves as an electron carrier on the substrate side of cytochrome bT. Kinetic capability of cytochrome bT is evidenced by its rapid electron transfer and energization time of less than 200 msec, its thermodynamic capability—by a 280 mV potential span suitable for providing one of the two electron transfer reactions required in ATP formation. Two secondary events of coupled electron flow may be identified with a charge separation across the lipid structure of the permeability barrier and a change in water structure; both events result in an increased 1-anilino-8-naphthalene-sulfonic acid (ANS) response to the altered environment.
TL;DR: The magnitude of the effect suggests that the contribution of substrate "freezing" to total rate enhancement by an enzyme can be considerably greater than previously supposed and may in fact be sufficient to complete the justification for enzyme catalysis.
Abstract: As a result of alkyl substitution in both aromatic ring and side chain, the rate constant for acid-catalyzed lactonization of hydrocoumaric acid is increased by factors as high as 1011 and, in comparison with the bimolecular esterification of phenol and acetic acid, by almost 1016. In the most favorable case studied, the half-life of the phenolic acid (imidazole buffer, pH 7, 30°C) is 6 sec, with 90% of the total rate being due to catalysis by the buffer species. The effect is attributed to a unique interlocking of methyl groups, which produces a severe conformational restriction of the side chain and increases greatly the population of the most productive conformer. This phenomenon is presented as a model for the conformational restraint imposed by an enzyme on its substrate. The magnitude of the effect suggests that the contribution of substrate „freezing” to total rate enhancement by an enzyme can be considerably greater than previously supposed and may in fact be sufficient to complete the justification for enzyme catalysis.
TL;DR: The ability of the 50 s ribosomal subunit to catalyze the peptidyl transferase reaction is absolutely dependent on the continued presence of certain monovalent cations in the ribosome medium and this ability is maintained as long as one of these cations is present and is lost if the cation is removed.
TL;DR: A rapid and convenient chemical assay for the enzyme d-glucose 6-phosphate-1l-myoinositol 1-ph phosphate cyclase is described and the K(m) of the enzyme, 7.5+/-2.5x10(-4)m), was identical with that measured by the radiochemical method.
Abstract: A rapid and convenient chemical assay for the enzyme d-glucose 6-phosphate-1l-myoinositol 1-phosphate cyclase is described. The 1l-myoinositol 1-phosphate formed enzymically was oxidized with periodic acid liberating inorganic phosphate, which was assayed. myoInositol 2-phosphate can be assayed in the same way. Glucose 6-phosphate and other primary phosphate esters gave only very small quantities of inorganic phosphate under the conditions described. The K(m) of the enzyme for d-glucose 6-phosphate, 7.5+/-2.5x10(-4)m, was identical with that measured by the radiochemical method. 2-Deoxy-d-glucose 6-phosphate was a powerful competitive inhibitor, K(i) 2.0+/-0.5x10(-5)m, but was not a substrate for the enzyme.
TL;DR: The authors showed that enzymes behave differently when attached to solid supports for four main reasons: their conformations when they are supported may differ from those in free solution, and they act upon substrate.
Abstract: Enzymes behave differently when attached to solid supports for four main reasons: (1) their conformations when they are supported may differ from those in free solution, (2) they act upon substrate...
TL;DR: It is proposed that the decreased activity of an insoluble chymotrypsin–Sephadex is due to diffusional effects shown by the gel matrix toward the substrate, and observed shifts in optimum pH are explained by accumulation of hydrogen ions in the gel.
Abstract: Catalytically active chymotrypsin derivatives can be synthesized from cyanogen bromide-activated Sephadex G200. In most cases the apparent catalytic activity of the covalently fixed enzyme appears to be considerably decreased in comparison to the activity of the free enzyme. However, by proper choice of the reaction conditions for the activation, enzyme conjugates with high activity, even toward a high molecular substrate, can be synthesized. These latter derivatives may be of practical value for the digestion of proteins. Crosslinked dextran as carrier was chosen because of the possbility, of digesting it enzymatically by dextranase. Sephadex G200, if activated at or below pH 10.3, will combine with chymotrypsin to yield digestable products. Changes of apparent kinetic properties of the fixed enzyme can accordingly be studied during the degradation process. On the solubilization of the insoluble conjugate, a total recovery of activity of the fixed enzyme can be obtained in cases the carrier has been activated by a sufficiently mild procedure. The high apparent Michaelis constant Km of insoluble chymotrypsin–Sephadex toward N-acetyl-L-tyrosine ethyl ester shifts back on solubilization to the value of free chymotrypsin. We therefore propose that the decreased activity of an insoluble chymotrypsin–Sephadex is due to diffusional effects shown by the gel matrix toward the substrate. Similarly observed shifts in optimum pH are explained by accumulation of hydrogen ions in the gel. The organic chemical reaction used for coupling the enzyme to the polymer can therefore be performed without decreasing the inherent catalytic activity of the enzyme. The route described for fixing chymotrypsin to Sephadex followed by solubilization of the products may be useful as a synthetic method for binding proteins, peptides, and other amino group-containing substances to soluble carriers, e.g., for the modification of pharmaceuticals.
TL;DR: The results show that the overall reaction rate may be restricted by the rate of diffusion of substrate into and product out of the insolubilised enzyme derivative.
Journal Article•
TL;DR: Investigations by means of thin-layer chromatography indicated that the addition of small amounts of this enzyme preparation to soy milk resulted in complete hydrolysis of galacto-oligosaccharides.
Abstract: SUMMARY
As part of a program to reduce the flatulence-inducing tendency of soy milk, a method for the enzymatic removal of galacto-oligosaccharides by means of an enzyme preparation from Aspergillus saitoi was investigated. It was found that a partially purified preparation possessing both α-galactosidase and invertase, yet free from protease, could be obtained easily from a commercial A. saitoi acid-protease product by means of a simple molecular sieving procedure. The α-galactosidase exhibited its optimum pH between 5.0 and 5.5, and seemed to be stable between pH 4.0 and 8.0. The optimum temperature was found at about 55°C; however, the enzyme itself was inactivated by maintaining it at 70°C for 30 min. These properties appeared suitable for the enzymatic treatment of soy milk. p-Chloromercuribenzoate, N-bromosuccinimide, HgCl2, AgNO3 or CuCl2 showed strong inhibitory effects on the enzyme. The presence of 1 x 10-2M galactose caused only slight inhibition. Km value of the enzyme with melibiose as a substrate was found to be 3.11 x 10-3M and the molecular weight of the enzyme estimated to be about 290,000 on the basis of a gel filtration technique. Investigations by means of thin-layer chromatography indicated that the addition of small amounts of this enzyme preparation to soy milk resulted in complete hydrolysis of galacto-oligosaccharides. The practicability of the present method was also discussed from an economic viewpoint.
TL;DR: The results suggest that the mechanism of DNA digestion by the enzyme appears to be the one-by-one type, which is similar to that observed with pancreatic DNase.
TL;DR: Two separate enzymes are probably involved in the hydroxylation of proline and in protocollagen, according to the results, which suggests that free sulphydryl groups are required for the activity of the enzyme.
TL;DR: O-Diphenol oxidase from potato tubers was isolated by a new approach that avoids the browning due to autoxidation as mentioned in this paper, which is not attacked by ribonuclease.
Abstract: 1. o-Diphenol oxidase was isolated from potato tubers by a new approach that avoids the browning due to autoxidation. 2. There are at least three forms of the enzyme, of different molecular weights. The major form, of highest molecular weight, was separated from the others in good yield and with high specific activity by gel filtration through Bio-Gel P-300. 3. The major form is homogeneous by disc electrophoresis but regenerates small amounts of the species of lower molecular weight, as shown by rechromatography on Bio-Gel P-300. 4. There is an equal amount of RNA and protein by weight in the fully active enzyme. The RNA cannot be removed without loss of activity, and is not attacked by ribonuclease. 5. The pH optimum of the enzyme is at pH5.0 when assayed with 4-methylcatechol as substrate. It is ten times more active with this substrate than with chlorogenic acid or catechol. The enzyme is fully active in 4m-urea. 6. A minimal molecular weight of 36000 is indicated by copper content and amino acid analysis of the protein component of the enzyme. 7. The protein contains five half-cystinyl residues per 36000 daltons, a value similar to that found in o-diphenol oxidase from mushrooms. It also contains tyrosine residues although, when pure, it does not turn brown by autoxidation.
TL;DR: The presence of this enzyme and of C55-isoprenoid alcohol in S. aureus accounts for the stimulation by ATP of peptidoglycan synthesis catalyzed by particulate enzyme from this organism.
TL;DR: The low activity of the modified enzyme was found to be due to a powerful substrate inhibition which is decreased in the presence of SO 4 2- and a reaction mechanism which explains the anomalous kinetics of sulphatase A has been proposed.