Showing papers on "Substrate (chemistry) published in 1978"

Multilayer film elements for clinical analysis: applications to representative chemical determinations.

Abstract: Using the general concept of a dry multilayer analytical element, we can change chemical procedures and configurations to assay several blood components. In the assay of serum urea nitrogen, urease in the reagent layer catalyzes the hydrolysis of urea. A semipermeable membrane excludes aqueous base, but allows ammonia to diffuse to an underlying indicator layer. For the amylase determination, the enzyme hydrolyzes a dyed-starch substrate coated on top of the spreading layer; this produces small fragments, which diffuse to a registration layer. The increase of absorbance at 540 nm is correlated with amylase activity. Bilirubin complexes with a cationic polymer at the interface between the spreading and reagent layers. The direct reading at 460 nm allows determination of total bilirubin in the range 1 to 500 mg/liter. Tirglycerides are hydrolyzed in the spreading layer, and the resulting soluble glycerol readily diffuses into the reagent layer, where it is phosphorylated and subsequently oxidized by glycerophosphate oxidase to yield dihydroxyacetone phosphate and hydrogen peroxide. Peroxidase catalyzes production of a color commensurate with the hydrogen peroxide produced.

Cellobiose Oxidase, Purification and Partial Characterization of a Hemoprotein from Sporotrichum pulverulentum

Abstract: An extracellular enzyme which utilizes molecular oxygen to oxidize cellodextrins to the corresponding aldonic acids has been isolated from culture filtrates of the white-rot fungus Sporotrichum pulverulentum. This enzyme, tentatively named cellobiose oxidase, has been highly purified by classical techniques and has been demonstrated to be a glycoprotein with a molecular weight of approximately 93000. Ultraviolet spectra of the enzyme in the presence and absence of substrate are characteristic of a hemoprotein. Acidic hydrolyses of the enzyme followed by a spectrofluorimetric investigation of the hydrolysate has demonstrated the presence of approximately one flavin component per enzyme molecule. The possible role of this complex enzyme in cellulose degradation is discussed.

P-Nitrophenol-alpha-D-glucopyranoside as substrate for measurement of maltase activity in human semen.

Abstract: Hitherto, seminal plasma maltase has been measured with maltose as substrate; this method is time consuming and lacks specificity. The use of a synthetic substrate, p-nitrophenol-alpha-D-glucopyranoside, allows accurate and rapid determination of this activity. When maltase is added to the incubation medium (the substrate and reduced glutathione in potassium phosphate buffer, pH 6.8), maintained at 37 degrees C, hydrolysis of the original substrate to p-nitrophenol goes at a constant rate during 4 h. Under optimal conditions of incubation, the Michaelis constant of the reaction, calculated by the Hanes method, was 2.92 +/- 0.84 (SD) X 10(-3) for six different semen samples. Isomaltase appeared to be absent from seminal plasma. The enzyme is stable to freezing and slow thawing and can be stored for at least 26 days at -80 degrees C. Its molecular weight is 259 000. Tris(hydroxymethyl)aminomethane (pH 6.8) exerts a noncompetitive inhibition on the enzyme activity. In 68 men 23 to 45 years old, whose semen analyses were normal, the seminal plasma maltase activity was 467 +/- 135 (SD) mU/g of protein. It was generally decreased in patients with infertility disorders.

The Mechanism of Substrate Activation of Pyruvate Decarboxylase: A First Approach

Abstract: The sigmoidal shape of the curve for v[S], characteristic of pyruvate decarboxylase, indicates that the catalytic activity of this enzyme is regulated by the substrate. The enzyme, which is inactive in the absence of its substrate, is activated not only by 2-oxo acids but also by 2-oxo acid amides, which cannot act as a substrate of the enzyme. Whilst the dissociation constant of the enzyme-activator complex depends on the electrophilic nature of the carbonyl group, the catalytic activity reached at saturation concentrations of the activator species is independent of the structure of the activator molecules. The mechanism of activation which proceeds via two reversible steps could be evaluated exactly by stopped-flow techniques. The kinetic parameters of the activation and deactivation reaction were estimated and the validity of the equations derived which describe the activation kinetics could be proved by comparing them with the measured data. Using glyoxylic acid as an irreversibly binding active-site marker, it could be shown that addition of the substrate to the enzyme-bound thiamin diphosphate is the step of the catalytic mechanism whose rate is controlled by the substrate (activator) molecule.

Analysis of double‐substrate limited growth

Abstract: Summary Mathematical models which relate the growth rate of a microorganism to a single limiting substrate concentration have long been established. In recent years, it has become apparent that, under certain conditions, the growth rate of an organism may be simultaneously limited by two or more substrates. Mathematical models of double-substrate limitation fall into two categories: interactive and noninteractive models. A discussion of both types of models is presented in both conceptual and mathematical terms. An analogous case of an enzyme which requires two different substrates to produce a single product is presented. This enzyme analog indicates that both types of double-substrate limitation models appear to be feasible under certain conditions. Based upon stoichiometry and specific growth rate-substrate concentration contour plots, a method for determining the operational conditions which will lead to double-substrate limitation is presented.

Enzyme deactivation during cellulose hydrolysis

Abstract: Hydrolysis of cellulose by Trichoderma viride cellulase reached a plateau after some 25 hr. If the initial enzyme-to-substrate ratio was low, resuspension of substrate in fresh enzyme or addition of enzyme resulted in further high rate hydrolysis. This did not occur if the initial ratio was high. Over 75% hydrolysis might be achieved in the former case, while less than 60% in the latter. A model postulating inactivation of adsorbed enzyme–substrate complex which blocked further hydrolysis was proposed, and it was found to fit the data well. The proposed model had five parameters, four of which could be checked by graphical methods, and all of which had physical meanings. The parameters were estimated by a nonlinear least-squares minimization FORTRAN computer program, using numerical integration and optimization of the parameters. The model was used to predict the resuspension data, powdered enzyme addition data, cellobiose addition data, and cellulose addition data; the deviations from the model are discussed. It was found that average values could be used for four out of the five parameters, while the fifth (initial enzyme concentration) did not correlate with independent measurements such as the filter paper activity or protein concentration.

Carbamoylphosphate synthetase I of rat-liver mitochondria. Purification, properties, and polypeptide molecular weight.

Abstract: An improved purification of carbamoylphosphate synthetase I from rat liver mitochondria is described. The enzyme is essentially homogeneous with enhanced specific activity and stability. The enzyme is stable at alkaline pH in concentrated solution of salts. Kinetic studies indicate two type of inactivation, reversible and irreversible, depending on pH. The monomeric unit of carbamoylphosphate synthetase I consists of one polypeptide chain. The protein migrates in dodecylsulfate/polyaacrylamide gel as a single component corresponding to a molecular weight of about 155000. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M guanidine hydrochloride containing dithioerythritol is 158000 ± 5000. The weight-average molecular weight of the native enzyme, 188000, suggests that the monomeric form is the predominant form of the enzyme, under the conditions described. Glutamine is inactive as a substrate. With ammonium, carbamoyl phosphate synthesis is optimal in the pH range 6.8–7.6. Mg2+ and Mn2+ are equally effective metal ions in the direction of carbamoyl phosphate synthesis, but excess Mn2+ is inhibitory. At less than saturating concentrations of substrate, considerable activation of the enzyme is observed when metal ions are in excess of ATP; when MgATP2− concentration is held constant at 1 mM and 10 mM, plots of v versus free Mg2+ concentration are hyperbolic, and extrapolate to zero. These results suggests that free divalent metal ion is required in the forward reaction. The apparent Km of the substrates is similar to other preparations of the enzyme. The determination of the maximal activity of carbamoylphosphate synthetase in rat liver mitochondria is reported, providing a measure of the total enzyme concentration that is about 1–1.5 mM. The purification procedure described also provides an easy method to obtain ornithine transcarbamylase in partially purified form.

A study of the mechanism of action of Taka-amylase A1 on linear oligosaccharides by product analysis and computer simulation

Abstract: The action pattern and mechanism of the Taka-amylase A-catalyzed reaction were studied quantitatively and kinetically by product analysis, using a series of maltooligosaccharides from maltotriose (G3) to maltoheptaose (G7) labeled at the reducing end with 14C-glucose. A marked concentration dependency of the product distribution from the end-labeled oligosaccharides was found, Especially with G3 and G4 as substrates. The relative cleavage frequency at the first glycosidic bond counting from the nonreducing end of the substrate increases with increasing substrate concentration. Further product analyses with unlabeled and end-labeled G3 as substrates yielded the following findings: 1) Maltose is produced in much greater yield than glucose from unlabeled G3 at high concentration (73 mM). 2) Maltooligosaccharides higher than the starting substrate were found in the hydrolysate of labeled G3. 3) Nonreducing end-labeled maltose (G-G), which is a specific product of condensation, was found to amount to only about 4% of the total labeled maltose. Based on these findings, it was concluded that transglycosylation plays a significant role in the reaction at high concentrations of G3, although the contribution of condensation cannot be ignored. A new method for evaluating subsite affinities is proposed; it is based on the combination of the kinetic parameter (ko/Km) and the bond-cleavage distribution at a sufficiently low substrate concentration, where transglycosylation and condensation can be ignored. This method was applied to evaluate the subsite affinities of Taka-amylase A. Based on a reaction scheme which involves hydrolysis, transglycosylation and condensation, the time courses of the formation of various products were simulated, using the Runge-Kutta-Gill method. Good agreement with the experimental results was obtained.

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.

Principles of Enzymatic Analysis

Abstract: Publisher Summary This chapter presents an overview of the principles of enzymatic analysis. The value of enzymes in analysis lies in their ability to react specifically with individual components of a mixture. The chapter highlights that the concentration of a substance which takes part in an enzymatic reaction can be determined in two ways: one, by physical, chemical, or enzymatic analysis of the product or unreacted starting material after completion of the reaction catalyzed by the enzyme; and two, from the rate of the enzyme reaction, which depends on the concentration of the substrate, cofactor, activator or inhibitor. The two methods are basically different. In the first case, the reaction should be completed as rapidly as possible. Relatively large amounts of enzyme and relatively small amounts of substrate are used. The measured values should be easily readable, not too small and not too large. In the second case, the substrate and enzyme concentrations are so arranged that the rate of the reaction, that is, the amount of substrate reacting per unit time, is not too fast and so can be measured accurately.

New substrates for the radioassay of angiotensin converting enzyme of endothelial cells in culture.

Abstract: To develop means of measuring angiotensin converting enzyme of endothelial cells in culture, we have synthesized benzoyl-Phe-Ala-Pro-OH (I), benzoyl-Pro-Phe-Arg-OH (II) and benzoyl-Gly-His-Leu-OH (III), each bearing a 3 H-atom on the para-position of its benzoyl moiety All three of the acylated tripeptides are substrates for the enzyme Substrate I exhibits the lowest Km (12·5 µ M) and yields the most sensitive assay: the enzyme of 10 6 cells can be measured in a 30 min incubation at 37°C Radiolabelled reaction product is separated from substrate by extraction of acidified reaction mixture with an organic solvent, and the rate of formation of product can be quantified by liquid scintillation counting of the organic phase Substrate III can also be used to measure angiotensin converting enzyme of cells but requires longer incubations (180-240 min) and high salt concentrations (0·75 M Na 2 SO 4 ) Substrate II is not specific: it is hydrolyzed by more than one enzyme of endothelial cells

Comparison of the molybdenum centres of native and desulpho xanthine oxidase. The nature of the cyanide-labile sulphur atom and the nature of the proton-accepting group.

Abstract: The non-functional form of xanthine oxidase known as the desulpho enzyme was compared with the functional enzyme in various ways, to obtain information on the structure of the molybdenum centre and the mechanism of the catalytic reaction. The desulpho enzyme, like the functional one, possesses a site for the binding of anions, presumably as ligands of molybdenum. Evidence is presented that in the Mo(V) e.p.r. signal from the desulpho-enzyme, as in that from the functional enzyme, a weakly coupled proton, in addition to a strongly coupled proton, interacts with the metal. Measurements were carried out by e.p.r. on the rate at which the proton strongly coupled to molybdenum exchanged, on diluting enzyme samples with 2H2O. For the desulpho enzyme the exchange rate constant was 0.40s-1, at pH 8.2 and 12 degrees C, and for the functional enzyme it was 85 s-1. It is shown that the great majority of reported differences between the enzyme forms are consistent with functional enzyme containing an (Enzyme)-Mo=S grouping, replaced in the desulpho form by (Enzyme)-Mo=O. Protonation of these groups, with pK values of about 8 and 10 respectively, would give (Enzyme)-Mo-SH and (Enzyme)-Mo-OH, these being the forms observed by e.p.r. The accepting group in the functional enzyme, for the proton transferred from the substrate while molybdenum is reduced in the catalytic reaction [Gutteridge, Tanner & Bray (1978) Biochem J. 175 869-878], is thus taken to be Mo=S.

The steady-state kinetics of yeast phosphoglycerate kinase. Anomalous kinetic plots and the effects of salts on activity.

Abstract: 1 A re-investigation of the kinetics of yeast phosphoglycerate kinase in the direction of 1,3-bis-phosphoglycerate formation has been carried out, covering a 1000-fold range in substrate concentrations. A variety of improved spectrophotometric and fluorimetric assay procedures have been used. 2 Kinetic plots proved to be non-linear for each variable substrate. A variety of checks have been carried out to show that this is not due to artifacts in the assay procedures or heterogeneity of the enzyme preparation. 3 The effects of a variety of salts on the activity of the enzyme have been examined. Most salts, especially those with multivalent anions, can cause activation of the enzyme, but inhibit at high concentration. 4 The salt effect is shown to be principally due to anions rather than cations, and not to ionic strength changes. Sulphate, as one of the most effective anions has been used in most comparisons. 5 Salt activation is steepest when the substrate concentrations are low; maximum activation has been about 5-fold with 0.2 mM MgATP and 0.2 mM 3-phosphoglycerate. Inhibition at the higher salt concentrations is strongest at the same substrate concentrations as when activation is steepest, indicating a link between the two effects. 6 The presence of 20 mM or more Na2SO4 converted non-linear kinetic plots to linear ones. A study of the kinetics in the presence of 40 mM Na2SO4 was interpreted in terms of a random sequential binding mechanism, with sulphate acting as a competitive inhibitor. 7 Possible explanations for these anomalous results are discussed in terms of several mechanisms which have been shown to apply in other systems.

GABA-transaminases of human brain and peripheral tissues--kinetic and molecular properties.

Abstract: — Kinetic experiments with 4-aminobutyrate-2-ketoglutarate transaminase (GABA-T), partially purified from human brain tissue, supported a Bi Bi Ping-Pong type of enzyme mechanism in which the enzyme oscillates between forms bound to pyridoxal phosphate and pyridoxamine phosphate. Extrapolated Km values were 0.31 mm for γ-aminobutyrate, 0.16 mm for α-ketoglutarate, and 3.8 μm for pyridoxal phosphate. Very similar kinetic parameters were observed with rat brain enzyme. Apparent molecular weight of human GABA-T by gel filtration was 70,000 ± 3000. Electrofucusing experiments indicated a single ionic form with isoelectric pH = 5.7. Enzyme activity was inhibited by Tris, halides, cadmium and cupric ions, and known GABA-T inhibitors. GABA-transaminating enzymes isolated from human kidney and liver were found to be similar to the brain enzyme with respect to substrate affinities, cofactor requirements, isoelectric pH values, molecular weights, and response to inhibitors.

Enzymatic Modification of Aminoglycoside Antibiotics: 3-N-Acetyltransferase with Broad Specificity that Determines Resistance to the Novel Aminoglycoside Apramycin

Abstract: Examination of a number of R-plasmid-containing bacterial isolates of animal origin has revealed the presence of a new aminoglycoside acetyltransferase (3- N ) with a broad substrate range that includes all the disubstituted 2-deoxystreptamine antibiotics and also the novel monosubstituted antibiotic apramycin. Antibiotic derivatives acylated with hydroxyaminobutyric acid at the 1-amino position were not modified by the enzyme.

An intramolecularly quenched fluorescent tripeptide as a fluorogenic substrate of angiotensin-I-converting enzyme and of bacterial dipeptidyl carboxypeptidase.

Abstract: The N-acyltripeptide 2-aminobenzoylglycyl-p-nitrophenylalanylproline was synthesized and applied as a substrate in the assay of angiotensin-I-converting enzyme from calf lung and human serum, and of the bacterial dipeptidyl carboxypeptidase from Escherichia coli. This compound belongs to a new class of substrates for proteolytic enzymes, having the general structure F–X–Q in which fluorescence of group F is quenched by intramolecular interaction with the group Q. Enzymatic cleavage of the peptide chain (X stands for one or more amino acid resiudes) generates the unquenched F-containing derivative and the resulting fluorescence is used for quantitative measurement of the hydrolysis rate. Cleavage of the Gly-Phe(NO2) peptide bond in the weakly fluorescent 2-amino-benzoylglycyl-p-nitrophenylalanylproline molecule results in appearance of the 71 times higher fluorescence (λmax= 415 nm) of 2-aminobenzoylglycine. Continuous recording of the rising fluorescence allows convenient, sensitive and specific determination of the enzymatic activity, applicable to crude enzyme preparations and human serum. The activity of the mammalian enzyme, measured by this method, is enhanced by Cl− ions and inhibited by low concentrations of EDTA and [Asn1, Val5]anmgiotenmsin II. Kinetic measurements showed Michaelis-Menten behavior, Km= 0.21 ± 0.1 mM and 0.16 ± 0.1 mM for the falf lung and the bacterial enzyme respectively.

The distribution of calcium-dependent phosphatidylinositol-specific phosphodiesterase in rat brain.

Abstract: — The properties of Ca2+-dependent phosphatidylinositol-phosphodiesterase in membrane fractions and supernatants prepared from rat brain have been examined with the aim of providing firm evidence for the existence of a membrane-bound activity distinct from the soluble enzyme found in the cytosol (EC 3.1.4.10). The soluble enzyme is either stimulated or inhibited at pH 7.0 by deoxycholate depending on the ratio of detergent to substrate. The effects of deoxycholate are pH dependent and result in a shift of the enzyme optimum to a higher pH if the enzyme is assayed in the presence of deoxycholate. The soluble enzyme cannot hydrolgse membrane-bound phosphatidylinositol (in 32P-labelled rat liver microsomes) unless deoxycholate is present. The pH optimum is 6.7 for this detergent-dependent hydrolysis and this is probably dependent on the ionization of deoxycholic acid. The lactate dehydrogenase (EC 1.1.1.27) content of rat brain membrane fractions has been measured to estimate the contamination of these fractions by supernatant phosphatidylinositol-phosphodiesterase. No evidence has been found for phosphatidylinositol-phosphodiesterase activities that cannot be explained by such contamination. It is concluded that all the properties of calcium-dependent phospha-tidylinositol-phosphodicsterase in rat brain can be explained by the existence of only the solublc cyto-plasmic enzyme: no evidence confirming a distinct membrane-bound activity has been obtained.

Rat liver cysteine dioxygenase (cysteine oxidase). Further purification, characterization, and analysis of the activation and inactivation.

Abstract: Rat liver cysteine dioxygenase has been purified to homogeneity. It is a single subunit protein having a molecular weight of 22,500 +/- 1,000, with a pI of 5.5. The enzyme purified was catalytically inactive and activated by anaerobic incubation with either L-cysteine or its analogues such as carboxymethyl-L-cysteine, carboxyethyl-L-cysteine, S-methyl-L-cysteine, D-cysteine, cysteamine, N-acetyl-L-cysteine, and DL-homocysteine. The enzyme thus activated with L-cysteine was rapidly inactivated under aerobic condition. This rapid inactivation was observed at 0 degrees C where no formation of either the reaction product cysteine sulfinate or the autoxidation product of cysteine, cystine, was detected. Further analysis shows that the inactivation of the activated enzyme was due to oxygen but unrelated to either the presence of substrate, enzyme turnover or accumulation of inhibitor produced during assay. A distinct rat liver cytoplasmic protein, called protein-A, could completely prevented the enzyme from the aerobic inactivation. The loss of activity during assay in the absence of protein-A was shown to be a first order decay process. From the plots of log(deltaproduct/min) versus time, the initial velocity (VO) and the velocity at 7 min (V7) were obtained. The apparent Km value for L-cysteine in the absence of protein-A was calculated from the initial velocity as 4.5 X 10(-4)M. Protein-A did not alter the apparent Km value for L-cysteine. The chelating agents such as o-phenanthroline, alpha,alpha'-dipyridyl, bathophenanthroline, 8-hydroxyquinoline, EGTA, and EDTA strongly inhibited the enzyme activity when these chelating agents were added before preactivation. The purified cystein dioxygenase contains 1 atom of iron per mol of enzyme protein. By the activation procedure, the enzyme became less susceptible to the heat denaturation, the inhibitory effects of chelating agents and the tryptic digestion.

Purification and Properties of 5′‐Nucleotidase from Lymphocyte Plasma Membranes

Abstract: 5′-Nucleotidase is purified from lymphocyte plasma membranes by teo affinity chromatog-raphies. The first one, on Lens culinaris lectin-Sepharose 4B yields a fraction of twelve lectin-binding alyvoproteins (lectin-receptor fractin). The second one on 5′-AMP-Sepharose 4B leads to pure enzyme. This enzyme is a glycoprotein with a molecular weigtht of 30000; it gives a single band in polyacrylamide/dodecylsulfte electrophoresis and displays a very high specific activity (2500–3000 μmol Pi h−1 mg−1). Some propertes of purified 5′-nucleotidase are similar to those of membrance-bound enzyme: substrate speciticity, temperature dependence, effects of ions and SH-blocking reagents. Others are completely differnt for the teo systems and these differences result form an interaction between the enzyme molecule and other Lens culinarid lectin binding proteins.