Showing papers on "Substrate (chemistry) published in 1981"

Assays for differentiation of glutathione S-transferases.

Abstract: Publisher Summary This chapter provides the spectrophotometric, titrimetric, nitrite, and cyanide assay for the differentiation of glutathione S-transferases. Spectrophotometric assays depend upon a direct change in the absorbance of the substrate when it is conjugated with glutathione (GSH). Because each of the reactions is catalyzed at a finite rate in the absence of enzyme, care is needed to reduce nonenzymatic catalysis by minimizing substrate concentrations and by decreasing pH wherever necessary. Titrimetric assay is based on the principle that the conjugation of alkyl halides with GSH can be measured titrimetrically. Although acid production accompanies many of the transferase catalyzed reactions in which thioethers are formed, titrimetry is only used when more convenient assays are not available. Nitrite assay is based on the principle that nitrite is released when GSH reacts with nitroalkanes or with organic nitrate esters. The nitrite can be assayed as the limiting factor in a diazotization reaction with sulfanilamide that produces a readily quantitatable pink dye. Cyanide assay is based on the fact that when glutathione transferases catalyze the attack of the glutathione thiolate ion on the electrophilic sulfur atom of several organic thiocyanates, it results in the formation of an asymmetric glutathionyl disulfide and cyanide. Cyanide can be readily quantitated by a calorimetric method.

Micellar solubilization of biopolymers in organic solvents. 5. Activity and conformation of .alpha.-chymotrypsin in isooctane-AOT reverse micelles

Abstract: The enzymatic activity of ..cap alpha..-chymotrypsin solubilized in reverse micelles formed in isooctane by bis(2-ethylhexyl)sodium sulfosuccinate and water (0.6 to 2.5% v:v) has been investigated with the use of n-glutaryl-l-phenylalanine p-nitroanilide as the substrate. The enzyme obeys Michaelis-Menten kinetics in the investigated concentration range, with Km values which are considerably higher than those in bulk water (when concentrations are referred to as water pools). Under certain conditions, there is an enhanced turnover number (up to a factor of 6) in micelles with respect to the aqueous solution. The pH profile of the enzyme activity in the hydrocarbon micellar solution is different from that in water, being shifted to higher pH values and the more so the lower the water content. Under conditions of low water content (0.6 to 1% v:v) the enzyme's stability is greater than in aqueous solution. Structure and activity changes are discussed in terms of the size and structure of the micellar aggregate. 29 references.

Colorimetry of angiotensin-I converting enzyme activity in serum.

Abstract: This simple, accurate, and reproducible colorimetric method for determining the activity of angiotensin-I converting enzyme is based upon colorimetry of the quinoneimine dye produced from the substrate p-hydroxyhippuryl-L-histidyl-L-leucine by action of this enzyme through the following series of reactions. The enzyme acts on the substrate to yield p-hydroxyhippuric acid and L-histidyl-L-leucine. The former is then hydrolyzed in the presence of hippuricase to produce p-hydroxybenzoic acid and glycine. Finally, oxidative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine is catalyzed by peroxidase in the presence of hydrogen peroxide, producing a quinoneimine dye, the concentration of which is measured at its absorbance maximum at 505 nm to evaluate the activity of ACE. The Km value for the above-mentioned substrate is 0.32 mmol/L, the optimum pH is 8.3. Results by the present method and Cushman and Cheung's method (Biochem. Pharmacol. 20: 1637, 1971) correlate closely (r = 0.986). The within-run CV is 2.93%.

Inhibition of catechol 2,3-dioxygenase from Pseudomonas putida by 3-chlorocatechol.

Abstract: Partially purified preparations of catechol 2,3-dioxygenase from toluene-grown cells of Pseudomonas putida catalyzed the stoichiometric oxidation of 3-methylcatechol to 2-hydroxy-6-oxohepta-2,4-dienoate. Other substrates oxidized by the enzyme preparation were catechol, 4-methylcatechol, and 4-fluorocatechol. The apparent Michaelis constants for 3-methylcatechol and catechol were 10.6 and 22.0 muM, respectively. Substitution at the 4-position decreases the affinity and activity of the enzyme for the substrate. Catechol 2,3-dioxygenase preparations did not oxidize 3-chlorocatechol. In addition, incubation of the enzyme with 3-chlorocatechol led to inactivation of the enzyme. Kinetic analyses revealed that both 3-chlorocatechol and 4-chlorocatechol were noncompetitive or mixed-type inhibitors of the enzyme. 3-Chlorocatechol (Ki = 0.14 muM) was a more potent inhibitor than 4-chlorocatechol (Ki = 50 muM). The effect of the ion-chelating agents Tiron and o-phenanthrolene were compared with that of 3-chlorocatechol on the inactivation of the enzyme. Each inhibitor appeared to remove iron from the enzyme, since inactive enzyme preparations could be fully reactivated by treatment with ferrous iron and a reducing agent.

Substrate Flux into Biofilms of Any Thickness

Abstract: A kinetic model is developed for determining substrate flux into biofilms with known thicknesses, even though the thickness may be of nonsteady state. The critical contribution of this model is that mass transport resistance is explicity included in order that the flux becomes a direct function of the bulk substrate concentration. Laboratory-scale, column reactors were utilized to culture steady-state biofilms of known thicknesses. Subsequently, the influent flow rate or substrate concentration was changed to determine the short-term kinetics of substrate utilization of biofilms of known thicknesses. The model successfully predicted the column performance, except for cases in which oxygen depletion caused dual-substrate limitation. Removal of substrate to very low levels was only possible during short-term experiments when the biofilm was in net decay.

Multilayer enzyme electrode membrane

Abstract: A contiguous multilayer membrane for use with an electrochemical sensor is prepared comprising a first relatively nonporous dense polymer layer, a second polymer layer less dense and more porous than the first layer, a third layer containing an enzyme and a fourth polymer layer less dense and more porous than the first layer The polymer is preferably cellulose acetate and the second and fourth layers are prepared with a solvent and nonsolvent for the polymer The membrane may contain multiple enzyme layers separated by a polymer layer The multilayer membrane provides advantages of higher substrate conversion, homogeneous distribution of enzyme and/or minimized interference with analyte diffusion

Diphosphatidylglycerol is required for optimal activity of beef heart cytochrome c oxidase

Abstract: Isolated beef heart cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) contains four or five molecules of tightly bound diphosphatidylglycerol per monomer (2-heme complex). This lipid could be removed in part, or wholly, by mixing the enzyme with high concentrations of Triton X-100 and then centrifuging the mixture through a glycerol gradient equilibrated in the same detergent. Cytochrome c oxidase retaining three or more diphosphatidylglycerol molecules per monomer was fully active when assayed in 1-oleoyl lysophosphatidylcholine. Upon removal of one or more of these diphosphatidylglycerols, enzymic activity was lost. Full activation could be obtained by adding diphosphatidylglycerol to the assay mixture along with lysophosphatidylcholine but not by adding phosphatidylcholine or phosphatidylethanolamine. Direct binding experiments, kinetic studies, and previous work using arylazidocytochrome c derivatives [Bisson, R., Jacobs, B. & Capaldi, R. A. (1980) Biochemistry 10, 4173-4178], indicate that diphosphatidylglycerol is involved in binding of substrate cytochrome c to cytochrome c oxidase.

Enzymatic hydrolysis of cellulose: Evaluation of cellulase culture filtrates under use conditions

Abstract: Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 hours. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given time was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme, but if high sugar concentrations are desired, very large quantities of enzyme are required. Since the slopes of these plots varied, the relative activity of cellulase on different substrates may be affected by enzyme concentration. (Refs. 28).

Adhesive substrates for fibronectin

Abstract: In order to promote cell attachment, fibronectin must first undergo activation by a suitable substrate. In this study, 52 materials have been surveyed for their ability (a) to bind fibronectin, (b) to activate the cell-adhesive property of fibronectin, and (c) to support the growth of cells. Many plastics, polysaccharides, metals, and ceramics were found to support cell growth as well as the fibronectin-dependent attachment of cells. Several other substrates have been identified that were inactive in promoting either cell attachment or growth. Hydrophobic substrates were found to be active in fibronectin activation, whereas hydrophilic substrates were found to be inactive. Since fibronectin binds to substrata of extremely varied chemical composition, it is clear that the binding of fibronectin to such substrata is nonspecific in nature. Since protein pretreatment of all substrata, except collagen and poly(L-lysine), abolished the physical binding of fibronectin, the binding of fibronectin to artificial substrata is probably ascribable to a nonspecific hydrophobic protein-substratum interaction. In contrast, several lines of evidence indicate that the interaction between fibronectin and collagen displays biological specificity. Poly(hydroxyethylmethacrylate)(poly(HEMA)), which has previously been shown to be nonadhesive for cells, is demonstrated here to be unique in its inability to bind fibronectin. Addition of one part per million of an adhesive polymer to poly(HEMA) permits fibronectin binding to occur.

Substrate specific galactose oxidase enzyme electrodes

Abstract: The relative specificity of the enzyme galactose oxidase for various substrates is controlled as a function of the electrical potential applied to the enzyme. The enzyme is incorporated into a thin layer, electrochemical cell laminate (40) having exterior membrane layers (42-44,42'-44') and an interior enzyme layer (46). An electrode (48) located within the enzyme layer applies varying electrical potentials to the enzyme. An intermediate electron transfer agent may be used to transfer electrons to and from the enzyme and electrode.

Mechanistic studies on the pyridoxal phosphate enzyme 1-aminocyclopropane-1-carboxylate deaminase from Pseudomonas sp

Abstract: The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACPC deaminase) from a pseudomonad is a pyridoxal phosphate (PLP) linked catalyst which fragments the cyclopropane substrate to alpha-ketobutyrate and ammonia [Honma, M., & Shimomura, T. (1978) Agric. Biol. Chem. 42, 1825]. Enzymatic incubations in D2O yield alpha-ketobutyrate with one deuterium at the C-4 methyl group and one deuterium at one of the C-3 prochiral methylene hydrogens. Stereochemical analysis of the location of the C-3 deuteron was accomplished by in situ enzymatic reduction to (2S)-2-hydroxybutyrate with L-lactate dehydrogenase and conversion to the phenacyl ester. The C-3 hydrogens of the (2S)-2-hydroxybutyryl moiety are fully resolved in a 250-MHz NMR spectrum. Absolute assignment of 3S and 3R loci was obtained with phenacyl (2S,3S)-2-hydroxy[3-2H]butyrate generated enzymatically by D-serine dehydratase action on D-threonine. ACPC deaminase shows a stereoselective outcome with a 3R:3S deuterated product ratio of 72:28. 2-Vinyl-ACPC is also a fragmentation substrate with exclusive regiospecific cleavage to yield the straight-chain keto acid product 2-keto-5-hexenoate. The D isomer of vinylglycine is processed to alpha-ketobutyrate and ammonia at 8% the Vmax of ACPC, while L-vinylglycine is not a substrate. It is likely that ACPC and D-vinylglycine yield a common intermediate--the vinylglycine-PLP-p-quinoid adduct--which is then protonated sequentially at C-4 and then C-3 to account for the observed deuterium incorporation. The D isomers of beta-substituted alanines (fluoroalanine, chloroalanine, and O-acetyl-D-serine) partition between catalytic elimination and enzyme inactivation. Each shows a different partition ratio, arguing against the common aminoacrylyl-PLP as the inactivating species.

Kinetic and optical spectroscopic studies on the purple acid phosphatase from beef spleen

Abstract: A new purification scheme has been developed for the purple acid phosphatase from beef spleen; typical yields are 8 mg of homogeneous enzyme per kg of spleen in only five steps. Kinetics studies have shown that the enzyme is strongly inhibited by fluoride, phosphate, and [p-(acetylamino)-benzyl]phosphonate, a nonhydrolyzable substrate analogue; the last two of these show simple competitive inhibition. In contrast, cyanide, azide, tartrate, and p-nitrophenol show no inhibition at concentrations up to 10 mM. Molecular weight estimations by gel electrophoresis and gel permeation chromatography give a value of 40 000 for the native enzyme, which is shown to consist of two subunits of apparent molecular weight 24 000 and 15 000. Careful metal analyses indicate the presence of 2.1 +/- 0.1 iron atoms per enzyme molecule, and less than 0.1 copper, zinc, nickel, or manganese atom per enzyme. The purple enzyme (lambda max 550 nm) is reversibly converted to a pink, active form (lambda max 505 nm) upon treatment with mild reducing agents (dithioerythritol or ascorbate). Addition of competitive inhibitors to the pink form causes rapid reversion to the purple form. Electron paramagnetic resonance spectroscopy at several temperatures showed only weak g = 4.3 signals (less than 0.1 spin/molecule) for the native, reduced, and inhibited forms of the enzyme.

Chemically modified electrodes for electrocatalysis

Abstract: At a modified electrode, electrocatalysis is accomplished by an immobilized redox substance acting as an electron transfer mediator between the electrode and a reaction substrate. Such mediated electrocatalysis is possible with monomolecular and multimolecular layers of the redox substance. The electron transfer mediation can assume several special forms; these are identified and experimental examples are given. The differences between electrocatalytic behaviour of monomolecular and multimolecular layers are discussed; electrocatalysis in the latter circumstance can include reaction rate elements of electrochemical charge and substrate migration through the multilayer in addition to the chemical rate. Theoretical ideas are presented that interconnect these three rate elements, to show that either all of the multilayer sites can participate in the electrocatalytic reaction, or only about the equivalent of a monolayer, depending on the relative rates of the electrochemical charge transport, the diffusion of substrate, and the chemical reaction rate.

Purification and properties of the insulin-stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes.

Abstract: The peripheral high-affinity cyclic AMP phosphodiesterase from rat liver plasma membranes was purified to apparent homogeneity. The procedure used involved the initial purification of liver plasma membranes and the solubilization of the enzyme by using a high-ionic-strength medium. This was followed by chromatography of the enzyme on DEAE-cellulose, Affi-Gel Blue, a novel affinity column and Sephadex G-100. A 9500-fold purification of the enzyme with a 24% yield was achieved by this procedure. The purified enzyme was apparently monomeric (Mr 52000) as it exhibited identical molecular weights on analysis by gel filtration, sedimentation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It is suggested that the non-Michaelis kinetics exhibited by the enzyme are due to it obeying a mnemonical mechanism, where it displays Km 0.7 micrometer, Vmax. 9.1 units/mg of protein and Hill coefficient (h) 0.62. Cyclic GMP acts as a poor substrate for the enzyme, with Km 120 micrometer and Vmax. 0.4 unit/mg of protein, and also as an inhibitor of the enzyme, with I50 (concentration giving 50% inhibition) 150 micrometer when assayed at 0.4 micrometer-cyclic AMP. Inhibition by 5'-AMP is unlikely to be of physiological importance, as it is only a weak inhibitor of the enzyme (I50 47 mM assayed at 0.4 micrometer-cyclic AMP).

Purification and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosus.

Abstract: Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) was purified from the culture filtrates of the thermophilic fungus Thermomyces lanuginosus and was established to be homogeneous by a number of criteria. The enzyme was a glycoprotein with an average molecular weight of about 57 000 and a carbohydrate content of 10-12%. The enzyme hydrolysed successive glucose residues from the non-reducing ends of the starch molecule. It did not exhibit any glucosyltransferase activity. The enzyme appeared to hydrolyse maltotriose by the multi-chain mechanism. The enzyme was unable to hydrolyse 1,6-alpha-D-glucosidic linkages of isomaltose and dextran. It was optimally active at 70 degrees C. The enzyme exhibited increase in the Vmax. and decreased in Km values with increasing chain length of the substrate molecule. The enzyme was inhibited by the substrate analogue D-glucono-delta-lactone in a non-competitive manner. The enzyme inhibited remarkable resistance towards chemical and thermal denaturation.

Method for enzymatic transesterification of lipid and enzyme used therein

Abstract: A method for the enzymatic transesterification useful for modification of a lipid, which comprises continuously or repeatedly contacting an enzyme or an enzyme preparation having transesterification activities with a fresh supply of a dried fatty ester substrate such as fats and oils of glycerides. The enzyme or the enzyme preparation has transesterification activities (Kr value) of 0.01 or more as well as lipolytic activities. The enzyme preparation is prepared by dispersing, adsorbing or bonding an enzyme having lipolytic activities in or to a carrier and drying the resulting mixture at an adequately slow initial drying rate to activate or increase the transesterification activities of the enzyme.