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PubMed1976

Substrate-specific enzyme variation in natural populations of Drosophila pseudoobscura.

By using a number of different alcohols as substrates, eight alcohol dehydrogenase loci were discovered in Drosophila pseudoobscura. Each of these loci can take more than one substrate. Several of these loci differed in their tissue specificities and activity patterns during development. The genic variation in natural populations was studied at four of these loci and three of them were polymorphic. A quantitative study of substrate-specific differences among alleles of the same locus produced negative results. This result appears to be typical of most studies done on this aspect. From this it was concluded that the substrate specificity of enzymes is not an important factor in determining the greater amount of genic variation at Group II loci than at Group I loci, as proposed by KOJIMA, GILLESPIE and TOBARI (1970). There are several observations which suggest a different explanation for the differences in the genic variability at Group I and Group II loci: (1) There are, on an average, more isozyme loci (loci with similar substrate specificity) for enzymes in Group II than in Group I; (2) The null alleles are far more common at Group II loci than at Group I loci; (3) There is significant heterogeneity in the number of alleles and the heterozygosities at loci within each of these two groups of enzymes; (4) Relatively higher levels of genic variation are observed at Group II loci even in populations which appear to be living in homogeneous environments; and (5) Some loci (e.g. esterases) are highly polymorphic in most species investigated by gel electrophoresis techniques. Based on these general observations, it is proposed that (1) the substrate-specific differences are between isozyme loci and not between alleles of a given locus, and (2) neutral alleles are proportionately far more common at loci at Group II than at loci in Group I, because the former is under less selection constraint than the latter.

Alcohol Oxidoreductases↗
PubMed1979

Substrate specificity of monoamine oxidase in pig liver mitochondria.

In pig liver both the A and the B form of monoamine oxidase (MAO) were found to be responsible for the oxidation of 5-hydroxytryptamine (5HT), a substrate oxidised by the A form alone in most other tissues. With increasing concentrations of this substrate, the percentage of the substrate oxidised by the B form increased. The Km value of the A and the B form of MAO for 5HT was 200 microns and 2.2 mM, respectively. It is suggested that the division of the monoamines into A and B form substrates should be done on the basis of the molecular turnover numbers rather than on their activities, and that the substrate specificities of the two forms of MAO should be determined over a large range of substrate concentrations.

Animals↗
PubMed1978

[Characterization of a protease from Thermoactinomyces vulgaris (thermitase). 3. Substrate specificity and properties of partially purified thermitase].

During the process of cultivation of Th. vulgaris several proteases are formed. In the present investigation the extensively purified major component was used. The substrate specificity was determined by means of 7 proteins, 7 amino acid esters, 5 fatty acid esters and 15 amino acid 4-nitroanilides. Among the protein substrates tested, urea denaturated hemoglobin was split best, followed by gelatin, casein, field bean protein, serum albumin and gluten. The weakest rate of hydrolysis was observed with elastin. In contrast to this acetyl-(L-ala)3-methylester, that is a substrate for elastase, was split best from all the esters tested. Only 8% of this activity could be found with the chymotrypsin substrates acetyl-L-tyr-ethylester and acetyl-L-phe-ethylester and 1% of the above activity with the trypsin substrates tosyl-L-arg-methylester and benzoyl-L-arg-methylester. The fatty acid esters and the p-nitroanilides were hydrolyzed much more slowly. The pH-optimum of thermitase was found in the weakly alkaline region of pH 7 to 9. There were only small differences between the individual high and low molecular substrates. The temperature optimum was between 60 and 75 degrees C for esters and p-nitroanilides as substrates and at 90 degrees C for casein. It should be mentioned that the enzyme was quickly inactivated at temperatures above 70 degrees C.

Hydrogen-Ion Concentration↗
PubMed1977

[Isolation and substrate specificity of neomycin (paromomycin)--phosphotransferase from Actinomyces fradiae, a producer of neomycin].

Neomycin (paromomycin) phosphotransferase was isolated from the mycelium and fermentation broth filtrates of Act. fradiae. The substance was partially purified by means of fractionation with ammonium sulphate followed by gel-filtration through Sefadex G-100. The extracellular and intracellular forms of the enzyme had the same substrate specificity and used only neomycin and paromomycin as substrates. The other aminoglycosides, including kanamycins A and B, lividomycin and ribostamycin were not used. The both forms had the same thermolability. The intracellular form of the enzyme was detected in the mycelium at the early stages of the organism development, while the extracellular form was found in detectable amounts in the culture medium only at the late stages of the actinomycete development. Therefore, the neomycin-producing organism, i.e. Act. fradiae had one enzyme which phosphorilated neomycin and paromomycin and was excreted from the mycellium into the culture medium during the fermentation process.

Adenosine Triphosphate↗
PubMed1979

The substrate specificity of yeast hexokinase: reaction with D-arabinose oxime.

By chromatography, electrophoresis, n.m.r. spectroscopy, and spectrophotometric assay, it has been shown that D-arabinose oxime acts as a weak substrate for yeast hexokinase. The enzyme-catalysed phosphorylation of the oxime, which exists as a mixture of E (80%) and Z (20%) acyclic forms in solution at equilibrium, is proposed to proceed via the transient formation of a furanoid species. Weak substrate-activity was also observed with 4-deoxy-D-xylo-hexose, but not with 5-deoxy-D-xylohexose. The relation of these and previous results concerning the carbohydrate-substrate specificity of yeast hexokinase in solution to X-ray crystallographic studies is discussed.

Arabinose↗
PubMed1975

Effect of fasting on substrate specificity of rat liver UDP-glucuronosyltransferase.

The effect of a 3-day period of complete starvation on the hepatic UDPglucuronosyltransferase activity was studied in the rat. The substrate specificity of the enzyme was assayed with bilirubin as a carboxylic acceptor, and phenolphthalein and p-nitrophenol as phenolic acceptors. Starvation increased the bilirubin UDPglucuronosyltransferase specific activity by 33%, whereas no increase in specific activities appeared when the phenolic substrates were used. However, on a total liver weight basis, all three activities were significantly lower than those of the controls. Kinetic studies of activated microsomal bilirubin UDPglucuronosyltransferase showed that apparent Km values were similar; fasting acted only by increasing V. The results suggest that the changes in bilirubin glucoronosyltransferase activity provoked by starvation may reflect actual enzyme induction; they favour the multiplicity of the UDPglucuronosyltransferase system.

Animals↗
PubMed1979

Modification of the substrate specificity of rat hepatic lipase by collagenase treatment.

Collagenase is currently used in the isolation of rat hepatocytes, but it rapidly inactivates the heparin-releasable triacylglycerol lipase of the liver. Since collagenase-isolated liver cells contain a heparin-releasable monoacylglycerol hydrolase, a study was made on the effect of collagenase treatment on the substrate specificity of purified heparin-releasable lipase of rat liver. Incubation of the purified lipase with collagenase selectively decreased the triacylglycerol lipase activity of the enzyme with no effect on the monoacylglycerol hydrolase activity. Gel filtration of the lipase before and after collagenase treatment indicated cleavage of a small molecular weight fragment from the enzyme. This resulted in a preparation with less triacylglycerol lipase activity but still capable of monoacylglycerol hydrolysis.

Animals↗
PubMed1978

Studies on the substrate specificity of Taka-amylase A1. XIV. Preparation of 6-deoxy-6-halogenomaltotrioses and their hydrolysis by Taka-amylase A.

1. O-6-Deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, O-6-chloro-6-deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, O-6-bromo-6-deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, and O-6-deoxy-6-iodo-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose were prepared, taking advantage of the substrate specificities of Taka-amylase A and glucoamylase, and the action of Taka-amylase A on these substrates was investigated. 2. The Michaelis constant Km and the molecular activity ko were determined at 37 degrees C and pH 5.2 using the modified maltotrioses. The values of Km and ko decreased upon modification of maltotriose and those of ko/Km were in agreement with the comparative initial rates for the corresponding derivatives of phenyl alpha-maltoside at low substrate concentrations. This result suggested that a subsite of the enzyme may have a specific interaction with halogen atoms in the substrate. 3. All halogenomaltotrioses examined showed substrate inhibition at high substrate concentrations.

Amylases↗
PubMed1979

Salmonella phage glycanases: substrate specificity of the phage P22 endo-rhamnosidase.

Interaction between phage P22 and phenol-water extracted lipopolysaccharides from sensitive Salmonella bacteria belonging to serogroups A, B and Di results in hydrolysis of the alpha-L-rhamnosyl linkages within the tetrasaccharide repeating unit of the O-antigenic polysaccharide chain. These O-antigens have identical structures except for the nature of the 3,6-dideoxy-hexosyl group linked to O-3 of the D-mannosyl residue. Removal of the dideoxysugar, or periodate oxidation followed by borohydride reduction of the L-rhamnosyl residue made the O chain resistant to the endo-rhamnosidase. Substitution of the D-galactosyl residue at O-4, but not at O-6, with an alpha-D-glucosyl group was compatible with hydrolysis. A number of Klebsiella pneumoniae and Shigella flexneri lipo- or capsular polysaccharides containing chain L-rhamnosyl residues were tested but none was sensitive to the P22 endo-rhamnosidase. The substrate specificity of the endo-rhamnosidase parallels the lytic specificity of the phage which suggests that the initial step in phage P22 infection is a P22 tail enzyme O-antigen substrate interaction. The main product of the hydrolysate was octa-, dodeca- and hexadecasaccharides. Treatment of phage FO resistant smooth strains of S. typhimurium with P22 tails removed O polysaccharide chains and made previously 'hidden' FO receptors accessible to the phage.

Glycoside Hydrolases↗
PubMed1977

Regulation of the fatty acid composition of alkyl ether phospholipid in Ehrlich ascites tumor cells. The substrate specificities of 1-O-alkylglycerol 3-phosphate and 1-O-alkylglycero-3-phosphocholine acyltransferases.

Activity for the acylation of 1-O-alkyl-GP was found in the microsomes of Ehrlich ascites tumor cells. The reaction product was shown to be 1-O-alkyl-2-acyl-GP by identifying the acetolysis product as 1-O-alkyl-2-acyl-3-acetylglycerol. The acyl transfer activity to 1-O-alkyl-GP was significantly lower than that to 1-acyl-GP. The substrate specificity of 1-O-alkyl-GP acyltransferase was rather broad as regards thiol esters. Similar specificity was observed with 1-acyl-GP acyltransferase. In contrast to these acyltransferase systems, the 1-acyl- and 1-O-alkyl-GPC acyltransferases were specific for polyunsaturated fatty acyl-CoA's. Since a high percentage of polyunsaturated fatty acid and a little palmitic acid were located at the 2-position of 1-O-alkyl-2-acyl-GPC(E), the observed specificities for acyl-COA's of these acyltransferase systems can be considered in relation to the fatty acid composition at the 2-position of 1-O-alkyl-2-acyl-GPC(E) in the cells.

Acyltransferases↗
PubMed1977

Substrate specificities of the two genetically distinct human brain beta-galactosidases.

The two human brain beta-galactosidases were solubilized and fractionated by Sephadex G-200 gel filtration, free from each other. Substrate specificities of the two enzymes were examined for galactosylceramide, lactosyl-[N-stearoyl]ceramide, lactosyl-[N-lignoceroyl]ceramide, galactosyl-N-acetylgalactosaminyl-[N-stearoyl]ceramide, lactosyl-[N-lignoceroyl]ceramide, galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]galactosyl-glucosylceramide (GMI-ganglioside), galactosyl-N-acetylgalactosaminyl-galactosyl-glucosylceramide (asialo GM1-ganglioside), and 4-methylumbelliferyl beta-galactoside. Under appropriately optimized conditions, either of the two beta-galactosidases could hydrolyze all of the substrates, although with widely varying rates. Relative specific activities of galactosylceramide beta-galactosidase toward galactosylceramide, lactosyl-[N-steroyl]ceramide, lactosyl-[N-lignoceroyl]ceramide. GM1-ganglioside, asialo GM1-ganglioside, and 4-methylumbelliferyl beta-galactoside were 100, 510, 250, 39, 41 and 120, respectively. Relative specific activities of GM1-ganglioside beta-galactosidase toward the same series of the substrates were 0.3, 78, 19, 100, 150 and 240; However, the optimal assay conditions for any given natural substrate were sufficiently different for each beta-galactosidase so that diagnostic assays for the two genetic diseases due to beta-galactosidase deficiencies could be carried out in whole tissues. Since the relative distribution of the two enzymes vary greatly in different tissues, contributions by the two enzymes to degradation of the natural glycosphingolipids in vivo may well vary in different organs. These findings may have an important bearing on the biochemical pathogenesis of these genetic disorders.

Adult↗
PubMed1979

Substrate specificity of the human lymphokine leucocyte migration-inhibitory factor (LIF): radioenzymic assay and inhibition by cGMP.

The human lymphokine, leucocyte migration-inhibitory factor (LIF), appears to be a serine esterase and protease by virtue of its susceptibility to the irreversible enzyme inhibitor, phenylmethylsulfonyl fluoride (PMSF), and by the ability of arginine esters and amides to protect LIF against PMSF-induced inactivation. In this paper, three methods are described by which putative substrates for LIF may be investigated. Thus, molecules satisfying the substrate specificities of this lymphokine should (1) protect LIF against inactivation by PMSF, (2) reduce LIF activity in vitro on polymorphonuclear leucocytes, and (3) reduce the esterolytic activity of purified LIF-rich supernatants. The first two reactions were tested by means of the leucocyte migration agarose technique; the third reaction was tested by a sensitive enzyme assay using tritiated tosyl arginine methyl ester as substrate. Guanosine 3',5'-cyclic monophosphoric acid, which is capable of protecting LIF against PMSF-induced inhibition, also inhibited the esterolytic activity of the purified LIF preparation. Four synthetic oligopeptide substrates for trypsin, thombin and plasmin were investigated. Only one, the thrombin- and trypsin-specific benzoyl-phenylalanyl-valyl-agarine-p-nitroanilide, possessed high affinity for the LIF molecule and may therefore prove to be a potent substrate for this lymphokine.

Anilides↗
PubMed1976

Human placental diamine oxidase. Improved purification and characterization of a copper- and manganese-containing amine oxidase with novel substrate specificity.

1. Isoelectric focusing studies of human placental diamine oxidase showed the pI value of the active enzyme to be 6.5. This information was used in modifying the enzyme purification by incorporating column chromatography on DEAE-Sephadex with ionic strength and pH gradient elution and this, together with affinity chromatography on concanavalin A--Sepharose, gave a highly purified preparation, with a specific activity of 7.0 units/mg. 2. The enzyme gave the expected stoicheiometry with p-dimethylaminomethylbenzylamine as substrate (Keq. 2700) and also oxidized [8-arginine]vasopressin, [8-lysine]vasopressin, collagen and tropocollagen. Polyacrylamide gel slices showed identical migration of diamine-oxidizing and [8-lysine]vasopressin-oxidizing activity. 3. The molecular weight, determined by ultracentrifugation, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, variable polyacrylamide-gel electrophoresis and Sephadex G-200 column chromatography, was estimated to be approx. 70000. 4. E.s.r. spectroscopy showed that copper and manganese were present in the purified enzyme. This result was confirmed by atomic absorption spectroscopy, which indicated a stoicheiometry for copper and manganese of approx. 1.0 and 1.2g-atom respectively/70000mol.wt. unit. 5. The e.s.r. spectral intensity did not decrease nor did the spectral line shape change when excess of p-dimethylaminomethylbenzylamine was added to the enzyme. 6. Addition of K13CN to the enzyme eliminated the copper e.s.r. signal without affecting the manganese signal. 7. The placental enzyme therefore appears to differ from other amine oxidases in terms of its metal cofactor requirement, molecular weight and substrate specificity, and possible roles in vivo for this enzyme are discussed.

Amine Oxidase (Copper-Containing)↗
PubMed1976

Phenylalanyl-tRNA and seryl-tRNA synthetases from baker's yeast. Substrate specificity with regard to ATP analogs and mechanism of the aminoacylation reaction.

Eighteen analogs of ATP have been tested in the aminoacylation reaction of phenylalanyl-tRNA and seryl-tRNA synthetases from baker's yeast. Four compounds are substrates for phenylalanyl-tRNA synthetase, five for seryl-tRNA synthetase, one compound is an inhibitor for both enzymes; their Km and Ki and V values have been determined. The substrate specificity shows that for the catalytic action of both enzymes with these substrates positions 6, 7, 8 and 9 of the purine moiety and positions 2' and 3' of the ribose moiety are important.

Adenosine Triphosphate↗
PubMed1975

alpha-Chymotryptic hydrolysis of derivatives of the specific substrates with substituents in the nucleus.

Steady state kinetic studies of alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis of nucleus-substituted derivatives of the specific substrates were made at pH 6.5 and 7.8. Ac-Trp(NCps)-OMe was hydrolyzed more readily than Ac-Trp-OMe owing to its smaller Km value. The kcat values of Ac-Trp(CHO)-OMe and Ac-Tyr(3-no2)-ome were higher than those of the corresponding unmodified substrates, suggesting that derivatives with a substituent as large as a formyl or nitro group at the epsilon-position are stereochemically favorable to the catalytic process. Derivatives of Ac-Phe-OMe with a chain of four atoms at the 3 or 4-position of the phenyl nucleus and 2,3-dihydropyrrolo[2,3-b]indoles derived from Ac-Trp-OMe were not hydrolyzed at all.

Chymotrypsin↗
PubMed1977

Substrate specificity of endo-polyguluronide lyases from Pseudomonas sp. on the basis of their kinetic properties.

Two endo-alginate lyases [EC 4.2.2.3] differing in their mode of degradation of substrates and practically free of polymannuronide lyase activity were partially purified from Pseudomonas sp. cells. Their substrate specificities were investigated for two different kinds of alginate fragments; a polyguluronide (SG) and a polyuronide consisting of mannuronic (M) and guluronic (G) acid residues (SMG). The effects of various salts and some organic compounds such as EDTA and p-chloromercuribenzoate on the degradation of the two substrates were similar. High concentrations of the substrates similarly inhibited the action ofthe lyases, giving a bell-shaped plot. A polymannuronide alginate fragment (SM) which was a substrate for polymannuronide lyase but was not attacked by these guluronide lyases also inhibited the degradation of SG and SMG. The overall degradation velocities of a mixture of SG and SMG by both lyases coincided with those calculated from the Michaelis-Menten formula. Based on the above results, it was concluded that SG and SMG are attacked by the same endo-polyguluronide lyase.

Alginates↗
PubMed1976

ATP-dependent reactions catalyzed by inner membrane vesicles of rat liver mitochondria. Kinetics, substrate specificity, and bicarbonate sensitivity.

Three ATP-dependent reactions catalyzed by the inner membrane of rat liver mitochondria and the ATPase reaction catalyzed by purified mitochondrial ATPase (F1), were studied with respect to kinetic properties, substrates specificity, and sensitivity to bicarbonate. The ATP-dependent transhydrogenase reaction (reduction of NADP+ by NADH) catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers, with Km (ATP) values of 0.035 mM and 0.054 mM respectively. The Vmax of transhydrogenase activity (25 nmol min-1 mg-1) is the same in Tris-bicarbonate or Tris-Cl buffer. ITP and GTP readily substitute for ATP in the transhydrogenase reaction. The ATP-P1 exchange reaction catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers with Km (ATP) values of 1.0 mM and 1.4 mM respectively. The Vmax of exchange (200 nmol min-1 mg-1) is the same in either buffer. ITP and GTP do not effectively replace ATP in the exchange reaction.

4-Nitrophenylphosphatase↗
PubMed1979

[Effect of alternating milieu changes on specific substrate consumption in microorganisms].

In technical microbiology microorganisms often undergo alternating milieu changes. For instance, this is the case in recirculation reactors. The organisms react on these changes with an increase of entropy production and in connection with this with increasing substrate consumption. This increasing substrate consumption contradicts the aim of an optimum yield from a given substrate. Thus, studies of the reaction of microbial growth to alternating milieu changes are of great importance. A simple model of the influence of alternating milieu changes on specific substrate consumption is given. In it the biological conversion of substances is built up by an irreversible consecutive reaction. After preliminary examinations on the analogue computer the reaction of the model to alternating perturbations is simulated on the digital computer. The results of the stimulations are compared with experimental data. The good agreement of experiment and model justifies the use of the simple formulation in the preparation of technical processes.

Candida albicans↗