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At least 19 recordsLinked to original sources
PubMed1978

Canine testicular 17beta-hydroxysteroid oxidoreductase activity. I. Substrate specificity.

The substrate specificity of 17beta-hydroxysteroid oxidoreductase (17beta-HOR) activity was investigated in microsomal preparations of canine testes. Enzyme activity was measured by quantitating the conversion of radioactive substrates to products. The apparent Michaelis constants were determined to be 1.3 x 10(-6)M for androstenendione, 3--10 x 10(-6)M for dehydroepiandrosterone and 25 x 10(-6)M for estrone. These data are similar to those reported for human testicular 17beta-HOR activity and suggest that the canine activity may serve as an animal model for the study of testicular 17beta-HOR.

17-Hydroxysteroid Dehydrogenases↗
PubMed1976

Mechanism of rat liver microsomal stearyl-CoA desaturase. Studies of the substrate specificity, enzyme-substrate interactions, and the function of lipid.

The three purified proteins which are required for microsomal stearyl-CoA desaturation, NADH-cytochrome b5 reductase, cytochrome b5, and desaturase, have been combined with egg lecithin or dimyristyl lecithin vesicles to reconstruct a functional electron transport system capable of utilizing NADH and O2 in the desaturation of stearyl-CoA. Such preparations appear to consist of phospholipid vesicles which contain the three proteins bound to the outer surface of the vesicles. Acyl-CoA derivatives containing 12 to 19 carbon fatty acyl chains are required for desaturase activity while derivatives containing 9 to 20 carbons are capable of binding to the enzyme. Shorter chain acyl-CoA derivatives, free CoA, and free fatty acids do not appear to bind to the enzyme. Inhibition and analog studies suggest that the methylene chain of stearyl-CoA assumes an eclipsed ("gauche") conformation at carbon atoms 9,10 in the enzyme-substrate complex. Furthermore, isotope rate effects obtained with deuterated stearyl-CoA derivatives indicate that hydrogen removal is the rate-limiting step of desaturation. Stearyl-CoA binds to pure liposomes and desaturase-containing liposomes, and it is this form of stearyl-CoA which appears to be the substrate for desaturase. The Arrhenius plots of desaturase activity obtained using desaturase bound to egg lecithin liposomes, in which the liquid crystalline to crystalline phase transition temperature is -5 degrees, was linear between 15 and 35 degrees, while that obtained using desaturase bound to dimyristyl lecithin liposomes showed a break at 24 degrees coinciding with the liquid crystalline to crystalline phase transition temperature for this lipid. The decrease observed in the deuterium isotope rate effect below the transition temperature indicates that a step in the reaction sequence other than hydrogen abstraction becomes rate-limiting when the lipid is in the crystalline state. In this system translational diffusion does not emerge as the rate-limiting step. The liposomes contained sufficient reductase and cytochrome b5 so that translational diffusion was not rate-limiting.

Animals↗
PubMed1975

Substrate specificity of carboxypeptidase from Watermelon.

The substrate specificity of carboxypeptidase (F-II) purified from watermelon for various synthetic peptides and esters was examined kinetically. The enzyme showed a broad substrate specificity against various carbobenzoxy- and benzyl-dipeptides. Peptides containing glycine or proline were hydrolyzed slowly by the enzyme. Peptides containing hydrophobic amino acids were hydrolyzed rapidly. The presence of hydrophobic amino acid residues, not only at the C-terminal position but also at the second position and probably the third position from the C-terminal resulted in an increase in the rate of hydrolysis. Inhibition studies with diisopropyl flurophosphate and diastereomers of carbobenzoxy-Phe-Ala demonstrated that the peptidase and esterase activities of the enzyme are both catalyzed by the same site of the enzyme molecule, but the binding sites for peptides and esters seem not to be the same. The enzyme also had amidase activity, which was optimal at pH 7.0.

Amidohydrolases↗
PubMedDate not supplied

[Substrate specificity of lipase from Pseudomonas fluorescens].

Substrate specificity of lipase isolated from the culture liquid filtrate of Pseudomonas fluorescens BKM-B-1151 was investigated with respect to vegetable oils and animal fats (olive, sunflower, cotton, mustard and soybean oils; beef and hog fats and their glycerides and fatty acid esters). The preparation showed a high specificity to the quantitative composition of the reaction mixture (substrate: enzyme ratio), chemical structure of the substrate, and the emulgator type (gelatine, gum arabic and Triton X-100). The lipase preparation hydrolyzed oils and water-insoluble fatty acid esters. The latter indicated an involvement of lipase.

Kinetics↗
PubMed1975

4-Nitrobenzoic acid reductase of Ascaris lumbricoides var suum. Substrate specificity and reaction products.

1. The substrate specificity of nitro-reductase from Ascaris lumbricoides varsum was determined. This enzyme reduced nitrobenzene, 4-nitrohippuric acid and the isomers of nitrophenol, nitroanisole, nitrobenzoic acid, nitrobenzaldehyde and nitrobenzyl alcohol. The same enzyme preparation reduced azobenzene, 4-dimethylaminoazobenzene and 1,2-dimethyl-4-(4-carboxyphenylazo)-5-hydroxybenzene. Nitrobenzaldehyde isomers were not reduced to the alcohols. 2. The products of nitro- and azo-reduction were the corresponding amines, no hydroxylamino or hydrazo compounds were detected. 3. The pH optima and cofactor requirements were the same for both azo- and nitro-reduction and neither reaction was inhibited by oxygen. 4. Ammonium sulphate fractionation failed to separate azo- and nitro-reductase activities. The molecular weight of both azo- and nitro-reductase was about 130 000.

Amines↗
PubMed1978

[Electrophoretic analysis of substrate specificity of wheat alcohol dehydrogenases].

Electrophoresis in polyacrylamide gel slabs has been used to study the isoform composition and substrate specificity of alcohol dehydrogenases in the embryo and young seedlings of the diploid wheat Triticum monococcum L., the tetraploid T. dicoccon (Schrank) Schuebl and the hexaploid T. spelta L. Three alcohol dehydrogenases of different substrate specificity and developmental pattern were distinguished: a) the NAD-dependent alcohol dehydrogenase, catalyzing the oxidation of different primary and secondary aliphatic and aromatic alcohols, as well as certain compounds with several hydroxyl groups (tris, triethanolamin) and revealing, after electrophoresis, one major band in the diploid wheat and three bands in both polyploid wheats; b) the NADP-dependent aromatic alcohol dehydrogenase (substrate--cinnamic alcohol), revealing, after electrophoresis, one major fast moving band in the diploid wheat and two bands in polyploid wheats; c) an aromatic alcohol dehydrogenase (2-3 bands after electrophoreis) with no specificity to the cofactors (NAD or NADP).

Alcohol Oxidoreductases↗
PubMed1979

Comparison of the substrate specificity of adenosine 3':5'-monophosphate- and guanosine 3':5'-monophosphate-dependent protein kinases. Kinetic studies using synthetic peptides corresponding to phosphorylation sites in histone H2B.

The substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinases have been compared by kinetic analysis using synthetic peptides as substrates. Both enzymes catalyzed the transfer of phosphate from ATP to calf thymus histone H2B, as well as to two synthetic peptides, Arg-Lys-Arg-Ser32-Arg-Lys-Glu and Arg-Lys-Glu-Ser36-Tyr-Ser-Val, corresponding to the amino acid sequences around serine 32 and serine 36 in histone H2B. Serine 38 in the latter peptide was not phosphorylated by either enzyme. Cyclic GMP-dependent kinase and cyclic AMP-dependent kinase catalyzed the incorporation of 1.1 and 2.0 mol of phosphate/mol of histone H2B, respectively. The phosphorylation of histone H2B, respectively. The phosphorylation of histone H2B by cyclic GMP-dependent kinase showed two distinct optima as the magnesium concentration was increased. However, the phosphorylation of either synthetic peptide by this enzyme was depressed at high magnesium concentrations. As the pH of reaction mixtures was elevated from pH 6 to pH 9, the rate of phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase continually increased. Acetylation of the NH2 terminus of the peptide did not qualitatively affect this pH profile, but did increase the Vmax value of the enzyme 3-fold. The apparent Km and Vmax values for the phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase were 21 microM and 4.4 mumol/min/mg, respectively. The synthetic peptide Arg-Lys-Glu-Ser36-Tyr-Ser-Val was a relatively poor substrate for cyclic GMP-dependent kinase, exhibiting a Km value of 732 microM, although the Vmax was 12 micromol/min/mg. With histone H2B as substrate for the cyclic GMP-dependent kinase, two different Km values were apparent. The Km values for cyclic AMP-dependent kinase for either synthetic peptide were approximately 100 microM, but the Vmax for Arg-Lys-Arg-Ser32-Arg-Lys-Glu was 1.1 mumol/min/mg, while the Vmax for Arg-Lys-Glu-Ser36-Tyr-Ser-Val was 16.5 mumol/min/mg. These data suggest that although the two cyclic nucleotide-dependent protein kinases have similar substrate specificities, the determinants dictated by the primary sequence around the two phosphorylation sites in histone H2B are different for the two enzymes.

Amino Acid Sequence↗
PubMed1977

Substrate specificity of antinuclear antibodies in scleroderma.

Studies of antinuclear antibodies (ANA) were carried out in 39 cases of systemic scleroderma and for comparison in 19 cases of systemic lupus erythematosus (SLE) and 4 of mixed connective tissue disease (MCTD) using indirect immunofluorescence (IF) methods under standard conditions. The results on three different substrates--monkey esophagus, guineapig lip and rat liver--are reported. In 48.7% of scleroderma cases ANA showed a substrate specificity. The highest percentage of positive results in scleroderma was obtained on monkey esophagus (97.4%) and the lowest on rat liver (61.5%). In SLE and MCTD, in contrast, only about 13% of the sera displayed such specificity. If only sera with substrate specificity are considered, the positive results on monkey esophagus and rat liver are 94.7% and 21.1%, respectively. Titers of sera reacting positively on 2 or 3 substrates were mostly in agreement, although some sera both in systemic scleroderma and SLE showed higher titers on monkey esophagus. The IF pattern was usually the same regardless of the substrate, Tests for ANA in scleroderma should be performed on at least 2 substrates simultaneously.

Adult↗
PubMed1976

Further characterization of phosphatase activities using non-specific substrates.

The demonstration of non-lysosomal acid phosphatase has been the subject of a number of recent investigations. In the present study we compared the enzyme activities in rat liver and kidney that are revealed after incubation in the presence of either beta-glycerophosphate, p-nitrophenylphosphate or phenylphosphate at varying pH. As seen by others, the activity towards p-nitrophenylphosphate at pH 5-6 was confined to lysosomes, Golgi apparatus, endoplasmic reticulum (ER), nuclear envelope and plasmalemma. The reactivity of the plasmalemma and the ER was increased at pH 7. The TER of Küpffer cells in the liver stained intensely in contrast to the ER of the parenchymal cells, which stained only weakly. In the presence of NaF, all sites except the plasmalemma became negative. Addition of a levamisole-analogue, L-p-bromotetramisole, which is a specific inhibitor of alkaline phosphatase, resulted in the disappearance of the plasmalemmal activity whereas the activity at the other sites appeared unaltered. The rather unusual locations of activities with so-called non-specific substrates were further compared with those obtained with specific substrates such as glucose-6-phosphate and thiamine pyrophospate. The possible implication of these data in relation to the specificity of marker-enzymes for subcellular organelles is discussed.

Animals↗
PubMed1977

New formamidase having substrate specificity for o-formylaminoacetophenone in pig liver.

The supernatant (13 000 x g, 20 min) of pig liver homogenate was filtered with Sephadex G-200 and formamidase (aryl-formylamine amidohydrolase, EC 3.5.1.9)activity in each fraction was measured. When formylkynurenine was used as substrate, two peaks of formamidase activity were observed but, with formylaminoacetophenone as substrate, only one peak was observed. Formamidase in the lower molecular weight fraction is known as kynurenine formamidase (FA I), formamidase found here in the higher molecular weight fraction has not been previously reported. This form, designated FA II has been purified about 160-fold from pig liver. The formamidase obtained has substrate specificity for o-formylaminoacetophenone only and could not hydrolyze formylkynurenine. The optimal pH was 8.5 and the Km for o-formylaminoacetophenone was 1.66-10(-3) M. This formamidase was considered to be a new enzyme and was different from FA I in molecular weight and substrate specificity. This new formamidase was present in pig, rabbit and guinea pig liver and not present in rat or mouse liver.

Acetophenones↗
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

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↗
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↗
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↗
PubMed1976

Alpha-N-Benzoylarginine-2-naphthylamide hydrolase (cathepsin BI?) from rat skin. III. Substrate specificity, modifier characteristics, and transformation of the enzyme at acidic pH.

Some properties of rat skin benzoylarginine-2-naphthylamide hydrolase types I (preparations I and AI) and II (preparations II and NII) were studied. Both types were activated by dithiothreitol and EDTA, but responded differently to 1 mM KCN, when benzoylarginine-2-naphthylamide (BANA) was used as a substrate: type I was inhibited, while type II was activated. When leucine-2-naphthylamide was used as a substrate, both types were activated by KCN. Thiol proteinase inhibiting substances, like heavy metals, iodoacetic acid, 4-chloromercuribenzoic acid, and tosyllysine chloromethylketone, inhibited the enzymes. Diisopropylfluorophosphate, phenylmethylsulfonyfluoride, 4-aminobenzamidine, and high-molecular-weight trypsin inhibitors were without effect. The substrate specificity of rat skin BANA hydrolase resembled that of an amino acid naphthylamidase, naphthylamides of methionine, lysine, arginine, and alanine being hydrolyzed most rapidly. The rate of hydrolysis of BANA was only 11% of that of methionine naphthylamide. Amino acid esters with a free alpha-amino group were also good substrates. The transformation of type II to type I at acidic pH was studied. During the transformation amino acids or peptides were formed and probably some inhibitor present in type II was destroyed proteolytically.

Animals↗
PubMed1976

Partial proteolysis of some cellulase components from Trichoderma viride and the substrate specificity of the modified products.

An endo-cellulase component [EC 3.2.1.4] or random type, F II, was obtained from "Cellulase Onozuka," a commercial product from Trichoderma viride, and was subjected to partial proteolysiats with a protease preparation of the same fungal origin. The resulting modified cellulase was fractioned by two steps of column chromatography, and the resulting patterns, together with the substrate specificity expressed in terms of the randomness of CMC hydrolysis and the immunological properties against anti-F II-rabbit se-um, were examined. The chromatographic patterns were very similar to those of cellulase subfractions without proteolytic treatment. Moreover, the immunological response of the modified cellulases from F II was mostly positive and their randomness of CMC hydrolysis was generally lower, compared with subfractions of F II which were not subjected to proteolysis. The subfractions of Peak III, which were obtained from F II by proteolysis, showed mostly negative immunological response and higher randomness of CMC hydrolysis compared with subfractions of Peak III which were not subjected to proteolysis. Thus, some limited proteolysis of cellulase components may, at least in part, be responsible for its multiplicity in vivo.

Amino Acids↗
PubMed2026

Proteome-wide Ubiquitinome Profiling Reveals Substrate-specific Dynamics Within the USP7 Network.

USP7 is a pleiotropic deubiquitylating enzyme that is involved in tumor suppression, (neuro) development, chromatin regulation and the DNA damage response. How USP7 regulates these diverse pathways is still unclear. Here, we report data-independent acquisition and label free quantitation mass spectrometry to profile the proteome-wide impact of USP7 on substrate de-ubiquitylation and overall protein abundance. First, we identified proteins associated with endogenous USP7 by immunopurification followed by data-independent acquisition and label free quantitation mass spectrometry. Integration of our new results with earlier interactomes of epitope-tagged USP7 yielded a consensus set of high-confidence protein targets. Domain mapping analysis revealed that, in addition to the TRAF domain, the ubiquitin-like domains of USP7 play a key role in substrate selection. Using specific enrichment of tryptic K-ε-GG peptides, we mapped proteome-wide changes in ubiquitinome dynamics following inhibition of USP7. Combining unbiased proteome-wide and targeted quantitative mass spectrometry revealed that deubiquitylation by USP7 can have different effects on the stability of distinct substrates, and suggests that USP7's activity profile is substrate-dependent rather than an intrinsic enzymatic property. Thus, in addition to providing a proteome-wide map of USP7 target sites, our multi-angle proteomics approach reveals that the effects of USP7-mediated deubiquitylation on its targets are remarkably variable and substrate-specific. Finally, based on these detailed molecular insights we show how USP7 connects various neurodevelopmental syndromes and tumor suppression pathways.

Ubiquitin-Specific Peptidase 7↗