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Biomedical subjects

A Guranowski

Publications and source records attributed to A Guranowski.

At least 37 records · Page 2Linked to original sources
PubMed1987

Phosphonate analogues of diadenosine 5',5'''-P1,P4-tetraphosphate as substrates or inhibitors of procaryotic and eucaryotic enzymes degrading dinucleoside tetraphosphates.

The substrate specificity of procaryotic and eucaryotic AppppA-degrading enzymes was investigated with phosphonate analogues of diadenosine 5',5'''-P1,P4-tetraphosphate (AppppA). App(CH2)ppA (I), App(CHBr)ppA (II), and Appp(CH2)pA (III), but not Ap(CH2)pp(CH2)pA (IV), are substrates for lupin AppppA hydrolase (EC 3.6.1.17) and phosphodiesterase I (EC 3.1.4.1). None of the four analogues is hydrolyzed by bacterial AppppA hydrolase (EC 3.6.1.41), and only analogue III is degraded by yeast AppppA phosphorylase (EC 2.7.7.53). The analogues are competitive inhibitors of all four enzymes. The affinity of analogue IV is 3-40-fold lower than that of analogues I-III for all four enzymes. Introduction of one methylene (as in I and III) [or bromomethylene (as in II)] group into AppppA results in a 3-15-fold increase of its affinity for lupin and Escherichia coli AppppA hydrolases. The same modifications only negligibly (10-30%) affect its affinity for yeast AppppA phosphorylase and decrease its affinity for lupin phosphodiesterase I about 2.5-fold. The data provide further evidence for the heterogeneity among catalytic sites of all four AppppA-degrading enzymes.

Acid Anhydride Hydrolases↗
PubMed1986

Diadenosine 5',5'''-P1, P4-tetraphosphate alpha, beta-phosphorylase from yeast supports nucleoside diphosphate-phosphate exchange.

Homogeneous diadenosine 5',5'''-P1,P4-tetraphosphate alpha, beta-phosphorylase (Ap4A-phosphorylase), the enzyme recently found in yeast (Guranowski, A., and Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547) catalyzes an exchange reaction between the beta-phosphate of nucleoside diphosphate (NDP) and orthophosphate from the medium (Pi). The common purine and pyrimidine ribonucleoside diphosphates as well as ADP analogs modified either in aglycone, sugar, or at the anhydride bond beta-position are substrates. The Km and rate values for the NDP-Pi exchange reaction were estimated at pH optima. These optima are 6.5 for UDP, 7.0 for ADP or CDP, and 8.0 for GDP. In the presence of 10 mM K2HPO4, 0.1 mM EDTA, and 100 mM Hepes/KOH (pH 7.0), the Km for ADP is 0.7 mM with a rate constant at saturating ADP of 96 s-1. The Km value for orthophosphate is 2 mM. In the NDP-Pi exchange reaction, phosphate can be substituted with arsenate and apparent arsenolysis of NDPs yields corresponding nucleoside monophosphates. The same pH optimum of 6.5 is found for arsenolysis of ADP, GDP, and CDP. Whereas the Ap4A phosphorylase sulfhydryl groups are essential for catalyzing the Ap4A phosphorolysis, the NDP-Pi exchange reactions, and the arsenolysis of NDPs, the divalent metal ions (Mg2+, Mn2+, Ca2+, Co2+, and Cd2+), which had been shown to be essential cofactors of the former reaction, are not required for the two latter ones. Used at concentrations which are optimum for Ap4A phosphorolysis, the cations (particularly Mg2+ and Cd2+) inhibit the NDP-Pi exchange and the arsenolysis of NDPs. Interestingly, the Ap4A phosphorylase exhibits higher specificity for adenosine 5'-phosphosulfate (APS) than for any other NDP tested. The V/Km ratio is almost 5-fold higher with APS than with ADP. However, in the presence of orthophosphate, the APS is irreversibly converted to ADP. Thus, the enzyme displays a property already attributed to ADP-sulfurylase (EC 2.7.7.5), (Grunberg-Manago, M., Del Campillo-Campbell, A., Dondon, L., and Michelson, A. M. (1966) Biochim. Biophys. Acta 123, 1-16; Nicholls, R. G. (1977) Biochem. J. 165, 149-155).

Adenosine Diphosphate↗
PubMed1986

Chromate, molybdate, tungstate and vanadate behave as substrates of yeast diadenosine 5',5'''-p1,p4-tetraphosphate alpha, beta-phosphorylase.

Diadenosine 5',5'''-p1,p4-tetraphosphate (Ap4A) alpha, beta-phosphorylase from yeast Saccharomyces cerevisiae catalyzes two reactions: Ap4A cleavage and nucleoside diphosphate--phosphate (NDP-Pi) exchange. In both reactions phosphate can be substituted by arsenate, chromate, molybdate, tungstate or vanadate. In the presence of each anion, nucleoside 5'-monophosphate (NMP) always accumulates as a product of the reaction. This indicates that an unstable NMP anion is formed as an intermediate.

Anions↗
PubMed1985

Phosphorolytic cleavage of diadenosine 5',5'''-P1,P4-tetraphosphate. Properties of homogeneous diadenosine 5',5'''-P1,P4-tetraphosphate alpha, beta-phosphorylase from Saccharomyces cerevisiae.

Novel enzymatic activity which splits diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorolytically has been found in extracts from Saccharomyces cerevisiae. One of the two alpha,beta-anhydride bonds between Ap4A phosphate residues undergoes phosphorolysis, and ATP (pppA) plus ADP (ppA) are the products of the reaction according to the equation: AppppA + P*i----pppA + p*pA The reaction is dependent on the presence of divalent metal ions; Mn2+ or Mg2+ sustain the greatest rates of reaction. Among analogues of the Ap4A substrate, Ap5A and Gp4G, but not p4A and Ap3A, are substrates, and corresponding products are p4A plus ADP, and GTP plus GDP, the phosphate being incorporated into the nucleoside 5'-diphosphates. In the reactions, phosphate can be substituted with arsenate. Arsenolysis of Ap4A, Ap5A, or Gp4G leads to ATP plus AMP, p4A plus AMP, and GTP plus GMP, respectively. The name diadenosine tetraphosphate alpha,beta-phosphorylase (ADP-forming) is proposed for the new enzyme. The phosphorylase has been purified to apparent homogeneity and behaves as a single polypeptide chain of Mr = 40,000. Optimum activity of the enzyme is at pH 8.0 and the sulfhydryl groups are essential for catalysis. At saturating Ap4A, the rate constant for the reaction is 36 s-1 and the Km value for Ap4A is 60 microM (37 degrees C, 50 mM Hepes/KOH (pH 8.2), 500 microM MnCl2, 10 mM K2HPO4, 1 mM 2-mercaptoethanol, and 2% glycerol). The Km values for phosphate and arsenate are 1 and 3 mM, respectively.

Adenine Nucleotides↗
PubMed1983

Catabolism of diadenosine 5',5"'-P1,P4-tetraphosphate in procaryotes. Purification and properties of diadenosine 5',5"'-P1,P4-tetraphosphate (symmetrical) pyrophosphohydrolase from Escherichia coli K12.

Enzymatic activity which hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) yielding ADP has been identified in extracts of eubacteria, Escherichia coli and Acidaminococcus fermentans, and of a highly thermophilic archaebacterium, Pyrodictum occultum. Specific Ap4A (symmetric) pyrophosphohydrolase from Escherichia coli K12 has been purified almost 400-fold. The preparation was free of phosphatase, ATPase, phosphodiesterase, AMP-nucleosidase, and adenylate kinase. The Ap4A pyrophosphohydrolase molecular weight estimated by gel filtration is 27,000 +/- 1,000. Activity maximum is at pH 8.3. The Km value computed for Ap4A is 25 +/- 3 microM. The sulfhydryl group(s) is essential for enzyme activity. Metal chelators, EDTA, and o-phenanthroline, inhibit Ap4A hydrolysis; I0.5 values are 3 and 50 microM, respectively. Co2+ is a strong stimulator with an almost 100-fold increase in rate of Ap4A hydrolysis and a plateau in the range of 100-500 microM Co2+, when compared with the nonstimulated hydrolysis. Other transition metal ions, Mn2+, Cd2+, and Ni2+, stimulate by factors of 8, 3.5, and 3.5, respectively, with optimal concentrations in the range 200-500, 2-5, and 4-8 microM, respectively. Zn2+, Cu2+, and Fe2+, up to 30 microM, are without effect and they inhibit at higher concentrations. Mg2+ or Ca2+, in the absence of other divalent metal ions, are weak stimulators (1.5-fold stimulation occurs at 1-2 mM concentration), but act synergistically with Co2+ at its suboptimal concentrations. Stimulation in the presence of 10 microM Co2+ and either 1 mM MgCl2 or CaCl2 increases up to 75-fold. The same degree of synergy is found at 10 microM Co2+ and either 2-5 mM spermidine or 0.5-1.5 mM spermine. Besides Ap4A, bacterial Ap4A pyrophosphohydrolase hydrolyzes effectively Ap5A and Gp4G, and, to some extent, p4A, Ap6A, and Ap3A yielding in each case corresponding nucleoside diphosphate as one of the products.

Acid Anhydride Hydrolases↗
PubMed1983

Enzymes hydrolyzing ApppA and/or AppppA in higher plants. Purification and some properties of diadenosine triphosphatase, diadenosine tetraphosphatase, and phosphodiesterase from yellow lupin (Lupinus luteus) seeds.

Three distinct enzymes hydrolyzing either ApppA or AppppA, or both, were separated and purified from yellow lupin seed extracts. Two of the enzymes were purified to homogeneity. These enzymes differ greatly in their catalytic and physical properties. One hydrolase, with a native molecular weight of 41,000, exhibits broad pH (from 5-8) optimum for activity, requires Mg2+ for activity, is inhibited by zinc ions (I0.5 = 25 microM) and hydrolyses ApppA (V = 1), ApppC (V = 0.38), ApppG (V = 0.2), and ribose(5')pppA (V = 0.2). The enzyme exhibits much lower activity with AppppA (V = 0.1), and ApppppA, AppppppA, ppppA, and ATP are hydrolyzed 25- to 100-fold slower then ApppA. ADP was always one of the products of the reactions catalyzed by the enzyme. AppA, NAD, NADP, FAD, cAMP, and p-nitrophenyl-thymidine 5'-phosphate were not hydrolyzed by the enzyme. The enzyme is diadenosine 5',5"'-P1, P3-triphosphatase. The second hydrolase, composed of one polypeptide chain of a molecular weight 18,000-18,500, exhibits optimal activity in the pH range from 7.5-9, requires Mg2+ for activity, is inhibited by calcium ions (I0.5 for calcium depends on the concentration of Mg2+ and is 35-180 microM in the presence of 0.5-10 mM Mg2+, respectively), and hydrolyzes AppppA (V = 1, Km = 1 microM), ApppppA (V = 0.42, Km = 1.8 microM), AppppppA (V = 0.34), AppppU (V = 0.73), AppppC (V = 0.67), AppppG (V = 0.27), and ppppA. ATP was always one of the products of the reactions catalyzed by the enzyme. Dinucleoside di- and triphosphates, ATP, cAMP, and p-nitrophenylthymidine 5'-phosphate were not hydrolyzed by the enzyme. This enzyme is diadenosine 5',5"'-P1,P4-tetraphosphatase (EC 3.6.1.17). The third hydrolase, composed of one polypeptide chain of a molecular weight of 56,000, exhibits maximal activity at pH 9-10.5, does not require Mg2+ ions for activity, is inhibited neither by divalent cations (Mg2+, Ca2+, Zn2+, Co2+, Mn2+, or Ni2+) nor by EDTA, and uses as substrates all compounds which are substrates for the diadenosine 5',5"'-P1,P3-triphosphatase and diadenosine 5',5"'-P1,P4-tetraphosphatase. In addition, the enzyme hydrolyzes p-nitrophenyl-thymidine 5'-phosphate, p-nitrophenylthymidine 3'-phosphate, bis-p-nitrophenylphosphate, ADP, AppA, NAD, NADP, and FAD, but not cAMP. With the exception of p-nitrophenylphosphate derivatives all other substrates of the enzyme yield AMP as one of the products of hydrolysis. This enzyme has a specificity similar to that of phosphodiesterases (EC 3.1.4.1) from other sources. With the lupin phosphodiesterase, ApppA (V = 1, Km = 2.2 microM) and AppppA (V = 1, Km = 2.0 microM) are better substrates than NAD (V = 0.8, Km = 9.6 microM), AppA (V = 0.4), ApppppA (V = 0.6), and AppppppA (V = 0.34).

Acid Anhydride Hydrolases↗
PubMed1983

Substrate specificity of S-adenosylhomocysteinase. Cysteine is a substrate of the plant and mammalian enzymes.

Substrate specificity of S-adenosylhomocysteinases (S-adenosyl-L-homocysteine hydrolase, EC 3.3.1.1) with respect to amino acid has been studied using homogeneous preparations of the enzymes from yellow lupin (Lupinus luteus) seeds and bovine liver. Both enzymes use cysteine, in addition to homocysteine, as a substrate. Homoserine, serine, pinicillamine, reduced glutathione and 2-mercaptoethanol are not substrates. In the presence of cysteine, the reaction of S-adenosylthio-amino acid synthesis is characterized by 20-40-fold lower kcat values (kcat = 0.23 s-1 or 0.11 s-1 in the presence of cysteine and either bovine or lupin enzyme) and 270-250-fold higher Km values (Km for cysteine is 15 mM and 35 mM with bovine and lupin enzyme, respectively) than the reaction in the presence of the normal substrate, homocysteine. In the reverse reaction, S-adenosylcysteine is hydrolyzed by the mammalian enzyme much faster than by the plant one. Specificity (kcat/Km) towards S-adenosylcysteine and S-adenosylhomocysteine is 0.9 M-1 . s-1 and 60 000 M-1 . s-1, respectively, with the plant enzyme and 15.3 M-1 . s-1 and 70 000 M-1 . s-1, respectively, with the mammalian enzyme. With plant enzyme, the reactions with cysteine and homocysteine are not competitive, i.e., cysteine does not inhibit the synthesis of S-adenosylhomocysteine, and homocysteine does not inhibit the synthesis of S-adenosylcysteine. This is consistent with independent binding of cysteine and homocysteine to both enzyme subunits. Using adenosine analogs and the mammalian S-adenosylhomocysteinase we were able to synthesize a number of novel S-adenosylcysteine analogs. These included: S-N6-hydroxyadenosyl-L-cysteine, S-2-aminoadenosyl-L-cysteine, S-nebularyl-L-cysteine, S-3-deazaadenosyl-L-cysteine, S-formycyl-L-cysteine, S-N6-methyladenosyl-L-cysteine and S-N1-oxideadenosyl-L-cysteine.

Adenosylhomocysteinase↗
PubMed1983

Plant 5-methylthioribose kinase: properties of the partially purified enzyme from yellow lupin (lupinus luteus L.) seeds.

Activity of 5-methylthioribose kinase, the enzyme which catalyzes the ATP-dependent formation of 1-phospho-5-methylthioribose, has been revealed in the extracts from various higher plant species. Almost 2,000-fold-purified enzyme has been obtained from yellow lupin (Lupinus luteus L. cv Topaz) seed extract. Molecular weight of the native enzyme is 70,000 as judged by gel filtration. The lupin 5-methylthioribose kinase exhibits a strict requirement for divalent metal ions. Among the ions tested, only Mg(2+) and Mn(2+) acted as cofactors. The curve of kinase initial velocity versus pH reaches plateau at pH 10 to 10.5. The K(m) values calculated for 5-methylthioribose and ATP are 4.3 and 8.3 micromolar, respectively.Among nucleoside triphosphates tested as potential phosphate donors, only dATP could substitute in the reaction for ATP. 5-Isobutylthioribose, an analog of 5-methylthioribose, proved to be the gamma-ATP-phosphate acceptor, too. The compound inhibits competitively synthesis of 1-phospho-5-methylthioribose (K(i) = 1.4 micromolar). Lupin 5-methylthioribose kinase is completely and irreversibly inhibited by the antisulfhydryl reagent, p-hydroxymercuribenzoate. As in bacteria (Ferro, Barrett, Shapiro 1978 J Biol Chem 253: 6021-6025), the enzyme may be involved in a new, alternative pathway of methionine synthesis in plant tissues.

Journal Article↗
PubMed1982

Metabolism of 5'-methylthioadenosine in Aspergillus nidulans. An alternative pathway for methionine synthesis via utilization of the nucleoside methylthio group.

Experiments in which 5'-methylthioadenosine was used as a culture supplement for methionine-requiring mutants of Aspergillus nidulans with various enzymatic lesions indicated that the methylthio group derived from the nucleoside can be recycled to methionine. The results strongly suggest that methionine may be synthesized in the reaction catalyzed by homocysteine synthase (EC 4.2.99.10) in which O-acetylhomoserine is an acceptor of the methylthio group. The first step on the salvage pathway of the methylthio group is, in Aspergillus nidulans, phosphorolytic cleavage of 5'-methylthioadenosine to adenine and 5-methylthioribose 1-phosphate catalyzed by a specific phosphorylase.

Adenosine↗
PubMed1982

Adenine and adenosine metabolizing enzymes in cell-free extracts from Euglena gracilis.

1. Activities of the following enzymes involved in adenine and adenosine metabolism were found in cell-free extracts from Euglena gracilis: acid phosphatase (EC 3.1.3.2), 5'-methylthioadenosine phosphorylase (EC 2.4.2.-), adenine deaminase (EC 3.5.4.2), adenine phosphoribosyltransferase (EC 2.4.2.7) and adenosine kinase (EC 2.7.1.20). 2. The activities occurred both in heterotrophic and photoautotrophic cells and their levels did not change during light-induced chloroplast development. 3. Neither S-adenosylhomocysteinase (EC 3.3.1.1), 5'-methylthioadenosine nucleosidase (EC 3.2.2.9) and nucleoside phosphotransferase (EC 2.7.1.77) nor adenosine degrading enzymes: adenosine deaminase (EC 3.5.4.4), adenosine nucleosidase (EC 3.2.2.7), and purine-nucleoside (adenosine) phosphorylase (EC 2.4.2.1) were found in the Euglena extracts. 4. Comparison of the adenine and adenosine metabolism in Euglena and in other organisms is comprehensively presented. The metabolism in Euglena gracilis differs from that in higher animals and plants.

Acid Phosphatase↗
PubMed1982

Purine catabolism in plants : purification and some properties of inosine nucleosidase from yellow lupin (lupinus luteus L.) seeds.

Inosine nucleosidase (EC 3.2.2.2), the enzyme which hydrolyzes inosine to hypoxanthine and ribose, has been partially purified from Lupinus luteus L. cv. Topaz seeds by extraction of the seed meal with low ionic strength buffer, ammonium sulfate fractionation, and chromatography on aminohexyl-Sepharose, Sephadex G-100, and hydroxyapatite.Molecular weight of the native enzyme is 62,000 as judged by gel filtration. The inosine nucleosidase exhibits optimum activity around pH 8. Energy of activation for inosine hydrolysis estimated from Arrhenius plot is 14.2 kilocalories per mole. The K(m) value computed for inosine is 65 micromolar.AMONG THE INOSINE ANALOGS TESTED, THE FOLLOWING NUCLEOSIDES ARE SUBSTRATES FOR THE LUPIN INOSINE NUCLEOSIDASE: xanthosine, purine riboside (nebularine), 6-mercaptopurine riboside, 8-azainosine, adenosine, and guanosine. The ratio of the velocities measured at 500 micromolar concentration of inosine, adenosine, and guanosine was 100:11:1, respectively. Specificity (V(max)/K(m)) towards adenosine is 48 times lower than that towards inosine.In contrast to the adenosine nucleosidase activity which is absent from lupin seeds and appears in the cotyledons during germination (Guranowski, Pawełkiewicz 1978 Planta 139: 245-247), the inosine nucleosidase is present in both lupin seeds and seedlings.

Journal Article↗
PubMed1981

S-Adenosylhomocysteinase from yellow lupin seeds: stoichiometry and reactions of the enzyme-adenosine complex.

Plant (Lupinus luteus) S-adenosylhomocysteinase, an alpha 2 dimer, forms a 1:2 enzyme-adenosine complex. The binding sites for adenosine are not equivalent. Binding of the first molecule of adenosine is fast (k greater than 7 x 10(5) M-1 s-1), whereas the second molecule of adenosine binds in a slow process with a half-life of 5 min. Adenosine in the 1:1 and 1:2 enzyme--substrate complexes reacts slowly (k = 0.05 min-1) to give finally free enzyme, adenine, and ribose. The enzyme does not lose its ability to catalyze the synthesis of S-adenosylhomocysteine during the reactions. The relevance of the data to the catalytic functioning of the plant S-adenosylhomocysteinase is discussed.

Adenosine↗
PubMed1981

Adenosine analogues as substrates and inhibitors of S-adenosylhomocysteine hydrolase.

In the reaction adenosine + L-homocysteine = S-adenosyl-L-homocysteine, catalyzed by S-adenosylhomo-cysteine hydrolase from beef liver (EC 3.3.1.1), 11 nucleosides are able to substitute for adenosine to generate their corresponding S-nucleosidylhomocysteine congeners: 3-deaza-adenosine, 2-aza-3-deazaadenosine, nebularine (purine ribonucleoside), formycin, N6-methyladenosine, 8-azaadenosine, adenosine N1-oxide, pyrazomycin, 8-aminoadenosine, inosine, and the carbocyclic analogue of adenosine [(+/-)-aristeromycin]. S-Adenosylhomocysteine hydrolase from lupin seeds is able to utilize all of these nucleosides except inosine to synthesize analogues of S-adenosylhomocysteine. There is no correlation between the ability of these nucleotides to function as substrates and their inhibitory potencies, except in the case of 3-deazaadenosine. The carbocyclic analogue of adenosine is the most potent inhibitor of S-adenosylhomocysteine hydrolase with a Ki of 5 X 10(-9) M. When incubated with 3T3-L1 fibroblasts, the carbocyclic analogue of adenosine caused a 20-fold increase in the cellular concentration of S-adenosyl-homocysteine. The cellular generation of S-2-aza-3-deaza-adenosylhomocysteine was observed when 3T3-L1 fibroblasts were incubated with 2-aza-3-deazaadenosine.

Adenosine↗
PubMed1979

Nucleoside phosphotransferase from yellow lupin seedling cotyledons.

Nucleoside phosphotransferase (nucleotide:3'-deoxynucleoside 5'-phosphotransferase, EC 2.7.1.77) from yellow lupin seedling cotyledons was purified and the active enzyme consists of a single polypeptide chain, Mr = 72 000 +/- 3000. In transphosphorylation, purine and pyrimidine nucleosides are good phosphate acceptors and 5'-nucleotides are effective phosphate donors. Among 2'- and 3'-nucleotides, only 3'-AMP and 3'-psi MP acted as phosphate donors, and p-nitrophenylphosphate appeared less active in this regard. The purine and pyrimidine bases inhibit transphosphorylation. The Km values determined for the inosine:5'-AMP pair were 400 micrometers for both the compounds. The enzyme showed optimum activity at pH 8.0 in mM Tris-HCl buffer. Antisulfhydryl reagents and EDTA did not affect enzyme activity.

Chemical Phenomena↗