Assuntos
Hidrolases Anidrido Ácido/química , Proteínas de Bactérias/química , Proteínas de Escherichia coli , Monoéster Fosfórico Hidrolases/química , Estrutura Secundária de Proteína , Hidrolases Anidrido Ácido/isolamento & purificação , Sequência de Aminoácidos , Sequência Conservada , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Placenta/enzimologia , Gravidez , Pirofosfatases , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de AminoácidosRESUMO
The cDNA and derived amino acid sequence of human diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase have been determined with the aid of the GenBank Expressed Sequence Tag database. This enzyme possesses a modification of the MutT sequence motif found in certain nucleotide pyrophosphatases. It is unrelated to the enzymes of diadenosine tetraphosphate catabolism found in prokaryotes and fungi.
Assuntos
Nucleotidiltransferases/genética , Pirofosfatases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Nucleotidiltransferases/análise , Nucleotidiltransferases/química , Placenta/enzimologia , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase and Ap4A pyrophosphohydrolase activities have been purified from extracts of the green alga Scenedesmus obliquus. Both activities were also detected in Scenedesmus brasiliensis, Scenedesmus quadricauda and in Chlorella vulgaris. This is the first time that both types of enzyme have been detected in the same species. The Ap4A phosphorylase has a molecular mass of 46-48 kDa, a broad pH optimum between 7.5 and 9.5, and requires a divalent ion for activity (Mg2+ > Co2+ > Ca2+ = Mn2+ = Cd2+ > Zn2+). It degrades substrates with at least four phosphate groups and always produces a nucleoside 5'-diphosphate product. The Km values for Ap4A and Pi are 5.3 microM and 160 microM, respectively, and kcat. = 1.8 s-1. Arsenate, vanadate, molybdate, chromate and tungstate can substitute for phosphate. The enzyme also catalyses Ap4A synthesis (Keq. = [Ap4A] [Pi]/[ATP][ADP] = 9 x 10(-4)) and ADP arsenolysis. The Ap4A hydrolase has a molecular mass of 26-28 kDa, an alkaline pH optimum of 8.8-9.8, and prefers Zn2+ as the stimulatory ion (Zn2+ > Mg2+ > Mn2+ > Co2+ > Cd2+). It degrades substrates with at least four phosphate groups, having a slight preference for Ap5A, and always produces a nucleoside 5'-triphosphate product. The Km value for Ap4A is 6.6 microM and kcat. = 1.3 s-1. It is inhibited competitively by adenosine 5'-tetraphosphate (Ki = 0.67 microM) and non-competitively by fluoride (Ki = 150 microM). A 50-54 kDa dinucleoside 5',5'''-P1,P3-triphosphate (Ap3A) pyrophosphohydrolase was also detected in S. obliquus, S. quadricauda and C. vulgaris. The corresponding enzyme in S. brasiliensis (> 100 kDa) may be a dimer
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Clorófitas/enzimologia , Nucleotidiltransferases/metabolismo , Hidrolases Anidrido Ácido/antagonistas & inibidores , Hidrolases Anidrido Ácido/isolamento & purificação , Nucleotídeos de Adenina/farmacologia , Difosfato de Adenosina/metabolismo , Cátions Bivalentes , Cromatografia Líquida de Alta Pressão , Fosfatos de Dinucleosídeos/biossíntese , Eletroforese em Gel de Poliacrilamida , Fluoretos/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/isolamento & purificação , Pirofosfatases , Especificidade por SubstratoRESUMO
1. A P1,P3-bis(5'-nucleosidyl)triphosphate pyrophosphohydrolase (Np3 Nase) has been partially purified from Artemia embryos. 2. The Np3 Nase has a native Mr of 115,000 and preferentially hydrolyses substrates of the form Np3 N. Relative rates of hydrolysis are Ap3A (Vrel = 1.0), Gp3G (Vrel = 0.71), Ap4A (Vrel = 0.08), Ap5A (Vrel = 0.09), Gp4G (Vrel = 0.3) and Gp5G (Vrel = 0.33). An NMP is always one of the products. 3. The Km values for Ap3A and Gp3G are 15 and 10 microM respectively. 4. Mg2+, Mn2+ and Ca2+ ions all stimulate the activity, while Zn2+, Co2+ and Ni2+ ions are inhibitory. 5. The activity of the Np3 Nase remains constant during pre-emergence development of encysted embryos but decreases slightly after hatching.
