IMP-GMP 5'-nucleotidase from rat brain: activation by polyphosphates.
IMP-GMP 5'-nucleotidase has been purified to homogeneity from total rat brain extracts. This preparation showed a unique band (Mr 54,000 +/- 1,509) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme presented the following properties: optimal pH value, 6.5-6.8; relative velocity measured in the presence of MgCl2, MnCL2, CoCl2, and NiCl2 (2 mM), 100, 60, 11, and <1, respectively; preferred substrates, IMP and GMP; and activation constant (Ka) found for Ap4A, Ap5A, and Ap6A, 83 +/- 38, 77 +/- 32, and 57 +/- 12 microM, respectively. Under assay conditions where activation by Ap4A was fivefold, the activation produced by dinucleotides was as follows: Ap4G (4.0), Ap4I (2.9), Ap4X (3.3), Ap4C (0.7), Ap4U (1.1), Ap4epsilonA (1.5), Ap4ddA (1.7), Gp4G (2.2), Ap3A (1.1), and Ap2A (1.2). Polyphosphates P18, P19, P20, and P35 were activators of the reaction with calculated Ka values of 3.5 +/- 0.5, 0.9 +/- 0.2, 0.6 +/- 0.2, and 1.3 +/- 0.5 microM, respectively. The following compounds, at 0.1 mM, were effectors of the phosphotransferase reaction producing the fold activation indicated: Ap4A (8.3), Ap5A (10.2), Ap6A (10.1), Ap4G (7.7), Ap4X (7.6), Ap4U (2.1), glycerate 2,3-bisphosphate (3.9), and unpurified P15 (7.6). Two enzyme forms of IMP-GMP 5'-nucleotidase were detected when the extracts from rat tissues or from the crustacean Artemia were subjected to chromatography on a Dyematrex Green A column. The ratio of the hydrolytic activities under both peaks (peak I/peak II) was as follows: brain (1.5), heart (1.9), liver (1.6), lung (2.0), testis (3.8), and Artemia cysts (2.0).
Marques
A F
AF
Laboratorio de Bioquímica, Faculdade de Farmácia, Universidade do Porto, Portugal.
Teixeira
N A
NA
Gambaretto
C
C
Sillero
A
A
Sillero
M A
MA
D015720:5'-Nucleotidase / Q000378:metabolism*; D000818:Animals; D001156:Artemia / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002021:Buffers; D002413:Cations, Divalent / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology; D002845:Chromatography; D004789:Enzyme Activation / Q000502:physiology; D006157:Guanosine Monophosphate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007291:Inosine Monophosphate / Q000378:metabolism*; D008297:Male; D009711:Nucleotides / Q000494:pharmacology; D010770:Phosphotransferases / Q000378:metabolism; D011122:Polyphosphates / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D002021:Buffers; D002413:Cations, Divalent; D002414:Cations, Monovalent; D009711:Nucleotides; D011122:Polyphosphates; D007291:Inosine Monophosphate; D006157:Guanosine Monophosphate; D010770:Phosphotransferases; D015720:5'-Nucleotidase