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THE PROPERTIES OF GOLGI-ASSOCIATED NUCLEOSIDE DIPHOSPHATASE AND THIAMINE PYROPHOSPHATASE: II. ELECTROPHORETIC SEPARATION AND IDENTIFICATION

JOHN M. ALLEN 1

1 Department of Zoology, University of Michigan, Ann Arbor, Michigan

Nucleoside diphosphatase and thiamine pyrophosphatase have been separated electrophoretically in 12.5% acrylamide gels. Identifications and characterizations of these enzymes was achieved by cytochemical means. Two basic reaction systems were used in these studies. System A contained 33 mM Tris, 15 mM calcium chloride, and 4 mM substrate material, and was adjusted to pH 9.5. System B constained 80 mM Tris-maleate buffer, pH 7.0, 3.6 mM lead nitrate, 5 mM manganous chloride, and 4 mM substrate material. Thiamine pyrophosphatase reacted most strongly with thiamine pyrophosphate, gave weak reactions with cytidine diphosphate, guanosine diphosphate, and inosine diphosphate, and did not react with uridine diphosphate and adenosine diphosphate. Nucleoside diphosphate reacted strongly with uridine diphosphate and inosine diphosphate, gave modest reactions with guanosinse diphosphate, reacted weakly with thiamine pyrophosphate and cytidine diphosphate, and was minimally reactive with adenosine diphosphate. Both enzymes were maximally activated by manganous ion and minimally activated by calcium ion. Thiamine pyrophosphatase was moderately activated by cysteine, glutathione, and potassium cyanide, was unaffected by uranyl nitrate, and was inhibited by sodium fluoride. Nucleoside diphosphatase was inhibited by cysteine, glutathione, and uranyl nitrate and was unaffected by sodium fluoride and potassium cyanide. These properties correspond very closely with those assigned to Golgi-associated nucleoside diphosphatase and thiamine pyrophosphatase in cytochemical studies. Nucleoside diphosphatase is of very general occurrence in mouse cells, whereas thiamine pyrophosphatase is of more limited distribution.

Submitted on December 31, 1962


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