title: Nucleoside diphosphate kinase from Xenopus oocytes; partial purification and characterization.
abstract: We have identified and partially purified a soluble nucleoside diphosphate kinase (NDP kinase) from Xenopus laevis oocytes. The enzyme preparation can catalyze the transfer of phosphate from ATP to all of the major oxy- and deoxynucleotides. It can also catalyze the transfer of a phosphorothioate group from gamma-S-ATP to an acceptor GDP forming gamma-S-GTP. Like NDP kinases from other sources, the catalytic mechanism appears to involve a phosphoenzyme intermediate which can be isolated. Transfer of phosphate from nucleoside triphosphates to protein is rapid, reaching saturation within 1 min following the addition of nucleoside triphosphates. The transfer of phosphate from phosphoprotein intermediate to nucleoside diphosphates is equally fast. While nucleoside diphosphate kinases are generally thought to require magnesium for activity, both the oocyte enzyme preparation and a commercial bovine liver enzyme preparation are only partially inhibited by short (10 min) exposures to 25 mM EDTA. Both enzyme preparations are, however, further inhibited by long incubations with this metal chelator (2 h, 70% inhibition). Zinc enhances the inhibition of NDP kinase by EDTA, but is ineffective on its own. Rapid phosphorylation in the presence of [gamma-32P]ATP and EDTA could be used to identify the phosphoenzyme intermediate in homogenates of Xenopus oocytes and facilitated its isolation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with autoradiography indicated the presence of only a single phosphorylated species of Mr 21,500 in supernatants of fresh oocyte homogenates. Partial purification of this protein utilizing salt precipitation, hydrophobic-interaction chromatography and an affinity step with Affi-Gel Blue Sepharose resulted in a 100-fold purification and a 29% overall yield of NDP-kinase activity. Size-exclusion chromatography of the purified preparation yielded two peaks containing enzyme activity. They eluted with apparent molecular weights of 45,000 and 70,000, suggesting a native enzyme that is multimeric or associated with other proteins.
authors.affiliation: Department of Chemistry, University of South Florida, Tampa 33620.
authors.forename: G
authors.initials: G
authors.lastname: Buczynski
authors.forename: R L
authors.initials: RL
authors.lastname: Potter
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009701:Nucleoside-Diphosphate Kinase / Q000302:isolation & purification / Q000378:metabolism*; D009865:Oocytes / Q000201:enzymology*; D014982:Xenopus laevis; D015032:Zinc / Q000494:pharmacology
chemical_list: D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D009701:Nucleoside-Diphosphate Kinase; D008274:Magnesium; D015032:Zinc