Nucleosidediphosphate kinase from Ehrlich ascites tumor cells.
A phosphate-incorporating protein has been highly purified from the cytosol of Ehrlich ascites tumor cells (EAT cells). The nitrocellulose membrane method was used to follow the progress of the purification by quantitation of the [32P]phosphorylated form of the protein. The purified protein was identified as an NDP-kinase since it exhibited NDP-kinase activity and had enzyme characteristics in common with other NDP-kinases from various mammalian cells. The purified NDP-kinase was found to have a molecular weight of approximately 76,000 daltons. Moreover, the enzyme appears to consist of two distinct polypeptides (18,000 and 20,000 daltons). This enzyme contained 19 amino acids, with high levels of glycine (9.8%) and lysine (9.0%). The enzyme rapidly formed a [32P]phosphoenzyme when incubated with [gamma-32P]ATP in the presence of Mg2+ (1 mM) at the optimum pH of 7.5 even at low temperature (below 4 degrees C). This phosphoenzyme is an enzyme-bound, high-energy-phosphate intermediate, because ATP was formed from it on incubation with ADP in the presence of Mg2+ (1 mM). This finding suggests that the phosphoenzyme functions as an intermediate in NDP-kinase action.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015470:Leukemia, Myeloid, Acute; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D009701:Nucleoside-Diphosphate Kinase / Q000302:isolation & purification* / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification*; D012265:Ribonucleotides / Q000494:pharmacology
D000596:Amino Acids; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; D012265:Ribonucleotides; D010770:Phosphotransferases; D009701:Nucleoside-Diphosphate Kinase