Physiological correlation between nucleoside-diphosphate kinases and the 21-kDa guanine-nucleotide binding proteins copurified with the enzymes from the cell membrane fractions of Ehrlich ascites tumor cells

Abstract

The physiological correlation between nucleoside-diphosphate kinases (NDP-kinases) and the 21-kDa guanine nucleotide-binding proteins (G1 and G2) which are copurified with the enzymes from the cell membrane fractions of Ehrlich ascites tumor cells has been biochemically investigated in vitro. We found that: (i) incubation of the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinases (F-I and F-II) with one of the nucleoside 5′-diphosphates in the presence of 1 mM Mg 2+ or 0.25 mM Ca 2+ results in the rapid formation of nucleoside 5′-triphosphates without strict base specificity; (ii) GDP on the guanine nucleotide-binding proteins (G1, G2 and recombinant v- ras H p21) acts as a phosphate acceptor for the high-energy phosphates of the phosphoenzyme in the presence of 0.25 mM Ca 2+; and (iii) [ 32P]GTP is preferentially formed from the 32P-labelled phosphoenzyme F-I and GDP-bound G1 or GDP-bound recombinant v- ras H p21 protein, even if any other nucleoside 5′-diphosphates are present in the reaction mixture. Although [ 32P]GTP formed was bound with the guanine nucleotide-binding proteins, it was immediately hydrolyzed by the proteins themselves in the presence of 5 mM Mg 2+, but not in the presence of 0.25 mM Ca 2+. Available evidence suggests that NDP-kinase may be responsible for the activation of the guanine nucleotide-binding proteins (G1, G2 and p21 proteins) through phosphate transfer by the enzyme.