A third type of nucleoside diphosphate kinase from spinach leaves: purification, characterization and amino-acid...

A third type of nucleoside diphosphate kinase (NDP kinase III), distinct from the previously described NDP kinases I and II (Nomura et al. (1991) Biochim. Biophys. Acta 1077, 47-55), was purified from spinach leaves to electrophoretic homogeneity. NDP kinase III was judged by SDS-PAGE and by gel filtration to have molecular masses of 17 kDa and 102 kDa, respectively, suggesting that it is composed of six subunits similarly to the other spinach isoforms, NDP kinases I and II. Amino-acid sequence analysis revealed the primary structure of NDP kinase III to be comprised of 153 amino-acid residues, the sequence of which exhibited 61% and 53% homology with those of NDP kinases I and II, respectively. In the reaction catalyzed by the three isoforms, the order of Km as phosphate acceptor was determined as GDP << ADP for NDP kinase III, different from those observed for NDP kinase I (ADP << GDP) and for NDP kinase II (GDP = ADP). These results suggest that the three isoforms may have distinct roles in regulating intracellular 5'-di- and 5'-triphosphonucleotide levels in spinach leaves.