Identification and purification of a cromoglycate-sensitive nucleoside diphosphate kinase from membranes of Cucurbita pepo

Labeling of membrane proteins by [gamma-S-35]GTP is often used as a measure of putative signal-transducing G-protein activity. In this paper, we use a filter binding assay to demonstrate that zucchini (Cucurbita pepo L.) microsomes are labeled by both [gamma-35S]GTP and [gamma-P-32]GTP, but not by [alpha-P-32]GTP. Autoradiography of microsomal proteins separated by SDS-PAGE shows covalent labelling of a 18 kDa polypeptide by [gamma-S-35]GTP and [gamma-P-32]GTP, but not by [alpha-P-32]GTP. A rapid two-step procedure utilizing anion exchange followed by a nucleotide affinity resin was developed to purify the membrane protein labeled by [gamma-S-35]GTP. Microsequence analysis of the purified polypeptide identified it as a nucleoside diphosphate kinase (NDP kinase, EC 2.7.4.6). This NDP kinase has a subunit size of 18 kDa, is capable of in-gel autophosphorylation and is inhibited by cromoglycate, an inhibitor of mammalian NDP kinase. The NDP kinase active site has equal affinity for adenine and guanosine nucleotides, and can hydrolyze [gamma-S-35]GTP. The zucchini NDP kinase is not found in plasma membranes purified by aqueous polymer phase-partitioning. These results suggest that some reports of [gamma-S-35]GTP binding to plant membranes presented as evidence for plant G-proteins may actually have been measurements of NDP kinase activity.