ACTIVATION OF GTP FORMATION AND HIGH-AFFINITY GTP HYDROLYSIS BY MASTOPARAN IN VARIOUS CELL-MEMBRANES - G-PROTEIN ACTIVATION VIA NUCLEOSIDE DIPHOSPHATE KINASE, A POSSIBLE GENERAL MECHANISM OF MASTOPARAN ACTION

The wasp venom, mastoparan (MP), is a direct activator of reconstitute d pertussis toxin-sensitive G-proteins and of purified nucleoside diph osphate kinase (NDPK) [E.C. 2.6.4.6.]. In HL-60 membranes, MP activate s high-affinity GTPase [E.C. 3.6.1.-] and NDPK-catalyzed GTP formation , but not photolabeling of G-protein alpha-subunits with GTP azidoanil ide; this suggests that the venom activates G-proteins in this system indirectly via stimulation of NDPK. Moreover, the MP analogue, mastopa ran 7 (MP 7), is a much more effective activator of reconstituted G-pr oteins than MP, whereas with regard to NDPK and GTPase in HL-60 membra nes, the two peptides are similarly effective. In our present study, w e investigated NDPK- and G-protein activation by MP in membranes of th e human neuroblastoma cell line, SH-SY5Y, the human erythroleukemia ce ll line, HEL, the rat basophilic leukemia cell line, RBI, 2H3, and the hamster ductus deferens smooth muscle cell line, DDT(1)MF-2. All thes e membranes exhibited high NDPK activities that were increased by MP. Compared to basal GTP formation races, basal rates of high-affinity GT P hydrolysis in cell membranes were low. MP activated high-affinity GT P hydrolysis in cell membranes but did not enhance incorporation of GT P azidoanilide into G-protein alpha-subunits. As with HL-60 membranes, MP and MP 7 were similarly effective activators of NDPK and GTPase in SH-SY5Y membranes. Pertussis toxin inhibited MP-stimulated GTP hydrol yses in SH-SY5Y- and HEL membranes, whereas NDPK activations by MP wer e pertussis toxin-insensitive. Our data suggest that indirect G-protei n activation via NDPK is not restricted to HL-60 membranes but is a mo re general mechanism of MP action in cell membranes. Pertussis toxin-c atalyzed ADP-ribosylation of alpha-subunits may inhibit the transfer o f GTP from NDPK to G-proteins. NDPK may play a much more important rol e in transmembrane signal transduction than was previously appreciated and, moreover, the GTPase of G-protein alpha-subunits may serve as GD P-synthase for NDPK.