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The wasp venom, mastoparan (MP), activates reconstituted pertussis toxin (PTX)-sensitive G-proteins in a receptor-independent manner. We studied the effects of MP and its analogue, mastoparan 7 (MP 7), on G-protein activation in HL-60 cells and a reconstituted system and on nucleoside diphosphate kinase (NDPK)-catalysed GTP formation. MP activated high-affinity GTP hydrolysis in HL-60 membranes with an EC50 of 1-2 microM and a maximum at 10 microM. Unlike the effects of the formyl peptide receptor agonist, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), on GTPase, those of MP were only partially PTX-sensitive. MP-induced rises in cytosolic Ca2+ concentration and superoxide-anion formation in intact HL-60 cells were also only incompletely PTX-sensitive. N-Ethylmaleimide inhibited MP-stimulated GTP hydrolysis to a greater extent than that stimulated by fMet-Leu-Phe. Unlike the latter, MP did not enhance incorporation of GTP azidoanilide into, and cholera toxin-catalysed ADP-ribosylation of, Gi-protein alpha-subunits in HL-60 membranes. By contrast to fMet-Leu-Phe, MP did not or only weakly stimulated binding of guanosine 5'-[gamma-thio]triphosphate to Gi-protein alpha-subunits. MP 7 was considerably more effective than MP at activating the GTPase of reconstituted Gi/G(o)-proteins, whereas in HL-60 membranes, MP and MP 7 were similarly effective. MP and MP 7 were similarly effective at activating [3H]GTP formation from [3H]GDP and GTP in HL-60 membranes and by NDPK purified from bovine liver mitochondria. Our data suggest the following: (1) MP activates Gi-proteins in HL-60 cells, but (2) the venom does not simply mimic receptor activation. (3) MP and MP 7 may activate GTP hydrolysis in HL-60 membranes indirectly through interaction with NDPK. (4) MP 7 is a more effective direct activator of PTX-sensitive G-proteins than MP, whereas with regard to NDPK, MP and MP 7 are similarly effective.

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Known nucleoside diphosphate kinases (NDPKs) are oligomers of 17±23-kDa subunits and catalyze the reaction N 1 TP + N 2 DP 3 N 1 DP + N 2 TP via formation of a histidine-phosphorylated enzyme intermediate. NDPKs are involved in the activation of heterotrimeric GTP-binding proteins (G-proteins) by catalyzing the formation of GTP from GDP, but the properties of G-protein-associated NDPKs are still incompletely known. The aim of our present study was to characterize NDPK in soluble preparations of the retinal G-protein transducin. The NDPK is operationally referred to as transducin-NDPK. Like known NDPKs, transducin-NDPK utilizes NTPs and phosphorothioate analogs of NTPs as substrates. GDP was a more effective phosphoryl group acceptor at transducin-NDPK than ADP and CDP, and guanosine 5 H - [g-thio] Keywords: G-protein b-subunit; guanine nucleotides; nucleoside diphosphate kinase; trans(thio)phosphorylation; transducin.Known nucleoside diphosphate kinases (NDPKs) are oligomeric enzymes consisting of 17±23-kDa subunits [1±4]. NDPKs are bivalent cation-dependent enzymes which catalyze the reaction N 1 TP + N 2 DP 3 N 1 DP + N 2 TP via formation of a histidinephosphorylated enzyme intermediate [5±9]. Another biochemical property of known NDPKs is that they are activated by the cationic-amphiphilic peptides mastoparan and mastoparan 7 [7,10±12]. NDPKs are crucial for homeostasis of cellular NDP and NTP levels, are involved in complex cellular processes such as differentiation and tumorigenesis and regulate activation of heterotrimeric GTP-binding proteins (G-proteins) by catalyzing the formation of GTP from GDP [5,13±16]. Although the functional role of trans(thio)phosphorylation reactions in G-protein activation has been well studied in numerous systems (see [13,15] for review), the biochemical properties of G-proteinassociated NDPKs are still incompletely known. The question whether (thio)phosphorylation of G-protein b-subunits is involved in NDPK reactions is unanswered, too [17±23].The aim of our present study was to learn more about the biochemical properties of a G-protein-associated NDPK. For the following reasons, we decided to use the retinal G-protein transducin as a model. First, transducin is a prototypical G-protein, and rod outer segments (ROS) and transducin preparations contain NDPK activity [24±27]. Second, transducin can be isolated in large amounts from ROS [28]. Third, transducin is a suitable system for studying (thio)phosphorylation of G-protein b-subunits [17,18]. [b,g-imido]triphosphate; ROS, retinal rod outer segment; transducin-a, a-subunit of transducin; transducin-b, b-subunit of transducin; transducin-bg, bg-complex of transducin; transducin-NDPK, operational term for the nucleoside diphosphate kinase activity present in soluble transducin preparations. Enzymes: guanosine 5 H -triphosphatase (EC3.6.1.-); nucleoside diphosphate kinase (EC2.7.4.6). MATERIALS AND METHODS Materials