Nucleoside diphosphate kinase activity in soluble transducin preparations biochemical properties and possible role of transducin-beta as phosphorylated enzyme intermediate.

Known nucleoside diphosphate kinases (NDPKs) are oligomers of 17-23-kDa subunits and catalyze the reaction N1TP + N2DP --> N1DP + N2TP 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'-gamma-thio]triphosphate (GTPS]) was a more effective thiophosphoryl group donor than adenosine 5'-gamma-thio]triphosphate (ATPS]). In contrast with their action on known NDPKs, mastoparan and mastoparan 7 had no stimulatory effect on transducin-NDPK. Guanosine 5'-beta, gamma-imido]triphosphate (pNH]ppG) potentiated 3H]GTPS] formation from 3H]GDP and ATPS] but not 3H]GTPS] formation from 3H]GDP and GTPS]. Depending on the thiophosphoryl group acceptor and donor, 3H]NTPS] formation was differentially regulated by Mg2+, Mn2+, Co2+, Ca2+ and Zn2+. gamma-32P]ATP and gamma-32P]GTP 32P]phosphorylated, and 35S]ATPS] 35S]thiophosphorylated, a 36-kDa protein comigrating with transducin-beta. pNH]ppG potentiated 35S]thiophosphorylation of the 36-kDa protein. 32P-labeling of the 36-kDa protein showed characteristics of histidine phosphorylation. There was no evidence for (thio)phosphorylation of 17-23-kDa proteins. Our data show the following: (a) soluble transducin preparations contain a GDP-prefering and guanine nucleotide-regulated NDPK; (b) transducin-beta may serve as a (thio)phosphorylated NDPK intermediate; (c) transducin-NDPK is distinct from known NDPKs and may consist of multiple kinases or a single kinase with multiple regulatory domains.