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 (GTP[S]) was a more effective thiophosphoryl group donor than adenosine 5'-[gamma-thio]triphosphate (ATP[S]). 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 (p[NH]ppG) potentiated [3H]GTP[S] formation from [3H]GDP and ATP[S] but not [3H]GTP[S] formation from [3H]GDP and GTP[S]. Depending on the thiophosphoryl group acceptor and donor, [3H]NTP[S] formation was differentially regulated by Mg2+, Mn2+, Co2+, Ca2+ and Zn2+. [gamma-32P]ATP and [gamma-32P]GTP [32P]phosphorylated, and [35S]ATP[S] [35S]thiophosphorylated, a 36-kDa protein comigrating with transducin-beta. p[NH]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.
Klinker
J F
JF
Institut für Pharmakologie, Freie Universität Berlin, Germany.
Seifert
R
R
D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D004492:Edetic Acid; D006153:Guanosine Diphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D009701:Nucleoside-Diphosphate Kinase / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides; D010766:Phosphorylation; D011487:Protein Conformation; D012374:Rod Cell Outer Segment / Q000737:chemistry / Q000201:enzymology; D012995:Solubility; D015293:Transducin / Q000302:isolation & purification* / Q000378:metabolism; D014862:Wasp Venoms / Q000494:pharmacology
D002413:Cations, Divalent; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014862:Wasp Venoms; D006153:Guanosine Diphosphate; C037500:mastoparan; D004492:Edetic Acid; D009701:Nucleoside-Diphosphate Kinase; D015293:Transducin