Nucleoside diphosphate kinase activity in soluble transducin preparations

Abstract

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

Regulation of G proteins by covalent modification

“…Sensitivity of the incorporated phosphate to HCl, hydroxylamine, and heat, and stability in NaOH, are consistent with a phosphoramidate linkage, implying histidine as the site of phosphorylation. The enzyme catalyzing the phosphorylation has not been identi®ed but may be a nucleoside diphosphate kinase (Klinker and Seifert, 1999). Thiophosphorylated bg can serve as an intermediate in the formation of GTPgS when presented with GDP, which might explain, if the GTPgS subsequently binds to Ga subunits, why it would decrease high-anity fMet-Leu-Phe binding to HL60 membranes (Wieland et al, 1991) or alternately stimulate or inhibit adenylyl cyclase in platelet membranes (Wieland et al, 1992).…”

Distinct Interactions of GTP, UTP, and CTP with GsProteins

“…UTPase and CTPase activities in Sf9 membranes were determined NDPK Activity-NDPK activity in S49 wild-type lymphoma membranes and Sf9 membranes was determined as described for soluble transducin preparations with modifications (28). Briefly, reaction mixtures contained S49 membranes (5.0 -10.0 g of protein/tube) or Sf9 membranes (0.5-1.0 g of protein/tube), NTPs at various concentrations, 0.1% (mass/volume) bovine serum albumin, 1 mM MgCl 2 , and 0.1 mM EDTA in 50 mM Tris-HCl, pH 7.4.…”

“…To obtain blank values, tubes containing all components described above except for membranes were processed in parallel. Stopping of reactions, separation of nucleotides on poly(ethyleneimine)-cellulose TLC plates (Schleicher & Schuell), elution of nucleotides, and counting of radioactivity were performed exactly as described (28).…”

Advances in development of new tools for the study of phosphohistidine

“…Two isoforms of nucleoside diphosphate kinase (NDPK): Nme1/NDPK-A [9][10][11][12][13] and Nme2/NDPK-B [14][15][16][17][18][19] have been characterized as mammalian His kinases. Other mammalian His kinases exist.…”