Activation of Heterotrimeric G Proteins by a High Energy Phosphate Transfer via Nucleoside Diphosphate Kinase (NDPK) B and Gβ Subunits: COMPLEX FORMATION OF NDPK B WITH Gβγ DIMERS AND PHOSPHORYLATION OF His-266 IN Gβ *

摘要: G protein betagamma dimers can be phosphorylated in membranes from various tissues by GTP at a histidine residue the beta subunit. The phosphate is high energetic and transferred onto GDP leading to formation of GTP. Purified Gbetagamma do not display autophosphorylation, indicating involvement separate kinase. We therefore enriched Gbeta-phosphorylating activity present preparations retinal transducin partially purified G(i/o) proteins bovine brain. Immunoblots, enzymatic measurements demonstrated nucleoside diphosphate kinase (NDPK) B both preparations, together with residual dimers. In NDPK B-enriched fractions, Gbeta-specific antiserum co-precipitated B, an against human Gbetagamma. addition, NDPK-containing fractions brain reconstituted phosphorylation For identification residue, G(t)betagamma were thiophosphorylated guanosine 5'-O-(3-[(35)S]thio)triphosphate, followed digestion endoproteinase Glu-C trypsin, separation resulting peptides gel electrophoresis pressure liquid chromatography, respectively, sequencing radioactive peptides. sequence information produced methods identified specific labeled fragments Gbeta(1) that overlapped heptapeptide, Leu-Met-Thr-Tyr-Ser-His-Asp (amino acids 261-267). conclude forms complexes contributes activation increasing transfer via intermediately His-266 subunits.