Demonstration of putative subunit(s) of a novel insulin receptor-associated 66 kDa GTP-binding protein, Gir

Biochem Mol Biol Int. 1993 Jul;30(3):517-23.

Abstract

A 66 kDa GTP-binding protein, Gir, and insulin receptor (IR) were copurified from human placental membrane by DEAE-Sephacel and Wheat Germ Agglutinin (WGA)-Sepharose affinity chromatography. The WGA-fraction containing IR and Gir (IR-Gir-fraction) was phosphorylated (95 kDa IR-beta and 66 kDa Gir) by IR-tyrosine kinase using [32P]ATP or photolabeled with [32P]8-azido-GTP (mainly 66 kDa), and was cross-linked with a bifunctional reagent, bis-[sulfosuccinimidyl] suberate (BS3). The Gir cross-linked with putative subunit(s) to form a 110 kDa complex. Phosphorylation as well as 8-azido-GTP binding to Gir was not affected by cross-linking indicating that like other G-Proteins, Gir may also have subunits and that cross-linking of Gir with its putative subunits(s) does not block GTP-binding and phosphorylation sites.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Azides / metabolism
  • Binding Sites
  • Chromatography, Affinity
  • Cross-Linking Reagents
  • GTP-Binding Proteins / chemistry*
  • GTP-Binding Proteins / isolation & purification
  • GTP-Binding Proteins / metabolism
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Guanosine Triphosphate / analogs & derivatives
  • Guanosine Triphosphate / metabolism
  • Humans
  • Phosphorylation
  • Pregnancy Proteins / chemistry*
  • Pregnancy Proteins / isolation & purification
  • Pregnancy Proteins / metabolism
  • Protein Conformation*
  • Protein-Tyrosine Kinases / metabolism
  • Receptor, Insulin / chemistry*
  • Receptor, Insulin / isolation & purification
  • Receptor, Insulin / metabolism

Substances

  • 8-azidoguanosine triphosphate
  • Azides
  • Cross-Linking Reagents
  • Pregnancy Proteins
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Guanosine Triphosphate
  • Protein-Tyrosine Kinases
  • Receptor, Insulin
  • GTP-Binding Proteins