Purification and characterization of a novel insulin-sensitive GTP-binding protein, G(IR), from human placenta

U. S. Singh, S. K. Srivastava

Research output: Contribution to journalArticle

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Abstract

Using minor modifications of the procedures developed to purify GTP binding protein (G-proteins) from rabbit liver, we have purified a novel G-protein [G(IR)] from human placental membranes. Placental membranes were solubilized and the known G-proteins such as G(s), G(i) and G(p) were eluted from the DEAE-Sephacel column by a gradient of sodium chloride (0-225 mM). Elution of G(IR) together with insulin receptor was accomplished at 1 M sodium chloride. Further purification of G(IR) and insulin receptor to apparent homogeneity was achieved by wheat germ agglutinin (WGA)-Sepharose, insulin-Sepharose, Sephadex G-200 and hydroxylapatite column chromatography. Molecular weight of G(IR) as determined by Ultrogel AcA34 filtration was found to be 150 kDa. By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the final G(IR) preparation separated into three major protein bands of molecular weights, 66 kDa, 64 kDa and 10 kDa. It was found to have GTPase (K(m) GTP = 225 nM and V(max) = 2.25 nmoles mg-1 hr-1) and GTPγS binding (K(D) 2 nM) activities. Binding of GTPγS to G(IR) (maximum binding at approximately 10 μM GTPγS) was dependent on magnesium chloride, and was inhibited significantly by 50 μM GTP, GDP and ITP. The guanine nucleotide binding site, as identified by a photolyzable GTP analogue, [32P] 8-azido GTP, was associated with the 66 kDa protein. Cholera toxin did not ADP-ribosylate G(IR) but pertussis toxin ribosylated G(IR) (66 kDa protein). Along with stimulating phosphorylation of 66 kDa protein, insulin also increased GTPγS binding, indicating a possible role of G(IR) in insulin signal transduction.

Original languageEnglish (US)
Pages (from-to)369-381
Number of pages13
JournalClinical Chemistry and Enzymology Communications
Volume3
Issue number6
StatePublished - May 14 1991

ASJC Scopus subject areas

  • Clinical Biochemistry

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