TY - JOUR
T1 - Purification and characterization of a novel insulin-sensitive GTP-binding protein, G(IR), from human placenta
AU - Singh, U. S.
AU - Srivastava, S. K.
PY - 1991
Y1 - 1991
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0025863132&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0025863132&partnerID=8YFLogxK
M3 - Article
AN - SCOPUS:0025863132
SN - 0892-2187
VL - 3
SP - 369
EP - 381
JO - Clinical Chemistry and Enzymology Communications
JF - Clinical Chemistry and Enzymology Communications
IS - 6
ER -