Purification and characterization of a low M(r) GTP-binding protein, c25KG, from human platelet membranes.
A low M(r) GTP-binding protein with a M(r) of 26,000 has been purified from a sodium cholate extract of human platelet membranes by using an antibody raised against a synthetic peptide of c25KG, which was previously purified from human platelet cytosol (Nagata, N., et al. (1989) J. Biol. Chem. 264, 17000-17005). The M(r) of membranous c25KG (m-c25KG) was slightly higher than that from cytosolic c25KG (M(r) 25,000) and calculated to be 26,000. It was suggested that m-c25KG contains an equimolar amount of GDP. The purified protein could bind approx. 1 mol of [35S]guanosine 5'-O-(thiotriphosphate)(GTP gamma S)/mol of protein, with a Kd value of 50 nM. [35S]GTP gamma S-binding to this protein was inhibited by GTP and GDP, but not by ATP and ADP, showing that the binding is specific for guanine. In the presence of 10 mM Mg2+, the dissociation of [8,5'-3H]GDP from the m-c25KG occurred with a rate of 0.01 min-1. The rate of release of Pi from [gamma-32P]GTP-bound m-c25KG was calculated to be 0.03 min-1. These results indicate that c25KG is also present in membrane fraction of human platelet which has very similar biochemical properties in those of the cytosolic type.
Nagata
K
K
Department of Biochemistry, Gifu University School of Medicine, Japan.
Hattori
T
T
Hachiya
T
T
Takahashi
T
T
Nozawa
Y
Y
D000918:Antibody Specificity; D001792:Blood Platelets / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000276:immunology / Q000302:isolation & purification*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification*
D008565:Membrane Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins