SMALL MOLECULAR-WEIGHT GTP-BINDING PROTEINS IN HUMAN-PLATELET MEMBRANES - PURIFICATION AND CHARACTERIZATION OF A NOVEL GTP-BINDING PROTEIN WITH A MOLECULAR-WEIGHT OF 22,000

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  • 25 January 1989
    • journal article
    • research article
    • Vol. 264  (3) , 1877-1881
Abstract
When guanosine 5''-(3-O-[35S]thio)triphosphate (GTP.gamma.S)-binding activity was assayed in the particulate and cytosol fractions of human platelets, most activity was found in the particulate fraction. GTP-binding proteins (G proteins) were extracted from the particulate fraction by sodium cholate and purified by several column chromatographies. At least three G protein with Mr values of about 21,000, 22,000, and 24,000 (21K G, 22K G, and 24K G, respectively) were separated in addition to the stimulatory (GS) and inhibitory (Gi) regulatory GTP-binding proteins of adenylate cyclase. Among them, the amount of 22K G was more than 10-fold of those of other G proteins. 22K G was purified to near homogeneity and characterized. 22K G specifically bound GTP.gamma.S, GTP, and GDP, with a Kd value for GTP.gamma.S of about 50 nM. [35S]GTP.gamma.S binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. 22K G hydrolyzed GTP to liberate Pi, with a turnover number of 0.01 min-1. 22K G was not copurified with the .beta..gamma. subunits of GS and Gi and was not recognized by the antibodies against the ADP-ribosylation factor for Gs and the ras protein. The peptide map of 22K G was different from those of the smg-25A and rho proteins, which we have purified from bovine brain membranes. 21K G was identified to be the c-ras protein, but 24K G was unidentified. These results indicate that there are multiple G proteins in platelet membranes and that a novel G protein (22K G) is a major G protein in platelets.