Specific binding of alpha-32PGTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C.

We have assessed the binding of alpha-32PGTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO4/PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with alpha-32PGTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-gamma-thiotriphosphate (GTPgamma S) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of alpha-32PGTP to the membrane-bound protein. GTPgamma S still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTPgamma S.