title: Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C.
abstract: We have assessed the binding of [alpha-32P]GTP 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-32P]GTP 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-thio]triphosphate (GTP[gamma 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-32P]GTP to the membrane-bound protein. GTP[gamma 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 GTP[gamma S].
authors.forename: E G
authors.initials: EG
authors.lastname: Lapetina
authors.forename: B R
authors.initials: BR
authors.lastname: Reep
mesh_terms: D001792:Blood Platelets / Q000201:enzymology*; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000097:blood*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010738:Type C Phospholipases / Q000097:blood*
chemical_list: D001798:Blood Proteins; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D006160:Guanosine Triphosphate; D010738:Type C Phospholipases