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Specific binding of (alpha-/sup 32/P)GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C

Journal Article · · Proc. Natl. Acad. Sci. U.S.A.; (United States)
;
We have assessed the binding of (alpha-/sup 32/P)GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO/sub 4//PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with (alpha-/sup 32/P)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-/sup 32/P)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).
Research Organization:
Burroughs Wellcome Co., Research Triangle Park, NC
OSTI ID:
6793275
Journal Information:
Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 8; ISSN PNASA
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BLOOD PLATELETS
BODY FLUIDS
CARBOXYLESTERASES
CELL CONSTITUENTS
CELL MEMBRANES
DAYS LIVING RADIOISOTOPES
ELECTROPHORESIS
ENZYME ACTIVITY
ENZYMES
ESTERASES
GUANYLIC ACID
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
LIPASE
MAMMALS
MAN
MATERIALS
MEMBRANES
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PRIMATES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
SERINE PROTEINASES
TRACER TECHNIQUES
TRYPSIN
VERTEBRATES