Guanine nucleotide and agonist-induced hydrolysis of phosphoinositides in isolated platelet membranes
Rabbit platelets, labelled by incubation in Ca/sup 2 +/-free Tyrode's solution containing myo-(/sup 3/H)inositol, were lysed by freezing and thawing in a medium (pH 7.4) containing 100 mM KCl, 25 mM Hepes, 2.5 mM EGTA, 1 mM MgATP and 0.5 mM Mg/sup 2 +//sub free/. The membrane fraction was washed twice at 4/sup 0/C in the same medium without KCl and then resuspended in medium with KCl. Samples were incubated for 10 min at 25/sup 0/C. Addition of CaCl/sub 2/ to give 0.1 ..mu..M Ca/sup 2 +//sub free/ caused the formation of (/sup 3/H)inositol monophosphate (IP) and bisphosphate (IP/sub 2/) in a ratio of about 1:3. These reactions were stimulated on average 3-fold by addition of 10 ..mu..M GTP..gamma..S (EC/sub 50/, 0.1 ..mu..M). GTP..gamma..S was ineffective in the absence of Ca/sup 2 +/. Omission of MgATP and Mg/sup 2 +/ from membrane preparations and incubations led to major losses of (/sup 3/H)polyphosphoinositides and marked decreases in the formation of both IP and IP/sub 2/ with GTP..gamma..S. Only trace amounts of inositol trisphosphate accumulated, whether Mg/sup 2 +/ was present or not. GTP (>4 ..mu..M) also increased the formation of IP and IP/sub 2/, but to a lesser extent than GTP..gamma..S. Thrombin (2 units/ml) and PAF (0.2 ..mu..M) had no effects alone, but in the presence of 1-4 ..mu..M GTP stimulated IP/sub 2/ formation to the same extent as 10 ..mu..M GTP..gamma..S. The results show that a G-protein mediates the stimulation of phosphoinositide breakdown by platelet agonists.
- Research Organization:
- McMaster Univ., Hamilton, Ontario
- OSTI ID:
- 6986065
- Report Number(s):
- CONF-8606151-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
AMINES
ANIMALS
AROMATICS
AZAARENES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BLOOD PLATELETS
BODY FLUIDS
CALCIUM COMPOUNDS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
DECOMPOSITION
ESTERS
GUANINE
HETEROCYCLIC COMPOUNDS
HYDROLYSIS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIPIDS
LYSIS
MAMMALS
MATERIALS
MEMBRANES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PHOSPHOLIPIDS
PURINES
RABBITS
REACTION KINETICS
SOLVOLYSIS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
VERTEBRATES