Rapid separation of cytosol and particle fraction of human platelets by digitonin-induced cell damage.
Current cell disruption and fractionation techniques are time consuming and unsuitable for metabolic studies. We have developed a rapid method for platelets in which separation of cytosol and particle fraction is obtained within 50 s. Isolated platelet suspensions were incubated with low concentrations of digitonin followed by separation of soluble and particle fraction by centrifugation through a phthalate layer. Cell disruption was 90.1+/-4.2% (mean+/-SD, n=18; lactate dehydrogenase leakage). Contamination of granules: acid hydrolase vesicles 16.2+/-3.6% (n=18, beta-N-acetylglucosaminidase), dense granules 7--9% (n=3, 14C-serotonin), mitochondrial matrix 0.6+/-0.1% (n=18, glutamate dehydrogenase). Low concentrations of digitonin did not affect sialic acid content, nucleoside diphosphate kinase and phosphodiesterase activity in isolated membranes. The method showed that most enzymes of glycolysis and hexose monophosphate shunt were localized in the cytosol except for hexokinase (96% particle bound), phosphoglucose isomerase (10% bound) and glutathion reductase (26% bound). About half the total ATP+ADP and most glycolytic intermediates were found partly particle bound, especially fructose 1,6-diphosphate (40% bound). The data suggest that in platelets glycolysis occurs in different cell compartments.
D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure*; D002458:Cell Fractionation / Q000379:methods*; D003600:Cytosol / Q000201:enzymology; D004072:Digitonin / Q000494:pharmacology; D006019:Glycolysis; D006600:Hexosephosphates / Q000097:blood; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000097:blood; D008854:Microscopy, Electron; D010976:Platelet Count
D006600:Hexosephosphates; D007770:L-Lactate Dehydrogenase; D004072:Digitonin