Rapid Separation of Cytosol and Particle Fraction of Human Platelets by Digitonin‐induced Cell Damage

Abstract

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.

Compartmentation of hexokinase in human blood cells characterization of soluble and particulate enzymes

“…Summarized, the results show that the digitonin-lysis technique works equally well for erythrocytes, lymphocytes and granulocytes as was documented for platelets [9].…”

“…The intracellular localization of hexokinase was studied by controlled digitonin induced cell lysis followed by discontinuous gradient centrifugation as described by Akkerman et al [9] for platelets. In this technique disturbances in metabolism after cell lysis are minimized by separating soluble and particulate fractions within 1 rain.…”

“…Freshly drawn human venous blood from healthy donors was collected in acid/ citrate/dextrose (final citrate concentration 13 mM) and centrifuged (10 rain, 200 x g, 22°C). Platelets were isolated by gelfiltration at room temperature in Ca-free tyrode solution [9]. After centrifugation in the presence of 9 mM EDTA (10 min, 800 X g, 22°C) the platelets were suspended in Ca-free Tyrode solution to concentrations varying between 2 and 10 x 105 cells/~tl.…”

“…Cytosolic and bound hexokinase were separated by discontinuous gradient centrifugation after controlled digitonin-induced lysis as described for human platelets by Akkerman et al [9]. This procedure enables the isolation of cytosol and particulate fractions within 1 rain after cell lysis.…”

“…damage to blood vessel walls. They have a high glycolytic activity in order to meet the energy demands of aggregation and secretion [5] and it was shown that virtually all hexokinase is localized in the particulate fraction of platelets [9]. Lymphocytes are relatively dormant cells and transform rather slowly to metabolically active lymphoblasts when they are stimulated.…”