Platelet Glycolytic Enzymes: Effect of Cellular Disruption Procedures on Activity

Abstract

S ummary The activities and cellular distribution of the following glycolytic enzymes were determined in human platelets: hexokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, glyceraldehyde 3‐phosphate dehydrogenase, 3‐phosphoglycerate kinase, phosphoglyceromutase, enolase, pyruvate kinase, lactate dehydrogenase and glucose 6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase. The effect of four different methods of cellular disruption on platelet ultrastructure and enzyme activity was determined. Freezing and thawing was found to inactivate phosphofructokinase markedly, and to inactivate hexokinase and glucose‐6‐phosphate dehydrogenase slightly. In addition, glucose‐6‐phosphate dehydrogenase was rapidly inactivated in cell lysates irrespective of the method of lysis. This process, which was considerably greater at room temperature than 4°C, was prevented by addition of 0.2 mM‐NADP to the medium before disruption of the platelets. Spontaneous loss of activity of phosphofructokinase, triosephosphate isomerase and pyruvate kinase in cell lysates was decreased by the presence of the reducing agent, dithiothreitol. Hexokinase was found in both the soluble and particulate fraction. The proportions of the enzyme distributed between these two fractions depended on the disruption procedure used.