Isolation and Characterization of Sertoli Cell Plasma Membranes and Associated Plasminogen Activator Activity 1

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Abstract
Plasma membranes were isolated from the cultured Sertoli cells of 20-day-old rat testes by differential centrifugation and sucrose density fractionation. The distribution and purity of subcellular components was determined by marker enzyme analysis of gradient fractions. The plasma membrane fraction showed an enrichment in 2 plasma membrane marker enzymes, 5''-nucleotidase and ouabain-sensitive Na+/K+-ATPase-specific activities, of 9- and 23-fold, respectively; 42% and 52% of the total cellular 5''-nucleotidase and ouabain-sensitive Na+/K+-ATPase activities, respectively, were found in the membrane fraction. The protein yield of plasma membrane was approximately 6% of the total cellular protein. Two-dimensional polyacrylamide gel electrophoresis was used to compare [35S]Met and [3H]glucosamine-labeled membrane proteins. The incorporation of [35S]Met and [3H]glucosamine was increased in several proteins when the cultured Sertoli cells were treated with FSH, insulin, retinol and testosterone. Isolated Sertoli cell membranes contained a membrane-associated form of plasminogen activator. Analysis of this plasminogen activator demonstrated that the membrane-associated enzyme existed priamrily as a single 38,000-40,000-Mr form.