Preparation and characterization of plasma membranes from bovine corpus luteum.

A rapid method for preparing plasma membranes from bovine corpus luteum is described. Various fractions are isolated by differential centrifugation and discontinuous sucrose density gradients. The plasma membrane fractions have a density in sucrose of 1.16 and 1.18. The yield is 0.5 to 0.75 mg per g of corpus luteum. This fraction is characterized by electron microscopy, enzymatic assay, and binding properties of lz51-luteinizing hormone. The plasma membrane fraction is free of nuclei and mitochondria when examined by electron microscopy. It is mainly vesicular in nature, with a unit membrane structure evident in most sections. The cholesterol to phospholipid ratio is 0.72. A small amount of RNA is found to be associated with the plasma membrane but no DNA is detected. 5’-Nucleotidase and ouabain-sensitive ATPase are concentrated in the plasma membrane. No more than 3 to 8% contamination by mitochondria is indicated by the activity of the mitochondrial markers: cytochrome c oxidase and succjnate dehydrogenase. The microsomal marker activities of glucose 6-phosphatase and glucose-6-P dehydrogenase are low, corresponding to 2 to 5 % contamination by microsomes. The binding of 1251-luteinizing hormone to the plasma membrane fraction is 20 times greater than its binding to the homogenate. The binding of 1251-luteinizing hormone to nuclei, mitochondria, and microsomes is low and parallels the 5’-nucleotidase activity of those fractions. It probably reflects their contamination by plasma membranes.