Characterization of Plasma Membrane Proteins in Mammalian Kidney

A simplified method has been developed for the separation of plasma membrane material from isotonic homogenates of rat kidney. When examined by electron microscopy, the preparation consists of membrane vesicles free of nuclei and essentially free of intact mitochondria. Membranes have been solubilized by sonic oscillation or with Lubrol W-X detergent. Gel filtrate chromatography of solubilized membranes on 6% agarose gel yields two major protein peaks. The first protein peak elutes in the void volume and contains macromolecules with a molecular weight exceeding 1,000,000. The second protein peak has an elution profile similar to proteins of approximately 45,000 molecular weight. Sodium-potassium-dependent, ouabain-sensitive ATPase activity is associated with fractions of the first peak. Protein material associated with the second peak is mostly insoluble when membranes are extracted with 1-butanol. The bulk of the protein material associated with the second peak is solubilized when membranes are extracted with NaI. When proteins eluted from agarose gel columns are subjected to polyacrylamide disc gel electrophoresis, a portion of the high molecular weight protein in the first column peak moves, separating into five protein bands. The sodium-potassium-dependent ATPase activity appears to be associated with the nonmobile portion. All proteins in the low molecular weight second peak move, separating into more than eight bands. Incorporation in vivo of radioactive leucine into membrane protein was measured. After column fractionation a considerable variation in radioactivity of membrane fractions was observed. The findings in this study indicate heterogeneity of membrane protein constituents.