Isolation and characterization of the urothelial lumenal plasma membrane

Abstract

The lumenal plasma membrane has been isolated from transitional epithelial cells (urothelium) lining the urinary bladder in sheep by a modified technique involving treatment with hypotonic thioglycolate. The isolated membranes, like those in situ, are distinguished morphologically by arrays of hexagonal particles (in plague regions) separated by smooth interplaque regions. These plaque regions, specifically, can be isolated from the lumenal plasma membrane. Of the proteins constituting the lumenal plasma membrane, five were found to characterize the plaque regions and, in particular, the 33,000-dalton species appears to be most heavily concentrated in the sodium dodecyl sulfate-polyacrylamide gel pattern of the isolated plaque regions. Lipid analyses showed that there are approximately 0.93 mg of phospholipid and 0.27 mg of cholesterol for each milligram of protein, giving a value of 55% lipids and 45% proteins for the composition of the lumenal plasma membrane. The total sialic acid content was measured to be approximately 0.038 micronmol/mg protein for the plasma membrane. Several plasma membrane marker enzymes were found to be associated with the lumenal plasma membrane fraction, but only the 5'-nucleotidase activity was found to be further enriched in the plaque region fraction. Amino acid analysis of the intrinsic proteins of the plaques indicated a polarity index of 45%.