Cell Surface Radiolabeling and Electrophoretic Analysis of Glycoproteins Exposed at the Blood-Sinusoidal Surface of Rat Hepatocytes

Abstract

Plasma membrane glycoproteins of rat hepatocytes that had been exposed to subendothelial spaces of Disse were selectively radiolabeled by reduction with tritiated sodium borohydride after oxidation with neuraminidase plus galactose oxidase. The labeled glycoproteins were separated by polyacrylamide gel electrophoresis and located by fluorography.Selective treatment with galactose oxidase and neuraminidase of glycoproteins and glycolipids exposed to spaces of Disse was made possible by perfusion of rat liver with an oxygenated phosphate-buffered saline containing the enzymes. After the perfusion, the blood-sinusoidal plasma membrane of hepatocytes was isolated from both the microsomal and nuclear pellets by the method of Touster et al. (J. Cell Biol. (1970) 47, 604-618). The carbon-6 aldehydes present in the plasma membrane subfraction were reduced back to galactose and N-acetylgalactosamine with tritiated borohydride.One-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulfate resolved many 3H-labeled components ; none of these components was labeled to a significant degree when galactose oxidase was not added to the perfusion medium. About forty [3H]glycoproteins were resolved by twodimensional polyacrylamide gel electrophoresis according to the method of Imada et al. (Biochim. Biophys. Acta (1978) 507, 459-469). These [3H]glycoproteins were fractionated by a modification of O'Farrell's method.