Effects of detergents on erythrocyte membranes: different patterns of solubilization of the membrane proteins by dihydroxy and trihydroxy bile salts.

enzymes is due to a more specific process than generalizedcell damage or loss (Holdsworth & Coleman, 1975). We have now attempted to make an initial separation of these enzymes from other biliary proteins, before investigating their relationship with the bile-canaliculus region of the hepatocyte plasma membrane. Fresh sheep bile was treated with solid protamine sulphate and centrifuged to remove the majority of pigments and lipids. The supernatant was concentrated tenfold with a Diaflo membrane and then applied to columns (1.2cm x 8.0cm) of concanavalin A-Sepharose 4B, equilibrated with 0.1 % deoxycholate in 0.1 M-Tris-HC1, pH 7.7. Almost all the biliary protein was removed by washing the columns with the equilibrating buffer. A small amount of protein containing the enzymes was eluted from the column with a-methyl D-glucoside, suggesting the presence of glycoproteins. The appearance of bands, stainable with periodic acid-Schiff reagent, after polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate, provided further evidence for the presence of glycoproteins. When a discontinuous gradient of a-methyl Dglucoside was used to elute the affinity column, 5’-nucleotidase and alkaline phosphatase were eluted before alkaline phosphodiesterase I and L-leucine 8-naphthylamidase. All four enzymes have been identified as glycoproteins in preparations containing liver plasma membranes (Evans, 1974; Evans & Gurd, 1973; Kaplan, 1972; Sternes & Behal, 1974). The glycoprotein nature of these same enzymes in bile not only gives a useful preliminary stage in their purification, but also indicates a possible relationship between the membrane-bound and biliary enzymes. Isolation and purification of some of these enzymes from liver plasma membranes involves an initial solubilization of the enzymes with detergents (Evans & Gurd, 1973; Evans, 1974; Widnell & Unkeless, 1968). It is possible that the release of enzymes into the bile is also related to a solubilization with detergents, since an important stage in bile formation is the output of bile salts from the liver cell and their accumulation in the bile canaliculus.