Isolation and characterization of proteoheparan sulfate from plasma membranes of an ascites hepatoma, AH 66

A proteoglycan was isolated from plasma membranes prepared from AH 66 cells by the following procedure. The plasma membranes were isolated from cells according to the method devised by Funakoshi and Yamashina (1976) J. Biochem. 80, 1185-1193), then the membranes were made lipid-free. The lipid-free membranes were solubilized with 5 mM sodium phosphate buffer, pH 7.0, containing 0.5% sodium dodecyl sulfate (SDS), then the solution was fractionated on a Sepharose CL 6B column. The proteoglycan eluted near the void volume fraction was further purified by repeated precipitation with cetylpyridinium chloride (CPC). The proteoglycan isolated was homogeneous on electrophoresis on a cellulose acetate strip and was identified as proteoheparan sulfate. The preparation contained 10.6% protein, its amino acid composition being characterized by high contents of glutamic acid, aspartic acid, proline, glycine, threonine, and serine.