A domain-specific marker for the hepatocyte plasma membrane. III. Isolation of bile canalicular membrane by immunoadsorption.

Abstract

Previous immunolabeling studies established leucine aminopeptidase (LAP) as a specific marker for the bile canalicular (BC) domain of the rat hepatocyte plasma membrane (PM). In this study, membrane from a sonicated PM vesicle fraction was isolated using anti-LAP-coated Staphylococcus aureus cells as a solid-phase immunoadsorbent. The extent and specificity of the immunoadsorption were assessed by following the behavior of LAP (the BC marker) and 32P-labeled membrane phospholipids (a uniform membrane marker). The BC fraction obtained was significantly enriched in LAP (yield: > 70% of PM-LAP). Alkaline phosphatase, 5''-nucleotidase and a 110,000-dalton glycoprotein, HA-4, were enriched in the BC fraction to the same extent as LAP (enzyme or antigen/LAP = 1.0). However, alkaline phosphodiesterase I was not enriched to the same degree (enzyme/LAP = 0.5). Contamination of this BC fraction by membrane derived from the sinusoidal domain and endoplasmic reticulum, as determined from the distribution of the asialoglycoprotein receptor and NADH cytochrome c reductase, respectively, was small (< 13%).