Isolation of rat hepatocyte plasma membranes. II. Identification of membrane-associated cytoskeletal proteins.

Abstract

Rat liver plasma membranes were isolated as presented in a previous paper and found to contain many filaments associated both with desmosomes along the lateral surface and with the cytoplasmic aspects of membranes comprising each of the 3 domains (lateral [LS], bile canalicular [BC] and sinusoidal [SF]). Exposure of the plasma membranes to alkaline media (up to pH 11) resulted in loss of recognizable filaments without loss of domain morphology or membrane enzyme activities. Electrophoretic analysis of solubilized components from control and alkaline-extracted plasma membranes revealed that 3 major polypeptides present at 43, 52 and 56 kdaltons in the control were were released by alkaline treatment (pH 11) and could be quantitatively recovered in the supernate. The 43-kdalton component was identified as cytoplasmic actin by comparison of its tryptic 125I-peptide map to those of muscle (.alpha.) and brush border (.beta., .gamma.) actins. The 52- and 56-kdalton polypeptides were identified as tonofilament components by their solubility properties and their ability to reassemble into 9.5-nm filaments from monomers present in an alkaline extract.