Isolation of bovine aortic endothelial cell plasma membranes: identification of membrane-associated cytoskeletal proteins.

The plasma membrane of bovine aortic endothelium was isolated, characterized, and found to contain at least four membrane-associated cytoskeletal proteins. Exposure of the plasma membranes to salt media (up to 1M KCl) resulted in the release of 30% of the total plasma membrane-associated proteins and extraction with 1% Triton X-100, 60%. At least four heavily glycosylated bands (185-, 165-, 150-, and 130,000 mol-wt) were evident. The Triton-insoluble pellet fraction contained several major polypeptides (30-, 43-, 58-, and 240,000 mol-wt), two of which were identified by immunoblotting as cytoplasmic actin (43,000 mol-st) and vimentin (58,000 mol-wt). Strikingly, vimentin and a 240,000 mol-wt polypeptide were routinely present in approximately a mole ratio of 4:1 in more than 60% of the plasma membrane preparations. We also report the presence of a 2.1-like and a 4.1-like protein associated with plasma membranes. The 2.1-like protein demonstrated similar solubilities and apparent molecular weight (210,000) as erythroid protein 2.1. Likewise, the endothelial 4.1-like protein exhibited similar solubilities and apparent molecular weight as erythroid protein 4.1. Immunofluorescence staining of fixed and permeabilized cultures with anti-2.1 antibodies showed a fibrillar pattern. In contrast, cells stained with anti-protein 4.1 were brightly fluorescent, bearing both a diffuse and punctate pattern. This paper presents several novel observations pertaining to the composition of bovine aortic endothelial cell plasma membranes, namely: the presence of two erythroid-like cytoskeletal polypeptides; the presence of vimentin and a 240,000 mol-wt polypeptide in a 4:1 mole ratio in more than 60% of the plasma membrane preparations and the co-elution in a 4:1 mol ratio with a protein perturbant; and the inability to release actin from the plasma membrane preparations, suggesting the association of actin with other molecules in the plasma membrane preparation.