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.

Abstract. Heat-shock proteins from confuent primary cultures of bovine aortic endothelial cells were analyzed by SDS-polyacrylamide gels. In addition to the increased synthesis of the classical heat-shock proteins, there is an increase of a 180,000-mol wt polypeptide in the growth media of heat-shocked cells. Immunoprecipitation with specific antiserum indicates that the 180,000-mol wt polypeptide is thrombospondin. Assay of mRNA levels coding for thrombospondin after brief hyperthermic treatment (45~ 10 min), followed by a recovery of 2 h at 37~ results in a twofold increase in mRNA abundance. In contrast, the activation level of the 71,000-mol wt heat-shock protein mRNA occurs at an earlier time than for thrombospondin mRNA. Immunofluorescence microscopy was used to study the intracellular and extracellular distribution of thrombospondin. Thrombospondin is localized to a prominent pattern of granules of intracellular fluorescence in a perinuclear distribution in cells not exposed to heat. Upon heat treatment, the pattern of granules of intracellular fluorescence appears more pronounced, and the fluorescence appears to be clustered more about the nucleus. There are at least three pools of extracellular forms of thrombospondin: (a) the fine fibrillar extracellular matrix thrombospondin; (b) the punctate granular thrombospondin; and (c) the thrombospondin found in the conditioned medium not associated with the extracellular matrix. When bovine aortic endothelial cells are exposed to heat, the extracellular matrix staining of a fibrillar nature is noticeably decreased, with an increase in the number and degree of fluorescence of focal areas where the punctate granule thrombospondin structures are highly localized. No gross morphological changes in extracellular matrix staining of fibronectin was noted. However, the intermediate filament network was very sensitive and collapsed around the nucleus after heat shock. We conclude that the expression of thrombospondin is heat-shock stimulated.

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Unlabeled WGA was obtained from Sigma and 3H-labeled WGA was from New England Nuclear. Bovine serum albumin was either ultra-pure, globulin-free or was the 96-99% fraction V from Sigma-both gave the same results.Preparation of Bilayer Structures. Six milligrams of cholesterol/dimyristoyl phosphatidylcholine (1.5:4 mol ratio) was dissolved in 1 ml of 2-chloroethanol (Eastman reagent grade). An equal volume of water containing either 0 or 3 mg of glycophorin was added dropwise to this solution. The solvent was removed on a rotary evaporator to produce a film on the inside of a round bottom flask, and this film was dried overnight in a desiccator connected to a rotary pump. Lipid films were hydrated overnight at 370C with phosphate-buffered saline (pH 7.4) containing Ca2+ and Mg2+. The flask periodically was shaken gently during several hours prior to liposome isolation by repeated differential centrifugation (supernatant material discarded) at 850 X g. When liposomes were to have a surface layer of albumin, 3 mg of it was included per ml in all buffers after the initial isolation.Liposome composition was monitored by the addition of After the incubation, N-acetylglucosamine (0.1 M) was added to one set of the duplicate samples. These were the controls. The incubation mixtures were then layered gently onto 10 ml of phosphate-buffered saline in Corning 15-ml disposable plastic centrifuge tubes. The tubes were centrifuged for 5 min at 850 X g and the pellets were washed twice more. Radioactivity in control pellets was substracted as nonspecific binding (which was never more than 10% of the specific binding). Plastic tubes used in the assay did not appreciably adsorb WGA with concentrations of total protein down to 1 ,ug/ml.

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