PROTEIN AND FATTY-ACID COMPOSITION OF CAVEOLAE FROM APICAL PLASMALEMMA OF AORTIC ENDOTHELIAL-CELLS

In endothelial cells (EC), caveolae or plasmalemmal vesicles (PVs) rep resent a structurally and biochemically specialized membrane microdoma in. Since few data are available on the biochemical composition of PVs of large vessel endothelium, we have designed experiments to isolate this domain and to analyze its chemical components. A highly purified apical membrane fraction was obtained from cultured bovine aortic EC b y using cationic colloidal silica (silica-ap), or the EC were surface- radioiodinated and a cell homogenate was prepared. Detergent treatment (Triton X-100; TX) and mechanical disruption of both the silica-ap fr action and cell homogenate followed by ultracentrifugation on a sucros e gradient gave detergent-soluble and detergent-insoluble membranous f ractions. The lowest density TX-insoluble fraction appeared morphologi cally as distinct vesicles (caveolae; 60 nm average diameter; PVs frac tion). Biochemical characterization of the PVs fraction (by comparison with the soluble fraction) revealed the presence, at high concentrati on, of specific caveolar markers, viz., caveolin (both isoforms, the 2 4-kDa form being conspicuously more abundant) and Ca2+-ATPase. By cont rast, angiotensin-converting enzyme and alkaline phosphodiesterase wer e present almost exclusively in the TX-soluble fraction. The glycoprot eins in the PVs fraction were of apparent molecular weights 52, 68, 95 , and 114kDa. Analysis of the fatty acid composition revealed more pal mitoleic and stearic acid in the PVs fraction then in the TX-soluble f raction. Thus, in comparison with the plasmalemma proper, the PVs frac tion (1) is detergent-insoluble; (2) contains caveolin in two isoforms ; (3) contains Ca2+-ATPase at high concentration; (4) contains a set o f specific glycoproteins; and (5) is enriched in palmitoleic and stear ic acids.