Immunoisolation and partial characterization of endothelial plasmalemmal vesicles (caveolae).
Plasmalemmal vesicles (PVs) or caveolae are plasma membrane invaginations and associated vesicles of regular size and shape found in most mammalian cell types. They are particularly numerous in the continuous endothelium of certain microvascular beds (e.g., heart, lung, and muscles) in which they have been identified as transcytotic vesicular carriers. Their chemistry and function have been extensively studied in the last years by various means, including several attempts to isolate them by cell fractionation from different cell types. The methods so far used rely on nonspecific physical parameters of the caveolae and their membrane (e.g., size-specific gravity and solubility in detergents) which do not rule out contamination from other membrane sources, especially the plasmalemma proper. We report here a different method for the isolation of PVs from plasmalemmal fragments obtained by a silica-coating procedure from the rat lung vasculature. The method includes sonication and flotation of a mixed vesicle fraction, as the first step, followed by specific immunoisolation of PVs on anticaveolin-coated magnetic microspheres, as the second step. The mixed vesicle fraction, is thereby resolved into a bound subfraction (B), which consists primarily of PVs or caveolae, and a nonbound subfraction (NB) enriched in vesicles derived from the plasmalemma proper. The results so far obtained indicate that some specific endothelial membrane proteins (e.g., thrombomodulin, functional thrombin receptor) are distributed about evenly between the B and NB subfractions, whereas others are restricted to the NB subfraction (e.g., angiotensin converting enzyme, podocalyxin). Glycoproteins distribute unevenly between the two subfractions and antigens involved in signal transduction [e.g., annexin II, protein kinase C alpha, the G alpha subunits of heterotrimeric G proteins (alpha s, alpha q, alpha i2, alpha i3), small GTP-binding proteins, endothelial nitric oxide synthase, and nonreceptor protein kinase c-src] are concentrated in the NB (plasmalemma proper-enriched) subfraction rather than in the caveolae of the B subfraction. Additional work should show whether discrepancies between our findings and those already recorded in the literature represent inadequate fractionation techniques or are accounted for by chemical differentiation of caveolae from one cell type to another.
Stan
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Division of Cellular and Molecular Medicine-0651, University of California, San Diego, La Jolla 92093-0651, USA.
Roberts
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Predescu
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Ihida
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Saucan
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Ghitescu
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Palade
G E
GE
D000042:Absorption; D000595:Amino Acid Sequence; D000818:Animals; D017306:Annexin A2 / Q000378:metabolism; D000918:Antibody Specificity; D001671:Biochemistry / Q000379:methods*; D015415:Biomarkers; D051242:Caveolin 1; D022461:Caveolins; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D004730:Endothelium, Vascular / Q000166:cytology*; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D008863:Microspheres; D008969:Molecular Sequence Data; D015388:Organelles / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012795:Sialoglycoproteins / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction; D013010:Sonication
D017306:Annexin A2; D015415:Biomarkers; D051242:Caveolin 1; D022461:Caveolins; D008565:Membrane Proteins; D011506:Proteins; D012795:Sialoglycoproteins; C001643:podocalyxin; D019204:GTP-Binding Proteins