Isolation and partial characterization of the luminal plasmalemma of microvascular endothelium from rat lungs.
This paper describes a procedure for isolating in high yield and at a high degree of purity the endothelial luminal plasmalemma from the microvasculature of the rat lung. The procedure relies on the modification of the density of the luminal plasmalemma obtained by coating it by perfusion in situ first, with cationized colloidal silica and then with Na polyacrylate. These steps generate a strongly adhering coat to the luminal plasmalemma that resists tissue homogenization to yield, upon repeated centrifugation through Nycodenz density gradients, a nearly homogeneous fraction of coated luminal plasmalemmal fragments still carrying their associated plasmalemmal vesicles. The fraction is enriched in the luminal plasmalemmal antigen, angiotensin converting enzyme, contains gp60, an antigen expected to occur on both plasmalemmal domains, is not enriched in either alkaline phosphatase or 5'-nucleotidase activity and is free of the mitochondrial and endoplasmic reticulum antigens so far tested. This procedure, that can be extended--in principle--to any vascular bed, obviates the use of cultured cells for studying the biochemistry of the endothelium, at least as far as the luminal endothelial plasmalemma is concerned.
Jacobson
B S
BS
Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla 92093-0651.
Schnitzer
J E
JE
McCaffery
M
M
Palade
G E
GE
D000818:Animals; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D003102:Colloids; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000648:ultrastructure; D007472:Iohexol; D008168:Lung / Q000098:blood supply*; D008297:Male; D008833:Microcirculation; D007703:Peptidyl-Dipeptidase A / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012822:Silicon Dioxide
D003102:Colloids; D007472:Iohexol; D012822:Silicon Dioxide; D007703:Peptidyl-Dipeptidase A