In situ iodination of angiotensin-converting enzyme and other pulmonary endothelial membrane proteins.
Biochemical responses of endothelial cells in culture to pharmacological or physiological stimuli are often extrapolated to define the behavior of the vascular endothelium in vivo. However, culture conditions cannot recreate the environment of endothelial cells in vivo. To compare cell functions in vivo and in vitro, we iodinated endothelial membrane proteins of both the perfused rabbit lung and cultured rabbit lung endothelial cells. Endothelial cell protein 125I-labeling in the perfused intact lung was catalyzed by lactoperoxidase and glucose oxidase immobilized on 3-10 microns polyacrylamide beads (Enzymobeads, Bio-Rad). Changes in 5-hydroxytryptamine uptake, angiotensin converting enzyme activity and perfusion pressure made before, during and/or after iodination were small, suggesting that the procedure does not grossly injury the lung. As confirmed by tissue autoradiography, iodination was confined to the vascular space. A subcellular "membrane" fraction of the whole homogenate was enriched for several iodinated proteins. Lectin binding further purified a library of putative iodinated endothelial membranes proteins, one of which was angiotensin-converting enzyme as shown by immunoprecipitation with goat anti-rabbit antibody to angiotensin-converting enzyme. Iodinated proteins of similar molecular weights were also isolated from cultured rabbit lung endothelium iodinated under the same conditions, thus confirming the endothelial lineage of proteins iodinated in the intact lung. We conclude that this technique labels endothelial surface proteins in the intact lung without causing observable tissue injury and thus should be valuable in the study of the physiology and pathophysiology of the vascular lining in vivo.
Merker
M
M
Department of Anesthesiology, Yale University School of Medicine, New Haven, CT 06510.
Carley
W W
WW
Gillis
C N
CN
D000818:Animals; D001345:Autoradiography; D002478:Cells, Cultured; D004727:Endothelium / Q000378:metabolism; D005949:Glucose Oxidase / Q000008:administration & dosage; D007455:Iodine / Q000008:administration & dosage* / Q000378:metabolism; D007467:Iodoproteins / Q000032:analysis*; D007784:Lactoperoxidase / Q000008:administration & dosage; D037102:Lectins / Q000378:metabolism; D008168:Lung / Q000098:blood supply / Q000201:enzymology* / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000276:immunology / Q000378:metabolism*; D010477:Perfusion; D011233:Precipitin Tests; D011817:Rabbits
D007467:Iodoproteins; D037102:Lectins; D008565:Membrane Proteins; C041744:enzymobeads; D007455:Iodine; D005949:Glucose Oxidase; D007784:Lactoperoxidase; D007703:Peptidyl-Dipeptidase A