A method for selective radiolabeling of lung endothelium plasmalemmal vesicles, in situ.
Albumin-gold complex (Alb-Au) was previously shown to bind selectively to plasmalemmal vesicles of capillary endothelium. Based on these findings, as well as on the ability of lactoperoxidase (LPO) to mediate the radioiodination of proteins, we have prepared a complex of gold particles bearing both albumin and anionized lactoperoxidase (Alb-Au-aLPO). The complex had a pI of 5.8, largely preserved aLPO enzymatic activity (approximately 74%), and was able to catalyze protein radioiodination. Upon washing out the blood, the complex was perfused in the mouse lung, the excess tracer removed, and a Na125I/H2O2 solution was introduced in the vasculature. After extensive washing, lung fragments were processed for either electron microscopy (EM), or to prepare a membrane-enriched fraction. In control experiments, lungs were perfused with native LPO (pI 9.3), or with a LPO-Affi Gel conjugate and further radioiodinated as described for Alb-Au-aLPO. By EM, it was found that both in tissue and in the isolated membrane fraction, only Alb-Au-aLPO labeled markedly and preferentially some uncoated pits and most plasmalemmal vesicles. Analysis by SDS-PAGE and autoradiography of a membrane-enriched fraction prepared from lungs perfused with Alb-Au-aLPO had some major identified 125I-labeled polypeptides of apparent molecular masses of 16, 18, 31, 36, 55, and 77 kDa. A different subset of polypeptides was labeled in lungs perfused with LPO, whereas after administration of LPO-Affi Gel the major radiolabeled polypeptides had a molecular mass of 33, 55 kDa and several peptides in the range of 77 to 160 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
Heltianu
C
C
Institute of Cellular Biology and Pathology, Bucharest, Romania.
Dobrila
L
L
Stanescu
M
M
Antohe
F
F
Simionescu
N
N
Silverstein
S C
SC
Simionescu
M
M
D000818:Animals; D002196:Capillaries; D002462:Cell Membrane / Q000648:ultrastructure*; D002712:Chlorides; D004705:Endocytosis; D004730:Endothelium, Vascular / Q000648:ultrastructure*; D005782:Gels; D017612:Gold Compounds; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D007784:Lactoperoxidase; D008297:Male; D008565:Membrane Proteins / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D008863:Microspheres; D010477:Perfusion; D011651:Pulmonary Artery / Q000648:ultrastructure*; D012710:Serum Albumin, Bovine
D002712:Chlorides; D005782:Gels; D017612:Gold Compounds; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D012710:Serum Albumin, Bovine; C024568:gold tetrachloride, acid; D007784:Lactoperoxidase