A METHOD FOR SELECTIVE RADIOLABELING OF LUNG ENDOTHELIUM PLASMALEMMALVESICLES, IN-SITU

Albumin-gold complex (Alb-Au) was previously shown to bind selectively to plasmalemmal vesicles of capillary endothelium. Based on these fin dings, as well as on the ability of lactoperoxidase (LPO) to mediate t he radioiodination of proteins, we have prepared a complex of gold par ticles bearing both albumin and anionized lactoperoxidase (Alb-Au-aLPO ). The complex had a pi of 5.8, largely preserved aLPO enzymatic activ ity (similar to 74 %), and was able to catalyze protein radioiodinatio n. Upon washing out the blood, the complex,vas perfused in the mouse l ung, the excess tracer removed, and a Na(125)L/H2O2 solution was intro duced in the vasculature. After extensive washing, lung fragments were processed for either electron microscopy (EM), or to prepare a membra ne-enriched fraction. In control experiments, lungs were perfused with native LPO (pI 9.3), or with a LPO-Affi Gel conjugate and further rad ioiodinated as described for Alb-Au-aLPO. By EM, it,vas found that bot h in tissue and in the isolated membrane fraction, only Alb Au aLPO la beled markedly and preferentially some uncoated pits and most plasmale mmal vesicles. Analysis by SDS-PAGE and autoradiography of a membrane- enriched fraction prepared from lungs perfused with Alb-Au aLPO had so me major identified I-125-labeled polypeptides of apparent molecular m asses of 16, 18, 31, 36, 55, and 77 kDa. A different subset of polypep tides was labeled in lungs perfused with LPO, whereas after administra tion of LPO-Affi Gel the major radiolabeled polypeptides had a molecul ar mass of 33, 55 kDa and several peptides in the range of 77 to 160 k Da. These results and the appropiate controls showed that Alb-Au-aLPO selectively iodinates membrane proteins of endothelial plasmalemmal ve sicles. This procedure can have a larger applicability using native LP O or anionized LPO conjugated with a specific ligand employed alone or complexed with gold particles for specific radioiodination and cellul ar localization of the molecular entities of interest.