Radio-iodination of plasma membranes of toad bladder epithelium

Abstract

The present report describes high yield enzymatic radio-iodination of the apical and basal-lateral plasma membranes of toad bladder epithelium, by a procedure that does not breach the functional integrity of the epithelium, as assessed by the basal and vasopressin-sensitive short-circuit current (SCC). Restriction of the label to the membrane surface was ascertained by light and electron-microscopic autoradiographs. On the apical surface, the grains were over the glycocalyx and the plasma membrane. Analysis of the labeled glycocalyx by agarose gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as enzymatic and pH-dependent hydrolysis indicated that the glycocalyx is a trichloro-acetic acid-soluble macromolecular complex of high molecular weight composed of a peptide moiety attached to large prosthetic groups (presumably carbohydrates) by O-glycosidic bonds. Analysis of the labeled apical plasma membrane components by agarose gel filtration and SDS-PAGE disclosed the presence of six major species of apparent molecular weights: 23,000, 28,000, 37,000, 44,000, 68,000, and 95,000. More than half of the membrane-associated radio-iodine was in two bands of molecular weights 37,000 and 44,000. Concentrations of vasopressin and cyclic AMP sufficient to increase the SCC significantly did not modify the extent of membrane labeling or the distribution of the label among the apical membrane components (presumably proteins) as assessed by SDS-PAGE. Iodination in the presence of amiloride inhibited incorporation but did not change the pattern of the distribution of the label among the components resolved by SDS-PAGE. Iodination of basal-lateral plasma membranes, at a yield comparable to that obtained with apical labeling, was attained after about 30 min of exposure of the intact bladder to the labeling solutions. Approximately 25% of the basal-lateral labeling was lost when the epithelial cells were harvested after collagenase treatment, implying that some iodination of the basement membrane had taken place. Less than 10% of iodination of the apical or basal-lateral surfaces was accounted for by lipid-labeling. Analysis of the labeled apical and basal-lateral species by enzymatic digestion and thin layer chromatography disclosed that virtually all the radioactivity was present as mono-iodotyrosine (MIT).