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Two methods were used to label pig kidney microvillar membrane proteins from the luminal and cytoplasmic surfaces of closed membrane vesicles. The first method was lactoperoxidase-catalysed radioiodination. The enzyme reagents, lactoperoxidase and glucose oxidase, were positioned inside the vesicles before sealing or externally after sealing, iodination being initiated by the subsequent addition of glucose and 125I-. After resolution of the labelled proteins by electrophoresis in the presence of dodecyl sulphate, asymmetric labelling patterns on radioautographs were observed. However, the major disadvantage of this method is the high degree of intramembrane labelling of the fatty acid chains of membrane lipids, a reaction that undermines any conclusions about the location of the label in that region of the protein supposedly exposed at the surface of the membrane. The second method overcame this disadvantage. A new hydrophilic photoreagent, 3,5-di[125I]iodo-4-azidobenzesulphonate, was synthesized via the intermediate, diazotized 3,5-di[125I]iodosulphanilic acid. It was transported by a Na+-dependent system into microvillar vesicles, thus permitting labelling from either side of the membrane when the vesicles were photolysed. The labelling of membrane lipids was less than with the first method and was essentially confined to the polar headgroups. The activity of several microvillar peptidases survived the labelling reaction and they could be identified in the immunoprecipitates after resolution of the detergent-solubilized membrane proteins by crossed-immunoelectrophoresis. Treatment with papain converted the detergent-solubilized form of susceptible enzymes into the proteinase-solubilized form, which lacked the intramembrane domain and any portion exposed at the cytoplasmic surface. Radioautography established that aminopeptidases M and A, dipeptidyl peptidase IV and neutral endopeptidase were transmembrane proteins. This novel approach to the investigation of membrane topology may be applicable to other complex membranes.

biochemical journal

Proteins of the kidney microvillar membrane. Asymmetric labelling of the membrane by lactoperoxidase-catalysed radioiodination and by photolysis of 3,5-di[125I]iodo-4-azidobenzenesulphonate