Proteins of the kidney microvillar membrane. Immunoelectrophoretic analysis of the membrane hydrolases: identification and resolution of the detergent- and proteinase-solubilized forms

Abstract

Antibodies raised in rabbits to detergent-solubilized pig kidney microvillar proteins were used to investigate the membrane hydrolases by crossed immunoelectrophoresis. Eight enzymes were detected by specific staining methods: aminopeptidase M, dipeptidylpeptidase IV, neutral endopeptidase, aminopeptidase A, carboxypeptidase P, .gamma.-glutamyltransferase, trehalase and phosphodiesterase I. The mobility of all the enzymes except trehalase and neutral endopeptidase was increased by treatment of the detergent-solubilized preparation with papain. The difference between the detergent and proteinase forms of these enzymes is attributed to the removal of a small, non-antigenic peptide to which detergent is bound in significant quantities. This interpretation was further supported by experiments in which the microvillus fraction was labeled with an intramembrane photolabeling reagent, 1-azido-4-[125I]iodobenzene. After photolysis, the radioactivity in the membrane could be solubilized by detergent treatment, but not by papain treatment. Radioautography after crossed charge-shift immunoelectrophoresis showed a good correlation between charge-shift (signifying the presence of detergent bound to hydrophobic domain) and the presence of the label.