Proteins of the kidney microvillar membrane. Reconstitution of endopeptidase in liposomes shows that it is a short-stalked protein

Abstract

Pig kidney microvillar proteins were extracted with octyl .beta.-glucoside and reconstituted in liposomes prepared from microvillar lipids of known composition. For peptidases, e.g., endopeptidase (EC 3.4.24.11), aminopeptidases N (EC 3.4.11.2) and A (EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), were reconstituted. At lipid/protein ratios greater than 4:1, about half the detergent-solubilized protein and nearly all of the activity of the 4 peptidases were reconstituted. Dissolution of the liposomes with Triton X-100 did not increase the activity of any of thes peptidases, a result consistent with an asymmetric, right-side-out orientation of these enyzmes. When purified, endopeptidase was subjected to the same procedure; the 2 amphipathic forms of the enzyme (the detergent form and the trypsin-treated detergent form) were fully reconstituted. The amphiphilic form, purified after toluene/trypsin treatment, failed to reconstitute. EM of microvilli showed that the appearance of the surface particles was profoundly altered by treatment with papain. Before treatment, the microvilli were coated with particles of stalk lengths ranging from 2.5-9 nm. After papain treatment nearly all the particles had stalks of 2-3 nm. Reconstituted microvillar proteins in liposomes showed the same heterogeneity of stalk length. In contrast, liposomes containing reconstituted endopeptidase revealed a very homogeneous population of particles of stalk length 2 nm. Since the smallest dimension of a papain molecule is 3.7 nm, the ability of papain, and other proteinases of similar molecular size, to release microvillar enzymes is crucially affected by the length of the junctional peptide that constitutes the stalk of this type of membrane protein.