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  • Title: Proteins of the kidney microvillar membrane. Reconstitution of endopeptidase in liposomes shows that it is a short-stalked protein.
    Author: Kenny AJ, Fulcher IS, McGill KA, Kershaw D.
    Journal: Biochem J; 1983 Jun 01; 211(3):755-62. PubMed ID: 6349616.
    Abstract:
    Pig kidney microvillar proteins were extracted with octyl beta-glucoside and reconstituted in liposomes prepared from microvillar lipids of known composition. Four peptidases, namely 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 shown to be reconstituted. At lipid/protein ratios greater than 4:1, about half the detergent-solubilized protein and nearly all of the activity of the four peptidases were reconstituted. Dissolution of the liposomes with Triton X-100 did not increase the activity of any of these peptidases, a result consistent with an asymmetric, 'right-side-out', orientation of these enzymes. When purified, endopeptidase was subjected to the same procedure; the two 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. Electron microscopy 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 to 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.
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