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. 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.

Genomic organization, exact localization, and tissue expression of the human CD26 (dipeptidyl peptidase IV) gene

“…Exon 2 contains the hydrophobic amino acids thought to comprise the NH2 anchoring domain (Ogata et al 1989). Exon 3 contains the area of the protein close to the cell surface which is easily cleaved at 39serine by papain and probably forms an extended stalk structure (Kenny et al 1983). Exons 4 through to 20 encode the large extracellular region of the CD26 protein which as yet has no known function.…”

Radiation inactivation analysis of kidney microvillar peptidases

“…This assumption has been supported by electron microscopy of a few enzymes that have been reconstituted into liposomes, e.g. aminopeptidase N (EC 3.4.11.2) [2], endopeptidase-24.11 (EC 3.4.24.1 I) [3] and dipeptidyl peptidase IV (EC 3.4.14.5) [4]. However, we know very little about the functional significance of the oligomeric state for these enzymes.…”

“…More importantly, the variance among the data from the samples in cups was significantly lower than those in vials. We conclude that in spite of the possible Dose ( M rad 1 The DR dose is the mean of 3, 6 or 9 determinations (assayed in duplicate), as is the coefficient of determination (3). The values given for the target Mr (X IO-') are means where more than one irradiation was performed.…”

Proteins of the kidney microvillar membrane. The amphipathic forms of endopeptidase purified from pig kidneys

“…A dimer therefore appears to bind one Triton X-100 micelle. Electron microscopy of the enzyme after reconstitution of the d-form [the following paper (Kenny et al, 1983)] also revealed dimers when viewed en face on the surface of liposomes. If we can assume that the two subunits are identical, each possessing an anchor, it follows that a single detergent micelle can accommodate both hydrophobic anchors.…”

“…In other examples this has been shown to be adjacent to the N-terminus, but this has yet to be established for the endopeptidase. The globular model has, however, been confirmed by reconstitution in liposomes [see the following paper (Kenny et al, 1983)1.…”