Proteins of the kidney microvillus membrane. Identification of subunits after sodium dodecylsullphate/polyacrylamide-gel electrophoresis

Abstract

The proteins of microvilli prepared from pig kidney were analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The typical pattern stained for protein revealed five major bands, four of which also stained for carbohydrate, and about 15 minor bands. For descriptive purposes the bands were designated numerically by their apparent molecular weights (X10(-3). Well-characterized proteins were identified with four of the five major bands. Dipeptidyl peptidase IV, a serine proteinase that may be specifically labelled with di-isopropyl [32P]phosphorofluoridate, was assigned to band 130. Aminopeptidase M was assigned to band 160, though when released from the membrane by a proteinase, this protein comprises three polypeptides each of lower apparent molecular weight than the native enzyme. Neutral endopeptidase can be assigned to band 95 and actin to band 42. The fifth major band (180) is an extrinsic glycoprotein that has not been identified with any microvillus enzyme activity. These four proteins contribute 21% of the microvillus-membrane protein. Kidney microvillus actin was characterized by a variety of properties and was similar to muscle actin. A computer analysis of the gel pattern indicates that it comprises 9.0% of the microvillus protein. Myosin is not present in the microvillus, but another protein associated with band 95, with properties that distinguish it from neutral endopeptidase, was tentatively identified as alpha-actinin. Alkaline phosphatase was identified as a monomeric polypeptide with an apparent molecular weight of 80000; it is a minor protein of the microvillus and is not discernible as a discrete band in the gel pattern. These and other results permit a model of the organization of the microvillus protein to be suggested. The computer program used has been deposited as Supplementary Publication SUP 50070 (12 pages) at the British Library Lending Division, Boston Spa. Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1976) 153,5.

An immunoradiometric assay for endopeptidase-24.11 shows it to be a widely distributed enzyme in pig tissues

“…In arbitrary units the values were 138 and 3520 units/mg of protein, an enrichment of 25.5-fold. This agrees well with estimates, derived from enzyme assays, that the enzyme constitutes about 4% of the microvillar membrane protein (Booth & Kenny, 1976;. The corresponding specific activities for the untreated homogenate and microvillar membranes were 167 and 854 units/mg of protein, an enrichment of 5.1.…”

Exendin-4, but not glucagon-like peptide-1, is cleared exclusively by glomerular filtration in anaesthetised pigs

“…Thus, GLP-1 is degraded initially by DPPIV and subsequently by other enzymes, including NEP [8,15]. Exendin-4, however, is reported to be more resistant to enzymatic degradation, and indeed, when incubated in the presence of kidney brush border membranes, which are especially rich in a variety of ectopeptidase activities [22], exendin-4 is remarkably stable, with a rate of proteolysis which is several orders of magnitude less than that of GLP-1 [15]. As a result of the presence of a penultimate Nterminal glycine in exendin-4, compared with an alanine in GLP-1, exendin-4 is not degraded significantly by DPPIV [15].…”

Studies on the Structure of the Rabbit Kidney Brush Border

“…If contraction of the microvilli occurs, mediated by the core filaments, it is reasonable to expect that a mechanical interconnection exists between the core and the membranes. It has been proposed that a-actinin may serve as cross-bridge between the membranes and the actin filaments [2,20] and the a-actinin presumably associates with integral proteins in the membrane. If this interaction is electrostatic then it is unlikely to be disrupted by a non-ionic detergent.…”

“…Analysis of the phospholipid composition of the supernatant and residue fractions after disruption with 1 (w/v) Triton X-100 showed that the acidic phospholipids (phosphatidylethanol-1 3 000 -70 000. 45.3 (2) amine, phosphatidylserine and phosphatidylinositol) were preferentially released, whilst phosphatidylcholine was largely retained in the residue (Table 4). After disruption of the brush borders with 1 % (w/v) Triton X-I 00 thin-section microscopy revealed core filaments in the residue which could be seen to radiate from terminal webs, the overlying membrane having apparently been removed ; desmosomes were also observed (Fig.…”

“…They comprise a lipoprotein membrane containing enzymes, a filamentous core containing actin [2,3], and a terminal web which, at least in the case of intestinal brush borders [4], contains myosin. Here we report attempts to 'dissect' renal brush borders using salts and detergents, correlating the release of brush border components with changes in morphology induced by these agents.…”