title: Proteins of the kidney microvillar membrane. Structural and immunochemical properties of rat endopeptidase-2 and its immunohistochemical localization in tissues of rat and mouse.
abstract: The phosphoramidon-insensitive endopeptidase-2 in rat renal brush borders was investigated by immunochemical approaches with a rabbit polyclonal antibody raised to the purified enzyme released from the membrane by papain. An immunoaffinity column successfully purified the detergent-solubilized form of endopeptidase-2. This preparation had an apparent subunit Mr of 80,000, and did not show the two subunits, of Mr 80,000 and 74,000, consistently found in the papain-solubilized forms, indicating that the latter resulted from proteolysis by papain. SDS/polyacrylamide-gel electrophoresis of non-reduced samples of the enzyme revealed a band of Mr 220,000, confirming the presence of disulphide-bridged subunits. Treatment with endoglycosidases H and F generated smaller molecular forms, indicating that endopeptidase-2 contained about 30% asparagine-linked carbohydrate and that a few of these oligosaccharide chains were of the high-mannose type. Treatment with phosphatidylinositol-specific phospholipase indicated that the enzyme did not possess a glycolipid membrane anchor. A survey of rat tissues examined immunohistochemically and by immunoblotting revealed that only the kidney and intestinal tract expressed the antigen in significant amounts. Although some weak staining was seen in salivary glands and thyroid, other organs and tissues including brain and spinal cord were negative by both immunochemical techniques. In the kidney the antigen was confined to the lumen of the proximal tubule and was seen mainly in the population of juxtamedullary nephrons. In the gut, luminal staining was observed throughout its whole length, from duodenum to rectum. Excellent cross-reactivity of the antibody with Balb/c mouse tissues was observed. Immunohistochemistry of mouse kidney and gut revealed a distribution identical with that observed in the rat. Immunopurification of the detergent-solubilized mouse kidney antigen showed it to be a protein containing disulphide-linked subunits of Mr 90,000. It possessed endopeptidase-2-like activity, but was more efficient in hydrolysing azo-casein and less efficient in hydrolysing a model substrate than the rat enzyme. The close similarity between rat endopeptidase-2 and mouse meprin is further supported by these results.
authors.affiliation: Department of Biochemistry, University of Leeds, U.K.
authors.forename: K
authors.initials: K
authors.lastname: Barnes
authors.forename: J
authors.initials: J
authors.lastname: Ingram
authors.forename: A J
authors.initials: AJ
authors.lastname: Kenny
mesh_terms: D000818:Animals; D000906:Antibodies; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006020:Glycopeptides / Q000494:pharmacology; D007123:Immunoelectrophoresis, Two-Dimensional; D007124:Immunoenzyme Techniques; D007668:Kidney / Q000166:cytology / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008666:Metalloendopeptidases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008861:Microsomes / Q000201:enzymology; D008871:Microvilli / Q000201:enzymology* / Q000648:ultrastructure; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D000906:Antibodies; D006020:Glycopeptides; D046911:Macromolecular Substances; D008666:Metalloendopeptidases; C053833:meprin A; C008890:phosphoramidon