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A monoclonal antibody to kidney endopeptidase-24.11. Its application in immunoadsorbent purification of the enzyme and immunofluorescent microscopy of kidney and intestine.

ABSTRACT: Hybridoma methodology has been used to produce a monoclonal antibody, GK 7C2, that binds specifically to microvillar endopeptidase-24.11 (EC 3.4.24.11). The antibody (an immunoglobulin G) was generated by fusion of mouse plasmacytoma cells with splenocytes from a Balb/c mouse immunized with pig kidney microvillar membranes. The identity of the antigen recognized by GK 7C2 was established by immuno-precipitation from detergent-solubilized pig kidney microvilli. The protein had an apparent Mr of 90 000 and contained endopeptidase activity sensitive to phosphoramidon. The identity was confirmed by immunoadsorbent purification of endopeptidase-24.11 by a column to which GK 7C2 had been attached. The endopeptidase, purified in a yield of 40%, was electrophoretically homogeneous and of specific activity comparable with that purified by other means. Fluorescence microscopy established that GK 7C2 bound specifically to the luminal membranes of kidney tubules and the intestinal mucosa. Thus endopeptidase-24.11 is located in the brush-border membranes of both cell types.

SUBMITTER: Gee NS

PROVIDER: S-EPMC1152258 | biostudies-other | 1983 Aug

REPOSITORIES: biostudies-other

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Endopeptidase-24.11 purified from pig intestine is differently glycosylated from that in kidney.

Project description:Endopeptidase-24.11 (EC 3.4.24.11) was purified from pig intestinal microvilli by immunoadsorbent chromatography, using antibodies raised to kidney endopeptidase-24.11. In many respects, the kidney and intestinal enzymes were indistinguishable, but some structural differences were demonstrated. In particular, the detergent form of the intestinal enzyme had an apparent subunit Mr of 95000, which, on treatment with trypsin, fell to a value of 89000, identical with that of the kidney form. The intestinal enzyme contained 3-4% more carbohydrate and many more fucose residues than that from kidney. Although these results show that post-translational processing was different in the two cell types, the possibility that the primary translation products also differed cannot be excluded.

| S-EPMC1152399 | biostudies-other

Deglycosylation by trifluoromethanesulphonic acid of endopeptidase-24.11 purified from pig kidney and intestine.

Project description:Endopeptidase-24.11, an integral microvillar membrane enzyme, exists in differently glycosylated forms when purified from pig kidney and intestine [Fulcher, Chaplin & Kenny (1983) Biochem. J. 215, 317-323]. When these glycoproteins, and another form of the kidney enzyme prepared from the Yucatan dwarf strain of piglet, were treated, under controlled conditions, with trifluoromethanesulphonic acid, the proteins were freed of carbohydrate and all had the same apparent subunit Mr (77 000) even though the untreated forms varied from Mr 89 000 to Mr 95 000.

| S-EPMC1144126 | biostudies-other

Purification of endopeptidase-24.11 ('enkephalinase') from pig brain by immunoadsorbent chromatography.

Project description:Membrane preparations from striatum of pig brain contain endopeptidase activity towards iodoinsulin B-chain. Only 50% of the hydrolysis of insulin B-chain is inhibitable by phosphoramidon, and DEAE-cellulose chromatography can resolve the phosphoramidon-sensitive and -insensitive activities. The former activity (now designated 'endopeptidase-24.11') is responsible for hydrolysis of [D-Ala2,Leu5]enkephalin and is identical with an enzyme in brain that has previously been referred to as 'enkephalinase'. Pig striatal endopeptidase-24.11 has now been purified to homogeneity in a single step by immunoadsorbent chromatography using a monoclonal antibody. The overall purification was 23 000-fold, with a yield of 30%. The brain enzyme appears to be identical with kidney endopeptidase-24.11 in amino acid composition as well as by immunological and kinetic criteria. However, it differs slightly in apparent subunit size (Mr = 87 000), attributable to differences in glycosylation.

| S-EPMC1152431 | biostudies-other

Kinetic analysis of the type-1 proinsulin endopeptidase by a monoclonal antibody-based immunoadsorbent assay.

Project description:A simple, rapid and sensitive assay for the type-1 endopeptidase (Arg-Arg cleaving) was developed by using an antiproinsulin monoclonal immunoadsorbent to separate reaction products from the substrate. The values obtained by this assay were identical with those obtained by an h.p.l.c.-based procedure and yielded similar values for the pH optimum (5.6) and Ca2+ activation (K0.5 = 2 mM). It was shown that the type-1 endopeptidase was readily solubilized by Triton X-114 (87 +/- 3%, n = 12) and partitioned principally into the aqueous phase at 30 degrees C (90.1 +/- 2.6%, n = 12). Activity was lost on gel filtration, but could be restored by adenosine 5'-[gamma-thio]triphosphate (K0.5 = 6 microM), 50 microM-dithiothreitol or 50 microM-Ca(2+)-trans-1,2-diaminocyclohexane-NNN'N'-tetra-acetic acid (CDTA), indicating that the enzyme was particularly sensitive to heavy metal ions. The Km obtained with proinsulin as substrate (13 +/- 1.7 microM) indicated that the enzyme works at close to its Vmax. in the nascent secretory granule. The Vmax. of the enzyme prepared from insulin granules (0.6% proinsulin converted/min) corresponded closely to the rate measured in vivo in rat islets. The type-1 endopeptidase also appears to be capable of binding to proinsulin in the region of the C-peptide/A-chain junction, since a peptide spanning this region was found to inhibit the 125I-proinsulin processing measured by this assay.

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Hydrolysis of rat melanin-concentrating hormone by endopeptidase 24.11 (neutral endopeptidase).

Project description:Melanin-concentrating hormone (MCH) is a cyclic peptide which behaves as an antagonist of the pituitary melanotropic hormone alpha-melanocyte-stimulating hormone in fishes. Cloning of the rat MCH cDNA precursor recently revealed the presence of an additional putative peptide named NEI. The present work examined the susceptibility of these novel peptides to hydrolysis by various purified exo- and endo-peptidases including endopeptidases 24.11 (NEP), 24.15, 24.16, angiotensin-converting enzyme, leucine aminopeptidase and carboxypeptidase A. NEP attacked MCH at three sites of the molecule with an apparent affinity of about 12 microM and a kcat. of 4 min-1. The first site of cleavage was at Cys-7-Met-8, i.e. within the peptide loop formed by the internal disulphide bridge. NEP could therefore be considered as an MCH-inactivating peptidase since the degradation products generated are probably devoid of biological activity. In contrast, NEI neither inhibited the degradation of the NEP chromogenic substrate glutaryl-Phe-Ala-Phe-p-aminobenzoate nor was susceptible to proteolysis by NEP. Unlike NEP, angiotensin-converting enzyme, endopeptidase 24.15 and endopeptidase 24.16 appeared totally unable to cleave MCH, whereas the peptide was readily degraded by aminopeptidase M and carboxypeptidase A.

| S-EPMC1133042 | biostudies-other

Purification and properties of rabbit brain and liver 4-aminobutyrate aminotransferases isolated by monoclonal-antibody immunoadsorbent chromatography.

Project description:The use of a monoclonal-antibody immunoaffinity column for the rapid isolation of 4-aminobutyrate aminotransferases (EC 2.6.1.19) from rabbit brain and liver is described. Homogeneous enzyme protein is eluted from the immunoadsorbent with 100mM-citrate buffer, pH5, and remains stable at 4 degrees C for several days. One such column (bed volume 8 ml) has been used 40 times in a 9-month period to isolate 10-15 units of enzyme activity (specific activity approx. 3.5-7.5 units/mg) per extraction. Kinetic and spectral analysis of the enzymes from the two tissues revealed a close similarity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed the isolated enzyme to have a monomeric Mr of 52 000, and this was confirmed by h.p.l.c. gel exclusion at pH 5.0. The results of Sephadex G-100 chromatography at different pH values are taken to indicate that the enzyme behaves as a dimer at pH 7.0 and above, but as a monomer at pH 5.0. 4-Aminobutyrate aminotransferase isolated from the brain by the procedure of Fowler & John [(1981) Biochem. J. 197, 149-152] is more stable than the immunoaffinity-purified material, and has been shown to contain a contaminant protein of Mr 84 000 that exhibits succinic semialdehyde dehydrogenase activity.

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The N-terminal amino acid sequence of pig kidney endopeptidase-24.11 shows homology with pro-sucrase-isomaltase.

Project description:Endopeptidase-24.11 (EC 3.4.24.11), a widely distributed ectoenzyme, was isolated from pig kidneys by detergent solubilization of membranes and immuno-affinity chromatography. In all, 12 preparations of the enzyme were submitted to solid-phase sequencing, yielding a consensus sequence of 25 amino acid residues of the N-terminal segment. Some samples were treated with either trypsin or Staphylococcus aureus V8 proteinase before sequencing. There were four lysine and one arginine residues in the first nine positions. This segment was susceptible to hydrolysis by trypsin and, in some samples, to endogenous proteinases. From residue 19 onwards, the sequence became intensely hydrophobic. There was a striking homology with the N-terminal sequence of pro-sucrase-isomaltase. From Lys7 to Leu20 there were seven identical amino acid residues and four conservative substitutions. We suggest that endopeptidase-24.11 is topologically similar to this glycosidase, the N-terminus at the cytoplasmic face and hydrophobic segment serving the roles of both signal peptide and hydrophobic anchor.

| S-EPMC1147413 | biostudies-other

Proteins of the kidney microvillar membrane. Biosynthesis of endopeptidase-24.11, dipeptidylpeptidase IV and aminopeptidases N and A in pig kidney slices.

Project description:An organ culture employing slices of renal-cortex tissue from piglets of the Yucatan strain was used to study the biogenesis of four microvillar peptidases: endopeptidase-24.11 (EC 3.4.24.11), dipeptidyl peptidase IV (EC 3.4.14.5), aminopeptidase N (EC 3.4.11.2) and aminopeptidase A (EC 3.4.11.7). The viability of the culture system was confirmed by the preservation of ultrastructural integrity and by an unchanged uptake of [3H]alanine into cells during the period of the experiments. After labelling with [35S]methionine, treatment with Mg2+ yielded two fractions, one containing microvilli and another, the Mg2+ pellet, containing intracellular and basolateral membranes. The labelled forms of the peptidases, isolated by immunoprecipitation, were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The Mg2+ pellet contained the earliest detectable forms of the enzymes. In each case, a polypeptide of lower Mr than the mature form and sensitive to treatment with endo-beta-N-acetylglucosaminidase H was the first form to be detected. These high-mannose forms were followed, about 30 min after the pulse, by a complex glycosylated form of higher Mr. Only the latter form was observed in microvilli and then only after 90 min of the chase period. A quantitative study of dipeptidyl peptidase IV showed that the forms observed in the Mg2+ pellet were precursors of those in the microvillar fraction. No labelled forms were observed in the cytosol. All four peptidases were thus synthesized within membrane compartments and glycosylated in two steps before assembly in microvilli.

| S-EPMC1144464 | biostudies-other

Characterization of neutral endopeptidase 24.11 in dog glomeruli.

Project description:Neutral endopeptidase (NEP; also known as neprilysin and enkephalinase; EC 3.4.24.11) is a cell-surface metallopeptidase that is present in many mammalian tissues. It is particularly abundant on the brush-border membranes of the kidney proximal tubule. In this paper, the presence of NEP in purified glomeruli from dog kidney was assessed by measuring phosphoramidon- and thiorphan-sensitive [D-Ala2,Leu5]enkephalin-degrading activity. Using this assay, the Km and kcat. of the glomerular enzyme were found to be identical to those of the tubular enzyme. By Western blotting the apparent M(r) of the glomerular enzyme was found to be 104,000, compared with 94,000 for the tubular enzyme. This might be due to a different glycosylation pattern, since endoglycosidase F treatment of NEP obtained from both tissues yielded deglycosylated enzymes with similar electrophoretic mobilities. The glomerular enzyme also appears to be membrane-bound, since it was retained in the detergent-rich phase after phase separation with Triton X-114. Autoradiography experiments performed with RB104, a new highly selective and potent NEP inhibitor, showed that NEP was expressed in both glomeruli and proximal tubules. The presence in glomeruli of NEP and some other brush-border peptidases (dipeptidyl-dipeptidase IV, aminopeptidase N and angiotensin I-converting enzyme) suggests that cell-surface peptidases might play an important role as regulators of plasma-derived peptides in this part of the nephron.

| S-EPMC1132435 | biostudies-other

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

Project description:An immunoradiometric assay for endopeptidase-24.11, which depended on the absorption by tissues of a monoclonal antibody, GK7C2, was established. The optimum conditions for the assay were defined and its correlation with an enzymic assay determined. The immunoassay was used to survey the endopeptidase in crude homogenates of various tissues of the pig. Detergent treatment decreased the sensitivity of the assay but did not invalidate it. Although the endopeptidase was found in many tissues, it was neither uniformly nor ubiquitously distributed. Kidney cortex was confirmed as the major location of the endopeptidase, containing 5000 ng/mg of protein. Lymph nodes were also very active (1370 ng/mg), followed by chondrocytes from articular cartilage (650 ng/mg). In the gut, the endopeptidase was concentrated mainly in the jejunum (130 ng/mg). Various glands (salivary, adrenal, anterior pituitary and pancreas) also contained the antigen in the range 20-55 ng/mg of protein. Lung contained only 5 ng/mg of protein and, in other tissues examined, little or none was detectable. In particular, other lymphoid tissues (spleen, thymus, tonsillar tissues) were relatively poor sources, and none was detectable in peripheral-blood leucocytes or in peritoneal macrophages.

| S-EPMC1144960 | biostudies-other

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