A neutral endopeptidase in the microvillar membrane of pig intestine. Partial purification and properties.
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ABSTRACT:
SUBMITTER: Danielsen EM
PROVIDER: S-EPMC1162257 | biostudies-other | 1980 Nov
REPOSITORIES: biostudies-other
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Project description:A second endopeptidase is present in the renal microvillar membrane of rats that can be distinguished from endopeptidase-24.11 by its insensitivity to inhibition by phosphoramidon. The purification of this enzyme, referred to as endopeptidase-2, is described. The enzyme was efficiently released from the membrane by treatment with papain. The subsequent four steps depended on ion-exchange and gel-filtration chromatography. These steps were monitored by the hydrolysis of various substrates: 125I-insulin B chain (the normal assay substrate), benzoyl-L-tyrosyl-p-aminobenzoate (Bz-Tyr-pAB), azocasein and benzyloxycarbonyl-L-phenylalanyl-L-arginine 7-amino-4-methylcoumarylamide (Z-Phe-Arg-NMec). All four assays revealed comparable stepwise increases in activity in the main stages of the purification, although it was apparent that the last-named fluorogenic assay depended on traces of aminopeptidase activity present in the preparation. The Km for 125I-insulin B chain was 16 microM and that for Bz-Tyr-pAB was 4.7 mM. Several experimental approaches confirmed that both peptides were hydrolysed by the same enzyme. The pH optimum was 7.3. Phosphate buffers were inhibitory and shifted the optimum to above pH 9. Zinc was detected in the purified enzyme; EDTA and 1,10-phenanthroline were strongly inhibitory. SDS/polyacrylamide-gel electrophoresis revealed polypeptides of equal staining intensity of Mr 80,000 and 74,000 in reducing conditions. In non-reducing conditions a single band of apparent Mr 220,000 was seen. Gel filtration yielded an Mr of 436,000. These results are consistent with an oligomeric structure in which the alpha and beta chains are linked by disulphide bridges. Endopeptidase-2 hydrolysed a number of neuropeptides. Enkephalins resisted attack, only the heptapeptide [Met]enkephalin-Arg6-Phe7 being susceptible to slow hydrolysis. Luliberin (luteinizing-hormone-releasing hormone) and bradykinin were rapidly hydrolysed. Neurotensin was shown to be slowly attacked at the Tyr3-Glu4 bond. Thus the specificity appears to be limited to the hydrolysis of bonds involving the carboxy group of aromatic residues, provided that this P1 residue is extended by additional residues, at least to the P3' position. The relationship of this membrane metalloendopeptidase to mouse meprin and human 'PABA peptidase' is discussed.
| S-EPMC1148152 | biostudies-other
Project description:Carboxypeptidase P has been purified by immunoaffinity chromatography from pig kidneys. A single-step assay with Z-Pro-Met (where Z represents benzyloxycarbonyl) as substrate was used, methionine being determined by using L-amino acid oxidase and horseradish peroxidase. The enzyme constitutes about 1.5% of the kidney microvillar proteins. Triton X-100-solubilized and papain-released forms of the enzyme were isolated. The former had an apparent subunit Mr of 135 000, and the latter form contained two polypeptide chains of Mr 128 000 and 95 000. The undenatured forms were dimeric proteins. In common with other microvillar hydrolases, carboxypeptidase P was a glycoprotein and each subunit contained one Zn atom. MnCl2 (1 mM) in the assay was necessary for maximum activity; in its absence, 0.5 mM-ZnSO4 produced a limited activation, but was inhibitory at higher concentrations. The Km for Z-Pro-Met, in the presence of MnCl2, was 4.1 mM, and the kcat. for freshly prepared enzyme was 1230 min-1. The enzyme lost activity during storage at -20 degrees C. In a limited survey of peptides, hydrolysis was observed only with substrates containing a proline, alanine or glycine residue in the P1 position, and these included angiotensins II and III. The best substrate in this series was Val-Ala-Ala-Phe.
| S-EPMC1145174 | biostudies-other
Project description:The purification of detergent-solubilized kidney microvillar endopeptidase (EC 3.4.24.11) by immuno-adsorbent chromatography is described. The product (the d-form) was 270-fold purified compared with the homogenate of kidney cortex and was obtained in a yield of 5%. It was free of other peptidase activities and homogeneous by electrophoretic analyses. It contained about 15% carbohydrate and one Zn atom/subunit. Two trypsin-treated forms were also characterized. One (dt-form) was obtained by treatment of the d-form. The other (tt-form) was the result of solubilizing the membrane by treatment with toluene and trypsin. All three forms had apparent subunit Mr values of approx. 89 000, but the d-form appeared to be slightly larger than the other two. Estimates of Mr by gel filtration showed that of the tt-form to be 216 000 whereas those of the other forms were 320 000. An estimate of the detergent (Triton X-100) bound to the d- and dt-forms accounted for this difference. By several criteria, including charge-shift crossed immunoelectrophoresis and hydrophobic chromatography, the d- and dt-forms were shown to be amphipathic molecules. In contrast, the tt-form was hydrophilic in its properties. Differences in ionic properties were also noted, consistent with the loss, in the case of the dt-form, of a positively charged peptide. The results indicate that the native endopeptidase is a dimeric molecule, each subunit being anchored in the membrane by a relatively small region of the polypeptide close to one or other terminus. The d- and dt-forms had similar enzyme activity when assayed by the hydrolysis of 125I-insulin B-chain. Chelating agents and phosphoramidon inhibited the endopeptidase. The kinetic constants were determined by a new two-stage fluorimetric assay using glutarylglycylglycylphenylalanine 2-naphthylamide as substrate and aminopeptidase N (EC 3.4.11.2) to hydrolyse phenylalanine 2-naphthylamide. The Km was 68 microM and Vmax. 484nmol X min-1 X (mg of protein)-1.
| S-EPMC1154422 | biostudies-other
Project description:Neurotensin (NT) endopeptidase (EC 3.4.24.16) has been purified about 800-fold from pig brain by four sequential chromatographic steps depending on ion-exchange and hydrophobic interactions. Two types of preparation were studied: one from a Triton X-100-solubilized membrane fraction, and the other from the soluble fraction containing 90% or more of the total activity in the homogenate. NT endopeptidase activity was monitored by high-precision liquid chromatography of the two peptide products, characterized as NT-(1-10) and NT-(1-8), resulting from cleavage of the Pro10-Tyr11 and Arg8-Arg9 bonds respectively. As purification proceeded, from both membranes and cytosol, the yield of the two products achieved a constant ratio of 5:1 and this ratio was reproduced in repeated purifications. However, a distinct peptidase which hydrolysed exclusively at the Arg8-Arg9 bond was partially resolved from NT endopeptidase by chromatography on hydroxyapatite, and this activity was further purified and assigned to endopeptidase-24.15 (EC 3.4.24.15). SDS/PAGE of both preparations of neurotensin endopeptidase revealed a major band of apparent Mr 75000, and treatment of the membrane-associated form with N-Glycanase gave no evidence that the enzyme was a glycoprotein. The membrane-associated and cytosol forms of NT endopeptidase activities, monitored for both NT-(1-10) and NT-(1-8) products, were compared in their responses to 1,10-phenanthroline, EDTA, dithiothreitol (DTT) and some synthetic site-directed inhibitors of endopeptidase-24.15 or peptidyl dipeptidase A. The effects revealed no significant differences between the two preparations, nor did the reagents discriminate between the activities generating the two NT fragments. The partially purified form of endopeptidase-24.15 was also included in this comparison: while some responses were similar, this peptidase was distinguishable in its activation by DTT and its relative resistance to inhibition by EDTA. Both forms of NT endopeptidase were found to hydrolyse other substrates, including Boc-Phe-Ala-Ala-Phe-4-aminobenzoate, bradykinin and substance P (these at faster rates than neurotensin), as well as dynorphin A-(1-8) and luliberin. The bonds hydrolysed in these neuropeptides, as well as in angiotensins I and II and alpha-neoendorphin, were defined. These studies confirm that NT endopeptidase is distinct from endopeptidase-24.15. They further show that the former is a soluble enzyme, not an integral membrane protein, that it is not peptide-specific and that it might be more appropriately named. enzyme, not an integral membrane protein, that it is not peptide-specific and
| S-EPMC1151045 | biostudies-other
Project description:1. Polynucleotide phosphorylase has been isolated and partially purified from crude preparations of guinea-pig liver nuclei. 2. The enzyme is particulate and associated with RNA and lipids characteristic of membranes. 3. It has phosphorolysis and exchange activities, but the latter may be due to a contaminating enzyme. 4. The phosphorolysis activity is dependent on bivalent cations, preferably Mg(2+), has a pH optimum between 8.6 and 9.2 and is inhibited by potassium chloride and sodium chloride. 5. The enzyme catalyses phosphorolysis of poly A, poly C, poly U, rRNA and tRNA. Poly G is only phosphorolysed to a very small extent and DNA is not a substrate. 6. The enzyme appears to lack nucleoside diphosphate polymerization activity.
| S-EPMC1179382 | biostudies-other
Project description:Sialorphin is an exocrine and endocrine signaling mediator, which has been identified by a genomic approach. It is synthesized predominantly in the submandibular gland and prostate of adult rats in response to androgen steroids and is released locally and systemically in response to stress. We now demonstrate that the cell surface molecule to which sialorphin binds in vivo in the rat kidney is the membrane-anchored neutral endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in nervous and peripheral tissues, as it turns off several peptide-signaling events at the cell surface. We show that sialorphin prevents spinal and renal NEP from breaking down its two physiologically relevant substrates, substance P and Met-enkephalin in vitro. Sialorphin inhibited the breakdown of substance P with an IC50 of 0.4-1 microM and behaved as a competitive inhibitor. In vivo, i.v. sialorphin elicited potent antinociceptive responses in two behavioral rat models of injury-induced acute and tonic pain, the pin-pain test and formalin test. The analgesia induced by 100-200 mcicrog/kg doses of sialorphin required the activation of mu- and delta-opioid receptors, consistent with the involvement of endogenous opioid receptors in enkephalinergic transmission. We conclude that sialorphin protects endogenous enkephalins released after nociceptive stimuli by inhibiting NEP in vivo. Sialorphin is a natural systemically active regulator of NEP activity. Furthermore, our study provides evidence that it is a physiological modulator of pain perception after injury and might be the progenitor of a new class of therapeutic molecules.
| S-EPMC166266 | biostudies-literature
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
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
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
Project description:Gastrointestinal infectious diseases remain an important issue for human and animal health. Investigations on gastrointestinal infectious diseases are classically performed in laboratory animals leading to the problem that species-specific models are scarcely available, especially when it comes to farm animals. The 3R principles of Russel and Burch were achieved using intestinal organoids of porcine jejunum. These organoids seem to be a promising tool to generate species-specific in vitro models of intestinal epithelium. 3D Organoids were grown in an extracellular matrix and characterized by qPCR. Organoids were also seeded on permeable filter supports in order to generate 2D epithelial monolayers. The organoid-based 2D monolayers were characterized morphologically and were investigated regarding their potential to study physiological transport properties and pathophysiological processes. They showed a monolayer structure containing different cell types. Moreover, their functional activity was demonstrated by their increasing transepithelial electrical resistance over 18 days and by an active glucose transport and chloride secretion. Furthermore, the organoid-based 2D monolayers were also confronted with cholera toxin derived from Vibrio cholerae as a proof of concept. Incubation with cholera toxin led to an increase of short-circuit current indicating an enhanced epithelial chloride secretion, which is a typical characteristic of cholera infections. Taken this together, our model allows the investigation of physiological and pathophysiological mechanisms focusing on the small intestine of pigs. This is in line with the 3R principle and allows the reduction of classical animal experiments.
| S-EPMC8382199 | biostudies-literature