Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neutral endopeptidase (NEP; E.C. 3.4.24.11) is a mammalian ectopeptidase identified as the common acute lymphoblastic leukemia antigen (CALLA or CD10). In order to investigate its cellular processing and its role in B lymphocyte differentiation, a fluorescent derivative of the mercapto NEP inhibitor thiorphan, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl) amino-1-hexyl]thiocarbamide (FTI), has been synthesized. The fluorescent characteristics of fluorescein were conserved in FTI after linkage with the thiol NEP inhibitor. FTI inhibited NEP with an IC50 value of 10 nM and a good selectivity compared to that of aminopeptidase N (greater than 100 microM) and angiotensin converting enzyme (32 microM). The FTI probe was shown to detect membrane-bound NEP using photomicroscopy on cultured cells or flow cytometry techniques. Using NEP-expressing MDCK cells and episcopic fluorescence microscopy, a specific labeling was obtained with 100 nM FTI which was completely displaced by 10 microM HACBOGly, a specific and potent inhibitor of NEP. Therefore, FTI can be considered a suitable tool for following cellular NEP traffic. In flow cytometry, the fluorescent probe FTI, used at concentrations as low as 1 nM with Reh6 cells, could be very useful for detecting NEP/CALLA on lymphoid cells. In addition, the recognition of FTI is independent of tissues and species, a major advantage of inhibitors over monoclonal antibodies.
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PMID:Detection of neutral endopeptidase-24.11/CD10 by flow cytometry and photomicroscopy using a new fluorescent inhibitor. 135 7

We previously reported that human theca interna cells and small luteal cells express membrane-bound aminopeptidase N, and suggested that membrane-bound peptidases are involved in folliculogenesis and luteal function by regulating extracellular peptide concentrations. In this study, we examined the expression of dipeptidyl peptidase IV (DPP IV), which is a membrane-bound peptidase and has its catalytic domain at extracellular sites, in human granulosa cells, thecal cells of growing, preovulatory, and atretic follicles, as well as corpora lutea. Indirect immunofluorescence staining of ovarian tissues with specific monoclonal antibodies revealed that DPP IV was present in large and small luteal cells in corpora lutea. DPP IV peptidase activity was also detected histochemically in corpora lutea. In growing, preovulatory, and atretic follicles, there was weak immunoreactivity and DPP IV peptidase activity on luteinized theca interna cells, but not on granulosa cells. The expression of DPP IV on the cell surface of large and small luteal cells was confirmed by indirect immunofluorescence staining of freshly isolated luteal cells. These results indicate that DPP IV is a useful surface differentiation marker of human luteal cells and suggest that peptidases are involved in luteal function.
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PMID:Human luteal cells express dipeptidyl peptidase IV on the cell surface. 138 70

Aminopeptidase activities were identified in extracts of kidney, ovotestis, head ganglia, heart and haemolymph of Aplysia californica. These enzyme preparations hydrolysed [3H][Leu]enkephalin at the Try-1-Gly-2 bond as determined by h.p.l.c. analysis of cleavage products. In all these tissues, enkephalin-degrading aminopeptidase activities were present both in membrane-bound and cytosolic fractions. The bivalent-cation-chelating agent, 1,10-phenanthroline, inhibited kidney membrane aminopeptidase activity with an IC50 of 30 microM, suggesting that this enzyme is a metalloproteinase. The aminopeptidase inhibitor amastatin was the most potent inhibitor of [Leu]enkephalin degradation (IC50 25 nM) by membrane-bound aminopeptidase, and bacitracin, bestatin and puromycin were about 100-1000 times less potent. In contrast with membrane-bound aminopeptidase, the cytosolic form is sensitive to puromycin. Angiotensin-converting enzyme inhibitor had no effect on [Leu]enkephalin degradation by kidney membranes, while the neutral endopeptidase inhibitors were poor inhibitors of the enzymes in this preparation. The Km values of the aminopeptidase in the kidney membranes and cytosolic fractions for the [Leu]enkephalin substrate were 2.4 and 7.4 microM respectively. The aminopeptidase present in the kidney membranes also hydrolysed endogenous Phe-Met-Arg-Phe-amide peptide at the Phe-1-Met-2 bond as well as synthetic alanine p-nitroanilide and leucine p-nitroanilide. When used in a competition assay, these substrates inhibited hydrolysis of [3H][Leu]enkephalin, suggesting that the same enzyme degraded all these substrates. Taken together, these results suggest that Aplysia tissues contain both a membrane-bound aminopeptidase related to the mammalian aminopeptidase N and a cytosolic puromycin-sensitive aminopeptidase.
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PMID:Identification and characterization of aminopeptidases from Aplysia californica. 141 57

Aminopeptidase N is a membrane-bound metalloprotease expressed on the surface of normal and malignant human myeloid cells, fibroblasts, hepatocytes, and the epithelial cells that form brush borders of the small intestine and kidney. Northern blot analysis of RNA extracted from these tissues revealed two distinct aminopeptidase N transcripts: a 3.7-kilobase (kb) transcript expressed by normal monocytes, myeloid leukemia cells, and fibroblasts and a 3.4-kb transcript expressed by intestinal epithelium and kidney cells. In intestinal epithelial cells, transcripts originated 47 base pairs upstream from the initiation codon and 22 base pairs downstream from a TATA box. By contrast, the longer transcripts found in myeloid cells and fibroblasts originated from several sites clustered in an upstream exon located 8 kb from the exon containing the initiation codon. Functional promoter activity was demonstrated by fusing sequences approximately 1 kb upstream from each transcription origin to bacterial reporter genes and transfecting the resultant constructs into murine NIH-3T3 fibroblasts. A novel feature of this system is that regulatory elements of the epithelial cell promoter, including the TATA box and transcription origin, are included within the 5'-untranslated region of the longer myeloid cell transcript. Both aminopeptidase N transcripts encode the same polypeptide, indicating that the physically distinct promoters must have evolved to regulate expression of this cell-surface peptidase by cells of different tissues.
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PMID:Separate promoters control transcription of the human aminopeptidase N gene in myeloid and intestinal epithelial cells. 167 38

We previously found that the myeloid cell surface glycoprotein CD13 (gp150) is identical to aminopeptidase N (EC 3.4.11.2), a widely distributed membrane-bound, zinc-dependent metalloprotease with an extracellular enzymatic domain that cleaves N-terminal amino acid residues from oligopeptides (J Clin Invest 83:1299, 1989). As a first step toward defining the function of this molecule on myeloid cells, we assessed cell surface-associated N-terminal peptidase activity by sensitive spectrophotometric measurements of the cleavage of p-nitroanilide amino acid derivatives. Aminopeptidase activity detected on the surface of normal and malignant hematopoietic cells coincided with the level of cell surface CD13 expression as measured by flow cytometry. The enzyme was specifically inhibited by the zinc-binding metalloprotease inhibitors, bestatin, 1,10-phenanthroline, or 2.2'-dipyridyl, but was not affected by several inhibitors of other classes of proteases. Aminopeptidase activity was demonstrated for CD13 molecules specifically immunoprecipitated from the surface of CD13-positive cells and was blocked by the metalloprotease inhibitor 1,10-phenanthroline. Moreover, cell surface aminopeptidase activity was partially inhibited when viable cells were incubated with two of a panel of 11 monoclonal antibodies (MoAbs) known to be specific for extracellular epitopes of human CD13. This inhibition was apparent in the absence of detectable downmodulation of CD13 molecules from the cell surface, suggesting that these MoAbs either physically interfere with substrate binding or alter the zinc-coordinating properties of aminopeptidase N molecules. Aminopeptidase N could play an important role in modulating signals generated by peptides at the surface of myeloid cells, either by removing key N-terminal residues from active peptides or by converting inactive peptides to active forms. The inhibitory antibodies used in this study should prove useful in delineating the physiologic roles of CD13/aminopeptidase N on normal and malignant myeloid cells.
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PMID:Metalloprotease activity of CD13/aminopeptidase N on the surface of human myeloid cells. 196 20

The uptake of beta-lactam antibiotics into small intestinal enterocytes occurs by the transport system for small peptides. The role of membrane-bound peptidases in the brush border membrane of enterocytes from rabbit and pig small intestine for the uptake of small peptides and beta-lactam antibiotics was investigated using brush border membrane vesicles. The enzymatic activity of aminopeptidase N was inhibited by beta-lactam antibiotics in a non-competitive manner whereas dipeptidylpeptidase IV was not affected. The peptidase inhibitor bestatin led to a strong competitive inhibition of aminopeptidase N whereas the uptake of cephalexin into brush border membrane vesicles was only slightly inhibited at high bestatin concentrations (greater than 1 mM). Modification of brush border membrane vesicles with the histidine-modifying reagent diethyl pyrocarbonate led to a strong irreversible inhibition of cephalexin uptake whereas the activity of aminopeptidase N remained unchanged. A modification of serine residues with diisopropyl fluorophosphate completely inactivated dipeptidylpeptidase IV whereas the transport activity for cephalexin and the enzymatic activity of aminopeptidase N were not influenced. With polyclonal antibodies raised against aminopeptidase N from pig renal microsomes the aminopeptidase N from solubilized brush border membranes from pig small intestine could be completely precipitated; the binding protein for beta-lactam antibiotics and oligopeptides of apparent Mr 127,000 identified by direct photoaffinity labeling with [3H]benzylpenicillin showed no crossreactivity with the aminopeptidase N anti serum and was not precipitated by the anti serum. These results clearly demonstrate that peptidases of the brush border membrane like aminopeptidase N and dipeptidylpeptidase IV are not directly involved in the intestinal uptake process for small peptides and beta-lactam antibiotics and are not a constituent of this transport system. This suggests that a membrane protein of Mr 127,000 is (a part of) the uptake system for beta-lactam antibiotics and small peptides in the brush border membrane of small intestinal enterocytes.
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PMID:Intestinal uptake of dipeptides and beta-lactam antibiotics. I. The intestinal uptake system for dipeptides and beta-lactam antibiotics is not part of a brush border membrane peptidase. 197 19

Recent studies have shown that some manifestations of cis-diamminedichloroplatinum (II) (cis-Pt) induced nephrotoxicity in animals may be exacerbated if the animals are nutritionally deprived of copper. The objective of the present study was to determine the effects of cis-Pt induced toxicity on enzyme activities in the kidney microvilli of rats with different copper statuses. Weanling male rats were fed copper-deficient (CuD) (less than 1 mg/kg Cu of diet) or copper-adequate (CuA) (5 mg/L of Cu in drinking water) regimens. After 24 days, rats were given i.v. injections of either cis-Pt (5 mg/kg BW) or saline in a 2 x 2 factorial design. At days 2 and 4 post-injection rats were killed and tubular microvilli isolated from the kidney cortex. Each preparation was assayed for the activities of 5 membrane-bound enzymes. Angiotensin-I converting enzyme (ACE) activity was 20 to 30% higher in the microvilli of CuD rats than in controls. Cis-Pt treatment enhanced ACE activity as well, and activity in treated rats was 60 to 110% higher than in controls. At day 2 there was a 20% greater increase in ACE activity in cis-Pt-treated CuD rats than in CuA rats. Aminopeptidase N activity was 35% lower in CuD rats than controls, but activity was not affected by cis-Pt. Gamma-glutamyltransferase activity was lowered by as much as 30% in cis-Pt-treated rats when compared to controls, but there was no effect of copper deficiency. Alkaline phosphatase and neutral endopeptidase 24.11 activities were significantly lower in microvilli of cis-Pt-treated rats than in those not treated.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of copper deficiency and cis-diamminedichloroplatinum (II) treatment on the activities of renal microvillar enzymes in rats. 198 71

By photoaffinity labeling of brush border membrane vesicles from rabbit small intestine with photoreactive derivatives of beta-lactam antibiotics and dipeptides, a binding protein for dipeptides and beta-lactam antibiotics with an apparent molecular weight of 127,000 was labeled. The labeled 127 kDa polypeptide could be solubilized with the non-ionic detergents Triton X-100, n-octyl glucoside or CHAPS. If the vesicles were solubilized prior to photoaffinity labeling, no clear incorporation of radioactivity into the 127 kDa polypeptide occurred indicating a loss of binding ability upon solubilization. By affinity chromatography of solubilized brush border membrane proteins on an agarose wheat germ lectin column, the binding protein for dipeptides and beta-lactam antibiotics of Mr 127,000 was retained on the column. With N-acetyl-D-glucosamine the photolabeled binding protein for beta-lactam antibiotics and dipeptides was eluted together with the brush border membrane-bound enzyme aminopeptidase N. Separation from aminopeptidase N and final purification was achieved by anion-exchange chromatography on DEAE-sephacel. Polyclonal antibodies against the purified binding protein were raised in guinea pigs. The photolabeled 127 kDa protein could be precipitated from solubilized brush border membranes with these antibodies. Incubation of brush border membrane vesicles with antiserum prior to photoaffinity labeling significantly reduced the extent of labeling of the 127 kDa protein. Treatment of brush border membrane vesicles with antiserum significantly inhibited the efflux of the alpha-aminocephalosporin cephalexin from the brush border membrane vesicles compared to vesicles treated with preimmune serum. These studies indicate that the binding protein for dipeptides and beta-lactam antibiotics of apparent molecular weight 127,000 in the brush border membrane of rabbit small intestinal enterocytes is directly involved in the uptake process of small peptides and orally active beta-lactam antibiotics across the enterocyte brush border membrane.
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PMID:Intestinal absorption of dipeptides and beta-lactam antibiotics. II. Purification of the binding protein for dipeptides and beta-lactam antibiotics from rabbit small intestinal brush border membranes. 226 92

To determine the primary structure of CD13, a 150-kD cell surface glycoprotein originally identified on subsets of normal and malignant human myeloid cells, we isolated the complete sequences encoding the polypeptide in overlapping complementary DNA (cDNA) clones. The authenticity of our cDNA clones was demonstrated by the ability of the coding sequences, subcloned in a retroviral expression vector, to mediate expression of bona fide CD13 molecules at the surface of transfected mouse fibroblasts. The nucleotide sequence predicts a 967 amino acid integral membrane protein with a single, 24 amino acid hydrophobic segment near the amino terminus. Amino-terminal protein sequence analysis of CD13 molecules indicated that the hydrophobic segment is not cleaved, but rather serves as both a signal for membrane insertion and as a stable membrane-spanning segment. The remainder of the molecule consists of a large extracellular carboxyterminal domain, which contains a pentapeptide consensus sequence characteristic of members of the zinc-binding metalloprotease superfamily. Sequence comparisons with known enzymes of this class revealed that CD13 is identical to aminopeptidase N, a membrane-bound glycoprotein thought to be involved in the metabolism of regulatory peptides by diverse cell types, including small intestinal and renal tubular epithelial cells, macrophages, granulocytes, and synaptic membranes prepared from cells of the central nervous system.
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PMID:Human myeloid plasma membrane glycoprotein CD13 (gp150) is identical to aminopeptidase N. 256 51

Using a polyclonal antibody, a partial cDNA clone for rat aminopeptidase M was identified in a lambda gt11 library from rat kidney. A synthetic oligonucleotide probe derived from the sequence of the insert was used to screen a randomly primed lambda gt10 library. This allowed the identification of several overlapping clones encoding the full sequence of the enzyme. The reading frame, 2898 base pairs in length, encodes a 966 amino acid polypeptide. A highly hydrophobic segment, 24 amino acids in length, located close to the aminoterminus, is proposed to serve as the membrane-spanning domain for this membrane-bound enzyme. The sequence includes nine potential N-linked glycosylation sites and one potential sulfation site. In addition, the rat aminopeptidase M sequence contains an eight amino acid consensus sequence believed to serve as the zinc binding domain in a family of zinc-metallohydrolases. Rat aminopeptidase M shows 77% similarity with the recently cloned human enzyme, as well as weaker but significant similarity with aminopeptidase N from E. coli (18%) and with human leukotriene A4 hydrolase (21%).
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PMID:Molecular cloning and amino acid sequence of rat kidney aminopeptidase M: a member of a super family of zinc-metallohydrolases. 256 64


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