Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.11 (CD10)
9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The activity of endopeptidase EC 3.4.24.15 (thimet oligopeptidase, EP 24.15), as measured by cleavage of a quenched fluorescent substrate, 7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Pro-D-Lys (2,4-dinitrophenyl), was increased 2-3 fold by the addition of 1 mM Mn2+ or of 10 mM Ca2+. The inhibitory capability of a specific EP. 24.15 inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, was also increased at similar concentrations of these metal ions. However, the hydrolysis of naturally-occurring peptides, thought to be the physiological substrates for EP 24.15, was not affected by either Mn2+ or Ca2+. These results suggest that the binding of synthetic analogs to the enzyme may differ significantly from the binding, and thus hydrolysis, of natural peptide substrates and caution against drawing conclusions about substrate interactions with the active site from data obtained with modified peptide ligands.
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PMID:Differential activation of endopeptidase EC 3.4.24.15 toward natural and synthetic substrates by metal ions. 895 29

Screening cultures of nonpathogenic microorganisms led us to a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051, which we purified and named BSase. The nucleotide sequence encoding BSase, with a molecular mass of 23,894 Da, completely agreed with that of the mpr gene, which had been reported by Rufo Jr. and Sloma et al. to encode a metalloprotease [J Bacteriol (1990) 172: 1019-1023 and 1024-1029 respectively]. However, enzymatic characterization revealed it to have the catalytic triad of a serine protease and not the consensus sequence of a metalloprotease, and it was inhibited by diisopropylfluorophosphate. We therefore consider BSase (mpr) to be a serine protease. In the alignment of the acidic-amino-acid-specific proteases, the proteases from bacilli have a highly conserved histidine residue, which is most important in the histidine triad in the proteases from streptomycetes. Furthermore, Ca2+ was necessary for its activity and stability. BSase cleaved the C-terminal glutamic acid with high specificity and was very stable over a wide pH range. On the basis of these properties, we tried to retrieve a bioactive peptide from a fusion protein by sequence-specific digestion, and succeeded in obtaining the bioactive peptide. BSase was found to be very useful as a tool for selective cleavage.
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PMID:Purification and characterization of a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051; application to the recovery of bioactive peptides from fusion proteins by sequence-specific digestion. 927 45

Ragweed (Ambrosia artemisiifolia) is clinically the most important source of seasonal aeroallergens, as it is responsible for the majority and most severe cases of hay fever (allergic rhinitis). Extracts from pollen grains have been shown to contain numerous proteins with various functions, including a novel serine proteolytic enzyme with chymotrypsin-like specificity that has been previously described (J. Biol. Chem. 1996; 271:26227-26232). We now report the isolation and properties of a second, trypsin-like enzyme with a molecular mass near 80 kD, from ragweed pollen extracts. This enzyme has a blocked N-terminus, a pH optimum near 9.0, and requires Ca2+ for stability and activity, but not reducing agents. The enzyme is inhibited by diisopropyl fluorophosphate, a general serine class proteinase inhibitor, and more specifically by N-p-tosyl-L-lysine chloromethyl ketone. Activity toward protein substrates was not detected, but various synthetic substrates and small biologically active peptides were efficiently cleaved, with a strong preference for Arg in the P1 position and either Arg or Gly in the P2 position. This specificity was confirmed through inhibition studies with both peptidyl chloromethyl ketone and organophosphate inhibitors. Significantly, atrial natriuretic peptide and angiotensin 2, whose degradation would amplify kinin activity and influence inflammatory diseases of the respiratory tract and nasal passages, were also rapidly hydrolyzed. Thus, the ragweed pollen endopeptidase may be involved in the inactivation of regulatory neuropeptides during pollen-initiated allergic reactions.
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PMID:Purification and characterization of an arginine-specific peptidase from ragweed (Ambrosia artemisiifolia) pollen. 949 Jun 54

Since calcium activated neutral proteinase (calpain) is present in the central nervous system (CNS) and degrades myelin proteins, this endopeptidase has been suggested to play a role in myelin destruction in demyelinating diseases such as multiple sclerosis (MS). In the present study, calpain immunocytochemical expression was examined in Lewis rats with acute experimental allergic encephalomyelitis (EAE), an animal model for MS and optic neuritis. To identify cells expressing calpain, we labeled rat optic nerve sections for calpain with a polyclonal myelin calpain antibody and with monoclonal antibodies for glial (GFAP, OX42) and inflammatory (CD2, ED2, ED1, IFN-gamma) cell-specific markers. The results showed increased calpain expression in microglia (OX42) and infiltrating macrophages (ED1,2) in EAE compared to normal controls. Astrocytes constitutively expressed calpain in controls and acute EAE. Reactive astrocytes in EAE located in or near inflammatory foci, exhibited markedly increased calpain expression. Most T cells in acute EAE showed low level calpain expression while activated IFN-gamma-producing lymphocytes in inflammatory foci exhibited elevated levels of calpain expression. Thus, our results demonstrate increased calpain expression (at transcriptional and/or translational levels) in a rat model of optic neuritis. A role for calpain in myelin destruction during optic neuritis may be relevant to the pathogenesis of this disorder.
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PMID:Increased calpain expression in experimental demyelinating optic neuritis: an immunocytochemical study. 951 58

Guinea pig intestinal phospholipase B is a calcium-independent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation of sn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.
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PMID:Ectopic epididymal expression of guinea pig intestinal phospholipase B. Possible role in sperm maturation and activation by limited proteolytic digestion. 959 72

Endothelin-3 (ET-3), a potent vasoactive peptide, is considered to be produced from big ET-3 by endothelin-converting enzyme (ECE) like the other members of the endothelin family (ET-1 and ET-2). We purified a novel ECE from bovine iris microsomes. The purified enzyme, a 140 kDa protein by SDS-PAGE analysis, converted big ET-3 to ET-3 but not big ET-1, with a Km value of 0.14 microM for big ET-3. The conversion to ET-3 was confirmed with sandwich EIA by monoclonal antibodies, the elution profile of HPLC, and intracellular calcium mobilization in CHO-K1 cells expressing recombinant human ET(B) receptors. The conversion activity was inhibited by an inhibitor of neutral endopeptidase 24.11 (NEP) phosphoramidon. These results show that ECE-3 purified from bovine iris is a novel metalloprotease totally different from ECE-1 or ECE-2, in that the enzyme is highly specific for big ET-3.
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PMID:Purification of a novel endothelin-converting enzyme specific for big endothelin-3. 965 54

Calcium-activated neutral proteinase (calpain) is a ubiquitous, cytosolic endopeptidase which is believed to play a role in many neural functions. In the present study, we examined the transcriptional and translational expression of microcalpain (microcalpain) and millicalpain (mcalpain) isoforms and the endogenous inhibitor calpastatin in rat and bovine spinal cord, brain stem, cerebellum, and cerebral cortex tissues using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. In rat central nervous system (CNS) samples, the microcalpain and mcalpain transcriptional expression was highest in white matter-enriched areas. Calpastatin mRNA expression demonstrated no significant differences among the CNS areas. Calpain and calpastatin translational expression levels were greatest in the spinal cord. In bovine CNS, microcalpain transcriptional expression was greatest in the spinal cord, while other CNS regions showed no significant differences. Bovine mcalpain transcriptional expression was similar among various CNS regions but marginally greater in the cortex. Translational expression of bovine calpain was greatest in the brain stem, while that of calpastatin was highest in the cerebral cortex. These results indicate that calpain expression varies among different CNS regions and is often highest in white matter-enriched areas.
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PMID:Calpain expression varies among different rat and bovine central nervous system regions. 971 Feb 68

Cell extracts of Trichoderma reesei exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences. This activity was stimulated by the presence of Ca2+ ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2. Analytical chromatofocusing revealed a single peak of activity. The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM p-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM. We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by T. reesei. Secretion of xylanase I (proprotein processing sequence -R-R- downward arrow-R- downward arrow-A-) and xylanase II (-K-R- downward arrow-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions. Secretion of cellobiohydrolase II (CBH II; -E-R- downward arrow-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected. In contrast, secretion of CBH I (-R-A- downward arrow-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected. Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein from Streptoalloteichus hindustanus, fused to a series of model proprotein-processing sequences downstream of the expression signals of the Aspergillus nidulans gpdA promoter. Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences. Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets. These data point to the existence of different endoproteolytic proprotein processing enzymes in T. reesei and demonstrate that dibasic processing is obligatory for the secretion of the proproteins containing this target.
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PMID:Role of endoproteolytic dibasic proprotein processing in maturation of secretory proteins in Trichoderma reesei. 972 60

Because mesangial cells (MC) are a target and a degradation site for angiotensin II (ANG II), we characterized the degrading enzymes and receptors of ANG IV, a metabolite of ANG II, on these cells. ANG IV was metabolized into its NH2-terminal deleted peptides, ANG II-(4-8), ANG II-(5-8), and ANG II-(6-8) by rat MC. Total protection of ANG IV was obtained only when PC-18, a specific aminopeptidase N (APN) inhibitor, and JFH-27A, a mixed inhibitor of dipeptidylaminopeptidase (DAP) and neutral endopeptidase (NEP), were simultaneously added. In contrast, thiorphan, an NEP inhibitor, was inactive. These results demonstrate the exclusive role of APN and DAP in ANG IV degradation. 125I-labeled ANG IV binding was studied in the presence of PC-18 and JFH-27A to suppress ligand degradation. Under these conditions, ANG IV-specific receptors could be demonstrated with a KD of 1.8 nM and a density of 55 fmol/mg. In contrast with MC, no evidence for ANG IV receptors could be obtained in freshly isolated glomeruli. ANG IV stimulated cytosolic calcium concentration in MC, whereas its NH2-terminal deleted metabolites were inactive. Therefore, ANG IV must be protected from degradation by APN and DAP in studies on the nonimmediate biological effects of this peptide.
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PMID:Characterization of angiotensin IV-degrading enzymes and receptors on rat mesangial cells. 975 25

Phosphopeptides that were derived from alpha s-CN or beta-CN were prepared with immobilized glutamic acid-specific endopeptidase, and their Ca2+ binding was characterized. alpha s-Casein or beta-CN was hydrolyzed in a fluidized bed bioreactor containing 2 ml of immobilized glutamic acid-specific endopeptidase by recirculating 20 ml of alpha s-CN or beta-CN solution (10 mg/ml in 50 mM Tris.HCl and 0.02% NaN3, pH 8.0) for 3 h at 20 degrees C. The molecular masses of casein peptides were monitored by SDS-PAGE. Each hydrolysate was applied to an anion-exchange column using stepwise elution with various concentrations of KCl to separate peptides. The casein phosphopeptide content of the elution profile was monitored by analysis of protein and P concentrations. Calcium binding in phosphopeptide-enriched fractions was determined by CaCl2 titration and measurement of free Ca2+ with a Ca-selective electrode. The electrophoresis patterns showed four major peptides having molecular masses of 10.8, 9.0, 6.6, and 3.6 kDa in the alpha s-CN hydrolysate and 9.3, 8.2, and 6.2 kDa in the beta-CN hydrolysate. The highest concentrations of P were detected in the fractions that eluted with 0.4 and 0.5 M KCl for the alpha s-CN hydrolysate and with 0.4 M KCl for the beta-CN hydrolysate. The calcium-binding ability was found only in the fraction that was eluted with 0.4 M KCl; the maximum Ca2+ binding and the apparent binding constant were 0.24 mmol/mg of protein and 75 M-1, and 0.14 mmol/mg of protein and 148 M-1, respectively. alpha s-Casein phosphopeptides had different patterns for Ca2+ binding than did beta-CN phosphopeptides as the total Ca concentration was increased. Calcium binding to these casein phosphopeptides differed from that previously characterized for the tryptic peptides.
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PMID:Preparation of phosphopeptides derived from alpha s-casein and beta-casein using immobilized glutamic acid-specific endopeptidase and characterization of their calcium binding. 983 27


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