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)

Chicken aminopeptidase H is a cysteine protease possessing endopeptidase as well as aminopeptidase activity [Rhyu, M. R., Nishimura, T., Kato, Y., Okitani, A. & Kato, H. (1992) Eur. J. Biochem. 208, 53-59]. This enzyme exhibits molecular masses of 400 kDa on gel filtration and 52 kDa on SDS/PAGE, indicating that it consists of eight subunits with the same molecular mass. In the current study, we cloned the cDNA for the catalytic subunit of chicken aminopeptidase H. The open reading frame of the aminopeptidase H gene consists of 1362 base pairs encoding a 52-kDa protein consistent with the molecular mass determined on SDS/PAGE; the deduced amino acid sequence contains all the partial sequences determined for the purified enzyme. The sequence is similar to that of the bleomycin hydrolase of rabbit lung, which has been partially determined. The recombinant 52-kDa protein expressed in COS7 cells exhibited both aminopeptidase and endopeptidase activities, which were inhibited by monoiodoacetic acid. Furthermore, the expression of aminopeptidase H in COS7 cells was also recognized on immunoblotting. This gene is the first one for aminopeptidase H in an animal tissue whose sequence has been completely determined.
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PMID:cDNA cloning and expression of chicken aminopeptidase H, possessing endopeptidase as well as aminopeptidase activity. 915 54

Capillary zone electrophoresis (CZE) was utilized to identify a synaptobrevin-thioredoxin fusion protein (TSB-51). TSB-51 is a substrate for cleavage by botulinum toxin B at the Q(76)-F(77) site. TSB-51 was derivatized with a fluorophore, CBQCA [3-(4-carboxy-benzoyl)-2-quinoline-carboxaldehyde], for 4 h at room temperature. Optimal conditions for CZE separation of the TSB-51-CBQCA complex were determined: buffer (sodium borate), pH (9.0), applied voltage (25 kV), temperature (25 degrees C) and forward polarity. SDS-PAGE showed that TSB-51 had a molecular mass of approximately 19 kDa. The protein was transferred to PVDF membrane and sequenced by the Edman degradation method verifying the first twelve amino acids as SDKIIHLTDDSF. TSB-51 was also collected during CZE separation and subsequently sequenced yielding the first three amino acids as SDK. This CZE-LIF method coupled with the CBQCA derivatization, fraction collection and Edman sequencing allowed for identification of the recombinant protein, a fast separation run time and utilization of small volumes of peptide (1.5 ng protein/23.6 nl injection). This method will be used for monitoring the endopeptidase activity of botulinum toxin B on TSB-51.
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PMID:Identification of a recombinant synaptobrevin-thioredoxin fusion protein by capillary zone electrophoresis using laser-induced fluorescence detection. 927 Nov 30

20 and 26 S proteasomes were isolated from rat liver. The procedure developed for the 26 S proteasome resulted in greatly improved yields compared with previously published methods. A comparison of the kinetic properties of 20 and 26 S proteasomes showed significant differences in the kinetic characteristics with certain substrates and differences in the effects of a protein substrate on peptidase activity. Observed differences in the kinetics of peptidylglutamyl peptide hydrolase activity suggest that the 26 S complex cannot undergo the conformational changes of 20 S proteasomes at high concentrations of the substrate benzyloxycarbonyl (Z) -Leu-Leu-Glu-beta-naphthylamide. Various inhibitors that differentially affect the trypsin-like and chymotrypsin-like activities have been identified. Ala-Ala-Phe-chloromethyl (CH2Cl) inhibits chymotrypsin-like activity assayed with succinyl (Suc) -Leu-Leu-Val-Tyr-AMC, but surprisingly not hydrolysis of Ala-Ala-Phe-7-amido4-methylcoumarin (AMC). Tyr-Gly-Arg-CH2Cl inhibits Suc-Leu-Leu-Val-Tyr-AMC hydrolysis as well as trypsin-like activity measured with t-butoxycarbonyl (Boc) -Leu-Ser-Thr-Arg-AMC, while Z-Phe-Gly-Tyr-diazomethyl (CHN2) was found to inhibit only the two chymotrypsin-like activities. Radiolabeled forms of peptidyl chloromethane and peptidyl diazomethane inhibitors, [3H]acetyl-Ala-Ala-Phe-CH2Cl, [3H]acetyl- and radioiodinated Tyr-Gly-Arg-CH2Cl, and Z-Phe-Gly-Tyr-(125I-CHN2), have been used to identify catalytic components associated with each of the three peptidase activities. In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3, 4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by two-dimensional polyacrylamide gel electrophoresis followed by autoradiography/fluorography and immunoblotting with subunit-specific antibodies. In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in different relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities. The results strongly suggest a relationship between trypsin-like activity and chymotrypsin-like activity. They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase activities.
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PMID:Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities. 931 91

Proteinase A is a papain-like cysteine endopeptidase of vetch (Vicia sativa L.) which was assumed to initiate storage-globulin breakdown just after the onset of seed germination. This enzyme was purified from cotyledons of vetch seedlings. On gelatin-containg SDS gels, active proteinase A migrated with an apparent molecular mass of 21 kDa, whereas after heat denaturation its molecular size on SDS/PAGE was 29 kDa. Although proteinase A is capable of hydrolyzing storage globulins in vitro it could not be localized in the protein-body fraction of cotyledons from germinating seeds. cDNA clones encoding proteinase A precursor have been obtained by PCR. The precursor is composed of an N-terminal signal sequence followed by a propeptide, the region encoding mature proteinase A, and a C-terminal KDEL sequence. Mature proteinase A with a derived molecular mass of 25,244 Da does not have the KDEL sequence. The derived amino acid sequence of the proteinase A precursor is 78.2% identical to sulfhydryl-endopeptidase (SH-EP), a cysteine endopeptidase from germinating Vigna mungo seedlings. Northern blot analysis indicated that proteinase A mRNA appears de novo in cotyledons of 1-day-germinated vetch seeds, where its amount increases up to day 6. No proteinase A mRNA was detected in other vetch organs, not even in the embryo axis, which contains stored globulins. By means of antibodies raised against the purified and against recombinantly produced proteinase A, the 29-kDa bands of mature proteinase A were detected in cotyledon extracts of 6-day-germinated seeds when globulin degradation has already far proceeded. The reported data do not agree with the proposed triggering role of proteinase A in storage-globulin breakdown during germination.
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PMID:Proteinase A, a storage-globulin-degrading endopeptidase of vetch (Vicia sativa L.) seeds, is not involved in early steps of storage-protein mobilization. 934 82

Neutral endopeptidase-24.11 (NEP; neprilysin; EC 3.4.24.11) and endothelin-converting enzyme (ECE) are related zinc metallopeptidases involved in the processing of biologically active peptides. Only ECE, however, exists as a disulphide-linked homodimer. The covalent linkage in rat ECE is between Cys412 in each subunit, which is equivalent to Glu403 in rabbit NEP. Here we report that directed mutagenesis of Glu403 to cysteine in rabbit NEP creates a disulphide-linked homodimer, as revealed by transient transfection in COS-1 cells and SDS/PAGE of a membrane fraction. Under reducing conditions, both the mutant (E403C) and the wild-type NEP migrate as a polypeptide of 92 kDa. However, under non-reducing conditions, the Mr of the wild type remains unchanged, whereas that of the mutant is doubled. Co-transfection of wild-type ECE and E403C NEP cDNA did not result in the production of a NEP-ECE heterodimer. Comparison of the kinetic constants for wild-type and E403C mutant NEP with either [D-Ala2,Leu5]enkephalin or 3-carb oxypropanoyl-alanyl-alanyl- leucine-4-nitroanilide(Suc-Ala-Ala-Leu-NH-Np) as substrate show a decrease of approx. 50% in Vmax/Km for the mutant form. The IC50 value for inhibition of the mutant by phosphoramidon or thiorphan is increased 3-fold and 5-fold respectively. Although NEP and ECE exhibit only about 40% identity and differ substantially in substrate specificity and some other characteristics, these data indicate that they have considerable similarity in three-dimensional structure, allowing dimer formation in the mutant NEP with the disulphide link probably occurring in a hydrophilic surface loop.
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PMID:Mutagenesis of Glu403 to Cys in rabbit neutral endopeptidase-24.11 (neprilysin) creates a disulphide-linked homodimer: analogy with endothelin-converting enzyme. 958 75

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

A membrane-bound protease induced by sulfur mustard in cultured normal human epidermal keratinocytes (NHEK) was purified and partially characterized. Maximum enzyme stimulation occurred at 16 hr after normal human epidermal keratinocytes were exposed to 300 microM sulfur mustard. Purification to homogeneity of the protease was accomplished by Triton X-100 solubilization, ultracentrifugation, and dialysis, followed by ion-exchange chromatography through DEAE-cellulose and finally hydrophobic column chromatography through phenyl Sepharose. Analysis of the purified enzyme by SDS-PAGE revealed a single polypeptide at the 80 kDa region. Further investigation of biochemical properties showed that a synthetic serine-specific Chromozym TRY peptide and the physiological protein laminin were good substrates for this enzyme. Moreover, this enzyme was inhibited mostly by the serine-protease inhibitors leupeptin and di-isopropyl fluorophosphate and not by the cysteine protease inhibitor E-64 or the metalloprotease inhibitor 1,10-phenanthroline (Component H, CH), indicating the serine protease nature of this enzyme. This enzyme had a pH optimum in the range of 7.0 to 8.0. Amino acid sequencing of the purified enzyme revealed that this enzyme belongs to the endopeptidase family (serine protease), and is homologous with a mammalian-type bacterial serine endopeptidase that can preferentially cleave K-X, including K-P. These results suggest that serine-protease stimulation may be one of the mechanisms of mustard-induced skin blister formation, and that some specific serine-protease inhibitors may be useful for the treatment of this sulfur mustard toxicity.
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PMID:Purification and characterization of protease activated by sulfur mustard in normal human epidermal keratinocytes. 976 22

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

An endopeptidase was purified from Archachatina ventricosa by chromatography on columns of gel filtration, DEAE-Sepharose and phenyl-Sepharose. The preparation was shown to be homogeneous by polyacrylamide gel electrophoresis and capillary electrophoresis. The purified enzyme displayed two protein bands on SDS-polyacrylamide gel electrophoresis with estimated molecular weights of 90,000 and 121,000. The protease exhibited maximum proteolytic activity at 55 degrees C and at pH 8.0, but it retained more than 85% of its activity in the pH range 7.5 to 8.5. It was completely inactivated by the chelating agents EDTA and 1,10-phenanthroline which are metalloprotease inhibitors. Studies on substrate specificity showed that only the amide bonds of peptide substrates having a threonine residue at the P1' position were hydrolyzed by the purified protease. This endopeptidase constitutes a novel tool for the study of proteins in view of its narrow and unique substrate specificity.
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PMID:A novel endopeptidase with a strict specificity for threonine residues at the P1' position. 1008 Sep 15

Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence [M. Matsushima et al. (1994) J. Biol. Chem. 269, 19976-19982]. In the present study, we purified the porcine enzyme approximately 2,200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.
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PMID:Purification and further characterization of enteropeptidase from porcine duodenum. 1022 May 88


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