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 cDNA libraries constructed from the human acute lymphoblastic leukemia cell line KM3 in the expression vector lambda gt11, were screened with the anti-CALLA (common acute lymphoblastic leukemia antigen) mAb (monoclonal antibody) J5. The selected J5-positive clone I containing a partial cDNA insert was isolated and sequenced. For completing the cDNA sequence the cDNA libraries were further screened by hybridization with the DIG (digoxigenin)-labelled DNA probe derived from clone I, the 5'-end region was analysed by 5'-RACE (rapid amplification of cDNA ends) using a sequence specific primer. In total a 1639 bp cDNA sequence was determined. The cDNA sequence contains a 1260 bp open reading frame and the untranslated 3'- and 5'-end sides. The 420 residue amino acid sequence, deduced from the cDNA sequence, unexpectedly differs fundamentally from CALLA (CD10) although clones I and II were J5-positive in immuno screening. The mature protein corresponding to the cDNA was isolated and characterized from the KM3 cells using polyclonal antisera raised against the in vitro expressed polypeptide from clone I. The protein is expressed on plasma membrane, in cytosol and is secreted into culture medium, its relative molecular mass was determined to be 55 kDa on SDS-PAGE. The deduced amino acid sequence from cDNA was confirmed by peptide sequences. The new protein contains a basic amino acid rich putative DNA binding domain (b) with a potential nuclear targeting signal, two helix-loop-helix (HLH) motif regions, concurrently EF-hand motifs, an acidic amino acid rich region (a) between the EF-hands, and a leucine zipper (Z) motif. This DNA binding protein therefore is characterized by a linked motif "b/HLH/a/HLH/Z". The protein was designated NEFA: DNA binding/EF-hand/acidic amino acid rich region.
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PMID:Human protein NEFA, a novel DNA binding/EF-hand/leucine zipper protein. Molecular cloning and sequence analysis of the cDNA, isolation and characterization of the protein. 781 91

In this paper, data are presented on purification and properties of a new serine endopeptidase (duodenase) isolated from bovine duodenum mucosa. The enzyme has been purified to homogeneity by combinations of ammonium sulphate fractionation, carboxymethyl-cellulose 52 chromatography, and affinity chromatography on Sepharose 4B with Kunitz soybean trypsin inhibitor as a ligand. Some physicochemical properties of this protease have been investigated. The molecular mass of the purified duodenase was determined to be 29 +/- 0.5 kDa by SDS/PAGE and G-2000 SW column chromatography. The enzyme molecule is a single chain and the native enzyme is a monomeric protein. Its isoelectric point was estimated to be 10 +/- 0.2. Duodenase has two forms (I and II) which possess similar properties but differ in their amino acid composition. The new protease is a glycoprotein and contains approximately 3.5% sugars. The enzyme displays trypsin-like and chymotrypsin-like activities and hydrolyzes the amide bonds of substrates having Lys, Arg, Tyr, Phe and Leu residues at the P1 position. Duodenase is most active at pH 7.9-8.2. Duodenase was irreversibly inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, indicative of an active-site serine in this protease. alpha-N-Tosyl-L-lysine chloromethane and alpha-N-tosyl-L-phenylalanine chloromethane, which react with an active His, caused marked inhibition of trypsin-like and chymotrypsin-like activities of duodenase. The enzyme activity was strongly suppressed by trypsin inhibitors from different sources (soybeans, bovine lungs and Lima beans). Chicken egg white ovomucoid had no effect on the duodenase activity. The N-terminal sequence of the native duodenase (24 amino acid residues) shows high similarity with those of human and murine cytotoxic T-lymphocyte granzymes, human leukocyte cathepsin G and rat mast cell chymases. The biological role of duodenase is discussed.
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PMID:Duodenase, a new serine protease of unusual specificity from bovine duodenal mucosa. Purification and properties. 786 48

Three subunits, Ac115, Ac39, and the proteolipid, were positively identified in the membrane sectors of V-ATPases from different sources. We searched for organelle-specific protein in purified preparations of V-ATPase from bovine chromaffin granules. A diffused protein band at a position of about 45 kDa was identified in SDS-polyacrylamide gels of the above preparation. Following digestion with endopeptidase Glu-C (V-8), a polypeptide of about 10 kDa was isolated and subjected to amino acid sequencing. Hence, the cDNA encoding the protein Ac45 was cloned from a bovine adrenal medulla library. The cDNA sequence contains an open reading frame encoding a protein of 468 amino acids with a calculated molecular mass of 51,786 daltons. A potential signal sequence comprised of the first 35 amino acids and a potential transmembrane domain at the C terminus of the protein were identified. There exist seven potential glycosylation sites between the aforementioned protein motifs. Experiments with a specific antibody against Ac45 demonstrated that it is copurifying with the V-ATPase from chromaffin granules. Immunological cross-reactivity was observed with purified V-ATPase from bovine kidney microsomes but not from plasma membranes of epithelial cells. Cell-free expression of the protein from synthetic mRNA produced a single protein band at about 50 kDa on SDS gels. Upon inclusion of dog pancreas microsomes in the reaction mixture, a slow migrating band sensitive to peptide:N-glycosidase F was observed.
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PMID:A novel accessory subunit for vacuolar H(+)-ATPase from chromaffin granules. 792 63

An extracellular endopeptidase (proteinase) from Serratia marcescens (Serratia marcescens extracellular proteinase, EC 3.4.24.4), purified to homogeneity, was analyzed for enzyme properties. The enzyme has a polypeptide chain molecular mass of 52 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme has an optimal temperature of 40 degrees C and an optimal pH of 7.0. Enzyme activity was enhanced over two times by the addition of Ca2+ and Mg2+ ions and eliminated almost completely by the presence of 0.2% SDS. The enzyme has broad substrate specificity and contains neither cysteine nor methionine. Low homology was found between the NH2-terminal amino acid sequence of the enzyme of this study and the NH2-terminal sequence of a proteinase from another strain of S. marcescens. Chemical modification with N-bromosuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and 8-anilino-1-naphthalene sulfonic acid and by photooxidation with methylene blue reduced enzyme activity considerably. The enzyme was shown to have broad peptide bond specificity judging from the contribution of 11 amino acids to the carboxyl side of the peptide bonds hydrolyzed.
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PMID:Characterization and primary specificity of an extracellular metalloproteinase from Serratia marcescens. 801 34

A cDNA encoding the Androctonus australis Hector insect toxin 1 (AaH IT1) was expressed in yeast leading to secretion of fully biologically active protein. Three different multicopy plasmids were constructed using PCR. Expression was directed by the strong PGK1 promoter of the yeast vector pMA 91. Plasmid pMA 91-AaH IT1 encodes AaH IT1 and its own signal peptide. In the two other constructions, the cDNA encoding the mature part of AaH IT1 is fused to the prepro-signal sequence of the yeast alpha-mating-factor precursor; the pBAL 7-alpha-KREAEA-AaH IT1 includes the cDNA sequence encoding the KR(EAEA) processing sequence of the alpha-mating factor, and pBAL 7-alpha-KR-AaH IT1 encodes the KR fused directly to the AaH IT1 gene. The yeast alpha-mating-factor signal peptide launched the pro-alpha-mating-factor-AaH IT1 fusion protein into the secretory pathway. The fusion proteins are expected to be cleaved in the Golgi by the KEX2 endopeptidase and the STE13 dipeptidyl aminopeptidase, leading to release of mature AaH IT1. Pulse/chase labelling of transformed yeast protoplasts, followed by SDS/PAGE analysis of proteins immunoprecipitated from either the lysate or the extracellular fluid, showed that AaH IT1 was produced. The highest concentration of recombinant AaH IT1 in the culture medium, as determined using a 125I-AaH IT1 specific radioimmunoassay, was 4 micrograms/l (0.5 nM). The recombinant toxin was fully biologically active against cockroaches as assessed by injection and comparison to native AaH IT1. Moreover, it competed with radiolabelled native toxin for its receptor on the voltage-sensitive Na+ channel with a dissociation constant of 0.5 nM.
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PMID:Production of active, insect-specific scorpion neurotoxin in yeast. 805 34

Proteinases are important factors in the pathogenicity of many parasitic diseases. In this study, the proteolytic activities of 10 trypanosomatids from five different genera (Crithidia, Phytomonas, Endotrypanum, Trypanosoma and Leishmania) were determined by SDS-PAGE containing copolymerized gelatin as substrate. In almost all species we could detect two proteolytic classes, cysteine- and metalloproteinases, based on the inhibition of their activities by E-64 and 1,10-phenanthroline, respectively. In all cases, the metalloproteinase activities did not change over a broad pH range (from 5.5 to 10). E. schaudinni, T. mega, T. dionisii, C. luciliae, C. fasciculata, C. oncopelti and C. guilhermei expressed one or two metalloproteinases of 45-66 kDa, whereas in P. serpens and P. hyssopifolia a double band of this endopeptidase was detected at 94 kDa. In contrast, no metalloproteinase activity was observed in L. tarentolae. The optimal pH for the cysteine-proteinase activities was acidic (about 5.5). In E. schaudinni, T. mega and in Crithidia sp., these proteinases had an apparent molecular weight of 66-94 kDa, while L. tarentolae expressed a broad band from 29 to 45 kDa. In Phytomonas sp., this class of endopeptidase showed a unique feature, in that major cysteine-proteinases were found at 29-66 kDa, but multiple, low-activity bands were detected from 116 to 200 kDa. The most striking characteristic, however, was the very intense cysteine-proteinase activity expressed by T. dionisii (29-66 kDa). We conclude that these differences in the proteolytic profiles could be useful markers to characterize and compare trypanosomatids.
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PMID:Characterization of proteinases in trypanosomatids. 808 Dec 71

The post-translational processing of chromogranin A (CGA) and the nature of the enzyme(s) involved were investigated in rat pancreatic islet and insulinoma tissue. Pulse-chase radiolabelling experiments using sequence-specific antisera showed that the 98 kDa (determined by SDS/PAGE) precursor was processed to an N-terminal 21 kDa peptide, a C-terminal 14 kDa peptide and a 45 kDa centrally located peptide with a rapid time course (t1/2 approx. 30 min) after an initial delay of 30-60 min. The 45 kDa peptide was, in turn, converted partially into a 5 kDa peptide with pancreastatin immunoreactivity and a 3 kDa peptide with WE-14 immunoreactivity over a longer time period. Incubation of bovine CGA with rat insulinoma secretory-granule lysate produced peptides of 18, 16 and 40 kDa via intermediates of 65 and 55 kDa. N-terminal sequence analysis indicated that cleavage occurred at the conserved paired basic sites Lys114-Arg115 and Lys330-Arg331, suggesting that cleavage of the equivalent sites (Lys129-Arg130 and Lys357-Arg358) in the rat molecule produced the initial post-translational products observed in intact pancreatic beta-cells. The enzyme activity responsible for the cleavage of bovine CGA co-chromatographed on DEAE-cellulose with the type-2 proinsulin endopeptidase and with PC2 immunoreactivity. The type-1 enzyme (PC1/3) appeared inactive towards CGA. The requirement for Ca2+ ions and an acidic pH for conversion was consistent with the involvement of a member of the eukaryote subtilisin family, and the composition of the released peptides in pulse-chase and secretion studies suggested that conversion occurred in the secretory-granule compartment. The overall catalytic rate as well as the relative susceptibilities of the Lys114-Arg115 and Lys330-Arg331 sites to cleavage were affected by pH, suggesting that the ionic environment of the processing compartment may play a role in the differential processing of CGA which is evident in various neuroendocrine cells.
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PMID:The post-translational processing of chromogranin A in the pancreatic islet: involvement of the eukaryote subtilisin PC2. 814 63

1. A kinin-inactivating chymotrypsin-like serine-endopeptidase was purified 202-fold from human urine by DEAE-cellulose chromatography, gel filtration, DEAE/HPLC chromatography and affinity chromatography. It hydrolyzed bradykinin at the Phe5-Ser6 peptide bond at a rate of 1.090 mumol min-1 mg protein-1 at pH 8.0 and 37 degrees C. The molecular weight of this endopeptidase H2, estimated by SDS-polyacrylamide gel electrophoresis and by gel filtration, was 60 kDa, and its optimum pH for bradykinin hydrolysis was near 8.5. 2. Bradykinin inactivating activity was inhibited 100% by the serine-proteinase inhibitor PMFS (1 mM) and the chymotrypsin inhibitor TPCK (5 mM). Reagents such as 2-mercaptoethanol (3 mM) and pOH-mercuribenzoate (3 mM) inhibited the enzyme by 100% and 67%, respectively. 3. Endopeptidase H2 hydrolyzes the Phe-Ser bond of peptides related to bradykinin and its activity appears to be limited to peptide chains of < or = 18 amino acid residues since it does not hydrolyze BAM 22, peptide E or kininogen. 4. The molecular size and inhibition profile suggested that endopeptidase H2 differs from the serine-proteinases previously described in rat liver, rat hepatic endothelium, rat and rabbit brain. 5. The physiological role of endopeptidase H2 may be a link between the kinin and neuropeptide systems in the control of water-electrolyte balance.
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PMID:Purification and characterization of endopeptidase H2, a kinin inactivating serine proteinase (kininase) from human urine. 822 Feb 64

Kidney plasma membranes of Aplysia californica were shown to contain an endopeptidase activity which cleaved [Leu]enkephalin (Tyr-Gly-Gly-Phe-Leu) and [Leu]enkephalinamide (Tyr-Gly-Gly-Phe-Leu-NH2) at the Gly3-Phe4 bond, as determined by reverse-phase h.p.l.c. analysis of metabolites. The optimal pH was shown to be 6.5. The bivalent cation chelating agent, 1,10-phenanthroline protected [Leu]enkephalin from degradation, suggesting that this enzyme is a metallopeptidase. The degradation of [Leu]enkephalin was also abolished by the neutral endopeptidase-24.11 inhibitors RB104 (2-[(3-iodo-4-hydroxyl)-phenylmethyl]-4-N-[3-(hydroxyamino-3-oxo-1- phenylmethyl)-propyl]amino-4-oxobutanoic acid), HABCO-Gly [(3-hydroxy-aminocarbonyl-2-benzyl-1-oxypropyl)glycine], phosphoramidon and thiorphan, with IC50 values of 1 nM, 1 microM, 20 microM and 30 microM respectively. By contrast, the angiotensin-converting enzyme inhibitor captopril and the serine proteinase inhibitor phenylmethanesulphonyl fluoride were without effect. Phase separation experiments using Triton X-114 showed that about 64% of the neutral endopeptidase activity in the Aplysia kidney membrane corresponds to an integral membrane protein. A specific radioiodinated inhibitor ([125I]RB104) was shown to bind the Aplysia endopeptidase with high affinity; the KD and Bmax. values were 21 +/- 5 pM and 20.3 +/- 5 fmol/mg of proteins respectively. This inhibitor was used to determine the molecular form of the enzyme, after separation of solubilized membrane proteins on SDS/PAGE and transfer on to nitrocellulose membranes. A single protein band with an apparent molecular mass of 140 kDa was observed. The labelling was abolished by specific neutral endopeptidase inhibitors. This study provides the first biochemical characterization of an endopeptidase with catalytic properties similar to those of neutral endopeptidase-24.11 in the mollusc Aplysia californica.
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PMID:Identification and characterization of a neutral endopeptidase activity in Aplysia californica. 825 38

Endopeptidase 24.16 was purified from rat kidney homogenate on the basis of its ability to generate the biologically inactive degradation products neurotensin (1-10) and neurotensin (11-13). On SDS gels of the proteins pooled after the last purification step, the enzyme appeared homogeneous and behaved as a 70-kDa monomer. The peptidase was not sensitive to specific inhibitors of aminopeptidases, pyroglutamyl aminopeptidase I, endopeptidase 24.11, endopeptidase 24.15, proline endopeptidase and angiotensin-converting enzyme but was potently inhibited by several metal chelators such as o-phenanthroline and EDTA and was blocked by divalent cations. The specificity of endopeptidase 24.16 towards peptides of the tachykinin, opioid and neurotensin families was examined by competition experiments of tritiated neurotensin hydrolysis as well as HPLC analysis. These results indicated that endopeptidase 24.16 could discriminate between peptides belonging to the same family. Neurotensin, Lys8-Asn9-neurotensin(8-13) and xenopsin were efficiently hydrolysed while neuromedin N and kinetensin underwent little if any proteolysis by the peptidase. Analogously, substance P and dynorphins (1-7) and (1-8) were readily proteolysed by endopeptidase 24.16 while neurokinin A, amphibian tachykinins and leucine or methionine enkephalins totally resisted degradation. By Triton X-114 phase separation, 15-20% of endopeptidase 24.16 partitioned in the detergent phase, indicating that renal endopeptidase 24.16 might exist in a genuine membrane-bound form. The equipotent solubilization of the enzyme by seven detergents of various critical miscellar concentrations confirmed the occurrence of a membrane-bound counterpart of endopeptidase 24.16. Furthermore, the absence of release elicited by phosphatidylinositol-specific phospholipase C suggested that the enzyme was not attached by a glycosyl-phosphatidylinositol anchor in the membrane of renal microvilli. Finally, endopeptidase 24.16 could not be released from these membranes upon trypsinolysis.
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PMID:Rat kidney endopeptidase 24.16. Purification, physico-chemical characteristics and differential specificity towards opiates, tachykinins and neurotensin-related peptides. 842 55


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