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)

Surface peptidase activities on the human monocytic lineage cell line U937 were characterized. Two diisopropyl phosphofluoridate (DFP)-inhibitable serine peptidases were identified by differences in their hydrolytic activities on chromogenic peptides: one removed tripeptides from the free NH2-terminal end of the synthetic peptide Ala-Ala-Phe-p-nitroanilide (pNA) and was not inhibited by inhibitors of metallo-, cysteic-, and aspartic-proteinases, or by those of elastase-, trypsin- and chymotrypsin-like enzymes, suggesting the presence of a hitherto unidentified serine tripeptidyl endopeptidase; the other peptidase catalyzed the release of Gly-Pro from Gly-Pro-pNA and was inhibited by DFP, phenylmethyl sulfonyl fluoride and diprotin A, thus resembling dipeptidyl peptidase IV (DPP IV) with respect to its substrate specificity and inhibitor profile. A group of N-exo-aminopeptidase activities specifically inhibited by bestatin, was also detected when Ala-, Leu-, Arg- and Lys-pNA were used a substrates. The activities were surface associated and not secreted as determined by extracellular location of product and enzymatic recovery in highly purified U937 cell membranes. Peripheral monocytes and macrophages were found to virtually exhibit identical levels of these two classes of peptidase activities when compared to those detected on U937 cells. The relative contributions of these hydrolytic enzymes to the cleavage of bioactive and radioiodinated cytokines including tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha and interferon-gamma was next examined. The results indicated that N-aminopeptidases do not appear to participate in the catabolism of any tested cytokine. In contrast, the most interesting finding was that both serine peptidases participate in TNF-alpha degradation. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native 17-kDa molecule TNF-alpha, and the concomitant release of biologically inactive fragments of less than or equal to 2 kDa. Together, these observations indicate new roles for both the DPP IV-like enzyme and the tripeptidyl endopeptidase located at the surface of human monocytic cells, including the regulation of the extracellular TNF-alpha concentration. Thus, the identification of functional ectopeptidases provides insight into their potential role in both normal and malignant monocytic function.
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PMID:Human U937 cell surface peptidase activities: characterization and degradative effect on tumor necrosis factor-alpha. 134 32

The cleavage of dynorphin and three analogs containing paired basic residues by several proteases was investigated. The cysteine protease of neuroblastoma cells cleaved only the bond between Arg-Arg residues. Submandibular arginyl-endopeptidase, however, cleaved bonds between both Arg-Arg and Arg-Lys residues, and pancreatic trypsin at the carboxyl sides of both arginine and lysine residues. This shows that the cysteine protease is highly specific for paired arginine residues.
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PMID:Dynorphin-degrading cysteine protease is highly specific for paired arginine residues. 134 63

Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones. This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways. Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions. A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates. Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities. Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant. This system also provides a tool for further studies of specific proteases of fungi.
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PMID:Proteolytic events in the processing of secreted proteins in fungi. 136 16

The Saccharomycopsis fibuligera alpha-amylase (Sfamy) gene was expressed in Saccharomyces cerevisiae. The highest productivity of Sfamy was 70 mg per liter of culture broth. We purified Sfamy from the culture broth and identified the NH2 terminal primary sequence. This sequence suggests that the Sfamy gene product is synthesized as a pre-pro-precursor, and the pro-sequence is cleaved after a Lys-Arg sequence with the calpain-like endopeptidase encode by the KEX2 gene, resulting in mature Sfamy protein composed of 468 amino acids. Furthermore, the enzyme Sfamy is a glycoprotein in which one N-linked sugar chain containing mannose residues is attached to the Asn residue at the 198 position. The Km and kcat values were 1.1 x 10(-4) M and 1.4 x 10(2) sec-1, respectively, using amylose (the degree of polymerization n = 18) as a substrate. Moreover, the secondary structure, the location of the secondary elements including alpha-helix, beta-sheet, and loop, and tertiary structure were predicted theoretically on the basis of the molecular structure of Aspergillus oryzae alpha-amylase. Taka-amylase A (TAA). These results indicate that Sfamy protein is composed of main (M) and C-terminal (C) domains. The molecular structure of M domain closely resembles that of TAA, but the C domain appears to be more compact than that of TAA because of deletions at three regions forming turns and one region forming alpha-helix.
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PMID:The enzymatic and molecular characteristics of Saccharomycopsis alpha-amylase secreted from Saccharomyces cerevisiae. 136 6

We investigated the amino acid sequence requirements for intracellular cleavage of the Rous sarcoma virus glycoprotein precursor by introducing mutations into the region encoding the cleavage recognition site (Arg-Arg-Lys-Arg). In addition to mutants G1 (Arg-Arg-Glu-Arg) and Dr1 (deletion of all four codons) that we have reported on previously (L. G. Perez and E. Hunter, J. Virol. 61:1609-1614, 1987), we constructed two additional mutants, AR1 (Arg-Arg-Arg-Arg), in which the highly conserved lysine is replaced by an arginine, and S19 (Ser-Arg-Glu-Arg), in which no dibasic pairs remain. The results of these studies demonstrate that when the cleavage sequence is deleted (Dr1) or modified to contain unpaired basic residues (S19), intracellular cleavage of the glycoprotein precursor is completely blocked. This demonstrates that the cellular endopeptidase responsible for cleavage has a stringent requirement for the presence of a pair of basic residues (Arg-Arg or Lys-Arg). Furthermore, it implies that the cleavage enzyme is not trypsinlike, since it is unable to recognize arginine residues that are sensitive to trypsin action. Substitution of the mutated genes into a replication-competent avian retrovirus genome showed that cleavage of the glycoprotein precursor was not required for incorporation into virions but was necessary for infectivity. Treatment of BH-RCAN-S19-transfected turkey cells with low levels of trypsin resulted in the release of infectious virus, demonstrating that exogenous cleavage could generate a biologically active glycoprotein molecule.
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PMID:Mutations within the proteolytic cleavage site of the Rous sarcoma virus glycoprotein define a requirement for dibasic residues for intracellular cleavage. 137 May 59

Neutral endopeptidase 24.11 contains an active site arginine believed to function in substrate binding. This arginine is thought to form an ionic interaction with the COOH-terminal carboxylate of NEP substrates. The functionality of arginine 102 has been investigated by using site-directed mutagenesis to produce mutants in which this residue was converted to a lysine, glycine, glutamine, or glutamate. All of the mutants exhibited essentially full activity as determined with a synthetic peptide amide, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. In contrast, activity was detected only with the wild-type enzyme and the lysine mutant using a synthetic substrate containing a free COOH-terminal carboxylate, dansyl-Gly-Trp-Gly. Inhibition studies with the physiologically active peptide substrates substance P, endothelin, and angiotensin I, as well as substance P free acid, [D-Ala2,Leu5]enkephalin, and [D-Ala2,Leu5]enkephalinamide indicated a lack of importance of arginine 102 in substrate binding. With [D-Ala2,Met5]enkephalin and the chemotactic peptide, N-formyl-Met-Leu-Phe, a significant decrease in affinity is observed with the arginine 102 mutants. These results suggest that the contribution of arginine 102 to substrate binding is dependent upon the strength of other subsite interactions. Examination of dipeptides as inhibitors indicates that the nature and orientation of the P'2 residue is important in determining the strength of the interaction of arginine 102 with its substrates.
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PMID:Analysis of the importance of arginine 102 in neutral endopeptidase (enkephalinase) catalysis. 137 21

Melittin possesses an amphiphilic property in the primary sequence in which hydrophilic residues are located at the C-terminal region from Lys-21 to Gln-26. A part of the hydrophilic sequence was cleaved off by endopeptidase Arg-C to obtain melittin 1-22. The affinity of melittin 1-22 for neutral phospholipid membrane was reduced to 1/3 that of melittin, indicating that the basic residues, Lys-23 and Arg-24, are important in binding of melittin to the membrane. The melittin 1-22 was extended toward the C-terminal end by connection of lactose (melittin-lac), the membrane affinity of which was slightly higher than the melittin 1-22, but lower than melittin. The leakage experiment of 5,6-carboxyfluorescein encapsulated in DPPC liposomes showed that the activities of melittin 1-22 and melittin-lac in membrane lysis were much lower than melittin. However, the melittin 1-22 formed a voltage-dependent ion-channel in an azolectin bilayer membrane. It is thus considered that Lys-23 and Arg-24 residues of melittin play an important role in binding to the polar region of membrane for lysis, but not for ion-channel formation.
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PMID:Interaction of melittin derivatives with lipid bilayer membrane. Role of basic residues at the C-terminal and their replacement with lactose. 138 6

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

Neuropeptides are synthesized as large precursor proteins that undergo posttranslational cleavages and modifications to produce bioactive peptides. Here, we have cloned two closely related precursor proteins for the sea anemone neuropeptide Antho-RFamide (<Glu-Gly-Arg-Phe-NH2) from Anthopleura elegantissima. The first precursor (435 amino acids long) contains 13 copies of immature Antho-RFamide (Gln-Gly-Arg-Phe-Gly) and nine other, Antho-RFamide-related neuropeptide sequences that are in the C-terminal part of the protein. The second precursor (429 amino acid residues) harbors 14 copies of immature Antho-RFamide and eight other related peptide sequences. Each copy of Antho-RFamide or Antho-RFamide-related peptide is followed, at its C-terminal side, by a single Arg residue, which is an established signal for posttranslational cleavage. At the N terminus of each Antho-RFamide sequence, however, basic residues are lacking, and instead one or more acidic residues occur. These acidic residues are the cleavage sites for a new type of processing enzyme occurring in neurons. This enzyme could either be an amino- or endopeptidase hydrolyzing at the C-terminal side of Asp or Glu residues. The N-terminal regions of the two precursor proteins harbor eight copies of the putative neuropeptide sequence Pro-Gln-Phe-Trp-Lys-Gly-Arg-Phe-Ser and three additional, closely related sequences. The total number of all established and putative neuropeptides that may be cleaved from the precursors is 33. Thus, the Antho-RFamide precursors beong to the most complex peptide precursor proteins known so far.
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PMID:Identification of a novel type of processing sites in the precursor for the sea anemone neuropeptide Antho-RFamide (<Glu-Gly-Arg-Phe-NH2) from Anthopleura elegantissima. 142 3

Three different types of biotinylated endothelin 1 (ET-1) derivatives, [Cys1]-biotinylated ET-1, [Lys9]-biotinylated ET-1, and [Cys1][Lys9]-dibiotinylated ET-1, were obtained when the biotinylation reaction was carried out with sulfosuccinimidyl-6-(biotinamido)hexanoate in an aqueous solvent. The binding of [Lys9]-biotinylated ET-1 to the ET receptor was as efficient as that of natural ET-1, whereas the binding of either [Cys1]-biotinylated ET-1 or [Cys1][Lys9]-dibiotinylated ET-1 was significantly reduced. When ET-1 was reacted with succinimidyl-6-(biotinamido)hexanoate in an organic solvent, ET-1 was exclusively modified at lysine 9. The ET receptor was then isolated from human placenta by affinity chromatography with [Lys9]-biotinylated ET-1 and avidin-agarose. The purified ET receptor was active in ET binding and was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptides with apparent molecular masses of 45 and 35 kDa. The NH2-terminal amino acid sequence indicated that the two polypeptides were from an identical subtype of the ET receptor (ETB, the ligand-nonselective type). A signal peptide from Met1 to Gly26 was missing from the 45-kDa ETB, whereas 64 amino acids at the NH2 terminus were missing from the 35-kDa ETB due to proteolytic cleavage which occurred between Arg64 and Ser65. Indeed, incubation of purified ETB with endopeptidase Arg-C resulted in degradation of the 45-kDa ETB, giving rise to the 35-kDa species by a specific cleavage at Arg64. The 35-kDa ETB was active in binding to ET-1, indicating that the NH2-terminal 64-amino-acid residues are not essential for ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biotin derivatives of endothelin: utilization for affinity purification of endothelin receptor. 145 57


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