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)

An acid protease produced by the thermophilic fungus Penicillium duponti K 1014 has been purified by consecutive ion-exchange and gel permeation chromatography, and crystallized from aqueous acetone solution. The purified endopeptidase gave a symmetrical schlieren peak by sedimentation velocity, and was found to be homogeneous upon disc gel electrophoresis at pH 9.5. The enzyme was most active at pH 2.5 against milk casein and showed high thermostability. An isoelectric point of 3.81 was found by isoelectric focusing. A minimum molecular weight of 41 590 was calculated from the amino acid composition, adopting an arginine content of one residue per mole of enzyme. This minimum molecular weight is in good agreement with the value of 41 000 previously found by gel permeation (Hashimoto, H., Iwaasa, T., and Yokotsuka, T. (1973), Appl. Microbiol. 25, 578). Besides the thermostability, the purified P. duponti protease differs from other well-characterized acid proteases in that it contains carbohydrate, 4.33% expressed as glucose. The enzyme was not affected by p-bromophenacyl bromide, but was completely inactivated by alpha-diazo-p-bromoacetophenone, diazoacetyl-DL-norleucine methyl ester, and diazoacetylglycine ethyl ester, in the presence of Cu2+. The complete inactivation of the protease by diazoacetyl-DL-norleucine methyl ester resulted in the specific incorporation of 1 mol of norleucine/mol of enzyme. On the basis of similar behavior of other acid proteases toward this inactivator, the results suggest the presence at the active site of an unusually reactive carboxyl group, involved in the catalytic function. The naturally occurring pepsin inhibitor of Streptomyces naniwaensis [Murao, S., and Satoi, S. (1970), Agric. Biol. Chem. 34, 1265] inhibited also the protease, at a threefold molar excess with respect to the enzyme.
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PMID:Purification and properties of the thermostable acid protease of Penicillium duponti. 0 87

Proteinase A obtained from the culture medium of Aspergillus niger var. macrosporus is a unique acid endopeptidase that is insensitive (or less sensitive) to specific inhibitors of ordinary acid or aspartic proteinases, such as pepstatin, diazoacetyl-DL-norleucine methyl ester, and 1,2-epoxy-3-(p-nitrophenoxy)-propane. In the preceding paper (Takahashi, K., Inoue, H., Sakai, K., Kohama, T., Kitahara, S., Takishima, K., Tanji, M., Athauda, S. B. P., Takahashi, T., Akanuma, H., Mamiya, G., Yamasaki, M. J. Biol. Chem. 266, 19480-19483), we reported the complete primary structure of the mature enzyme determined at the protein level. The enzyme has a unique two-chain structure with a 39-residue light (L) chain and a 173-residue heavy (H) chain linked noncovalently. As an extension of this study, we isolated genomic and cDNA clones encoding this proteinase and determined their nucleotide sequences. To isolate a genomic clone, the genomic DNA was selectively amplified by polymerase chain reaction using mixed oligonucleotide primers designed from the amino acid sequence of the H chain, and a specific probe thus generated was used for screening a lambda gt10 genomic library. A cDNA for the enzyme was also selectively amplified by polymerase chain reaction using primers synthesized based on the sequence of the genomic DNA. Sequencing of the cloned genomic DNA and cDNA revealed the nucleotide sequence of the structural gene for the enzyme of 846 base pairs without introns. It encodes the precursor form of proteinase A, including an NH2-terminal preprosequence of 59 residues, the L chain of 39 residues, an intervening sequence of 11 residues, and the H chain of 173 residues linked in that order. Thus, proteinase A is thought to be synthesized as a single peptide chain preproenzyme of 282 residues, which is processed to generate the mature two-chain form.
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PMID:The gene and deduced protein sequences of the zymogen of Aspergillus niger acid proteinase A. 191 60

The distribution of proteases potentially reactive with peptide sequences containing pairs of basic amino acids or single basic amino acids was studied in subcellular fractions of a transplantable rat insulinoma using the affinity probes 125I-Tyr-Ala-Lys- ArgCH2Cl and 125I-Tyr-Ala-norleucine- ArgCH2Cl . Both probes labeled predominantly proteins of Mr = 39,000, 31,500, and 25,000. The Mr = 25,000 component appeared to be of lysosomal origin, while the Mr = 39,000 and 31,500 proteins were present in both the lysosomes and insulin granules. The Mr = 39,000 and 31,500 proteins were identified as precursor/product forms of the cysteine protease cathepsin B, while assays performed with fluorigenic peptide substrates suggested that the Mr = 25,000 protein was probably cathepsin L and/or H. The greater reactivity of the Mr = 39,000 form with the dibasic probe suggests that the relative proportions of the Mr = 39,000 and 31,500 forms of cathepsin B in different organelles may determine the extent to which the enzyme expresses activity as a specific (prohormone processing) endopeptidase or a more general (degradative) peptidase.
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PMID:Cathepsin B-related proteases in the insulin secretory granule. 632 60

Two distinct enzymes with aryl-l-aminoacylamidase activity were found in cellular extracts of Streptococcus durans. One of these enzymes was strictly an arylamidase lacking any observable N-terminal exopeptidase activity. The other enzyme functioned as an aminopeptidase capable of catalyzing the hydrolysis of a variety of l-peptide and arylamide substrates. The arylamidase (molecular weight, 80,000) purified 425-fold to homogeneity preferred arylamides containing large hydrophobic side chains, whereas the partially purified aminopeptidase (molecular weight, 300,000) preferred substrates with small nonpolar or basic side chains. Neither enzyme contained any endopeptidase or carboxypeptidase activity. The purified arylamidase was unaffected by metal chelators, but Mn(2+) and Mg(2+) did act as nonessential activators exclusively affecting the maximal velocity. The arylamidase-catalyzed hydrolysis of l-leucyl-p-nitroanilide exhibited a bell-shaped pH dependence for log V(max)/K(m) (pK(1) of 7.2; pK(2) of 8.5), whereas the log V(max)-versus-pH profile showed only an acid limb (pK of 6.8). The ionizable group responsible for the basic limb of the log V(max)/K(m)-versus-pH profile corresponded to the alpha-amino group of the substrate l-leucyl-p-nitroanilide (pK(a) = 8.5). Diazoacetyl-dl-norleucine methyl ester (2 mM) in the presence of 100 mM Cu(2+) caused a rapid inactivation of the enzyme (t((1/2)) of 24 min). Neither parachloromercuribenzoate (0.5 mM) nor N-ethylmaleimide (50 mM) had any effect on the arylamidase activity. Reversible noncompetitive inhibition was observed for iodoacetate (K(i) of 30 mM), N-acetylimidazole (K(i) of 4.0 mM), and ethyl acetimidate (K(i) of 45 mM), although time-dependent irreversible inactivation was not observed with these reagents.
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PMID:Aryl-L-aminoacylamidase activities in extracts of Streptococcus durans. 706 33

1. We have examined several phosphorus-containing peptides as potential mixed inhibitors of two neurotensin-degrading zinc metallopeptidases, endopeptidase 3.4.24.15 and endopeptidase 3.4.24.16. 2. Among a series of 13 phosphonamide peptides, N-(2-(2-naphtyl)ethylphosphonyl-glycyl-prolyl-norleucine (phosphodiepryl 08) was found to inhibit potently the hydrolysis of neurotensin by purified endopeptidase 3.4.24.15 and 3.4.24.16 with an identical Ki value of 0.4 nM. 3. Phosphodiepryl 08 displayed a strong selectivity towards the two peptidases since it failed to inhibit several other zinc-containing peptidases such as endopeptidase 3.4.24.11, angiotensin-converting enzyme, aminopeptidase M, leucine aminopeptidase and carboxypeptidases A and B. 4. The protective effect of phosphodiepryl 08 on neurotensin degradation was examined in vitro and in vivo in central and peripheral bioassays. 5. Phosphodiepryl 08 virtually abolished neurotensin degradation by 4-day-old plated pure cultured neurones from mouse embryos and greatly potentiated neurotensin-induced antinociception in the mouse hot plate test. 6. In the periphery, phosphodiepryl 08 inhibited neurotensin degradation by membranes prepared from isolated longitudinal smooth muscle of guinea-pig ileum and greatly potentiated the neurotensin-induced contraction of the same longitudinal smooth muscle preparation. 7. Our study indicates that phosphodiepryl 08 behaves as a potent and selective mixed inhibitor of endopeptidase 3.4.24.15 and 3.4.24.16 and can be used as a powerful agent to prevent neurotensin degradation, in vitro and in vivo, in central and peripheral assays.
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PMID:Phosphorus-containing peptides as mixed inhibitors of endopeptidase 3.4.24.15 and 3.4.24.16: effect on neurotensin degradation in vitro and in vivo. 758 3

The aim of this study was to identify the role of chymase in the conversion of exogenously administered Big endothelin-1 in the mouse in vivo. Real-time polymerase chain reaction analysis detected mRNA of mucosal mast cell chymases 4 and 5, endothelin-converting enzyme 1a, and neutral endopeptidase 24.11 in pulmonary, cardiac, and aorta homogenates derived from C57BL/6J mice, with the latter tissue expressing the highest levels of both chymase isoforms. Furthermore, hydrolysis of a fluorogenic peptide substrate, Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, was sensitive to the chymase inhibitors Suc-Val-Pro-Phe(P)(OPh)(2) (200 microM) and chymostatin [(S)-1-carboxy-2-phenylethyl]-carbamoyl-alpha-[2-iminohexahydro-4(S)-pyrimidyl]-(S)-Gly-X-Phe-al, where X can be the amino acid Leu, Val, or Ile) (100 microM) in supernatants extracted from the same tissue homogenates. In anesthetized mice, Big endothelin-1, endothelin-1 (1-31), and endothelin-1 triggered pressor responses (ED(50)s, 0.67, 0.89, and 0.16 nmol/kg) that were all reduced or potentiated by selective endothelin ET(A) or ET(B) receptor antagonists, respectively, BQ-123 (cyclo[D-Asp-Pro-D-Val-Leu-D-Trp]) or BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-l-leucyl]-1-(methoxycarbonyl)-D-tryptophyl]-d-norleucine sodium salt), each at 1 mg/kg. The pressor responses to big endothelin-1 were significantly reduced by the neutral endopeptidase inhibitor thiorphan (dl-3-mercapto-2-benzylpropanoylglycine) (1 mg/kg) or the endothelin-converting enzyme inhibitor CGS 35066 [alpha-[(S)-(phosphonomethyl)amino]-3-dibenzofuranopropanoic acid] (0.1 mg/kg). In contrast, the responses to endothelin-1 (1-31) were abolished by thiorphan but unaffected by CGS 35066. In addition, Suc-Val-Pro-Phe(P)(OPh)(2) (20-40 mg/kg) reduced, by more than 60%, the hemodynamic response to big endothelin-1 but not to endothelin-1 (1-31) and endothelin-1. Finally, intravenous administration of big endothelin-1 induced Suc-Val-Pro-Phe(P)-(OPh)(2)-sensitive increases in plasma-immunoreactive levels of endothelin-1 (1-31) and endothelin-1. The present study suggests that chymase plays a pivotal role in the conversion and cardiovascular properties of big endothelin-1 in vivo.
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PMID:Chymase-dependent conversion of Big endothelin-1 in the mouse in vivo. 1898 1