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)

Three endopeptidases, proteinases A, B, and Y, were purified from baker's yeast, Saccharomyces cerevisiae. Two molecular forms of proteinase A (PRA), Mr 45,000 and 54,000, (estimated on SDS-PAGE) were obtained. Both forms were inhibited by pepstatin and other acid proteinase inhibitors. The enzyme digested hemoglobin most rapidly at pH 2.7-3.2 and casein at pH 2.4-2.8 and 5.5-6.0. The optimum pH for hydrolysis of protein substrates could be shifted to about 5 with 4-6 M urea. Urea also stimulated the enzyme activity by 30-50%. As other acid proteinases, the enzyme preferentially cleaved peptide bonds of X-Tyr and X-Phe type. A proteinase B (PRB) preparation of approximately Mr 33,000 possessed milk clotting activity and showed an inhibition pattern typical for seryl-sulfhydryl proteases. The purified enzyme could be stabilized with 40% glycerol and stored at -20 degrees C without significant loss of activity for several months. The third endopeptidase, designated PRY, of Mr 72,000 when estimated by Sephadex G-100 gel filtration, had properties resembling PRA and PRB. Similar to PRB, it could be inhibited by up to 90% with phenylmethylsulfonyl fluoride and para-chloromercuribenzoate and preferentially hydrolyzed the Leu15-Tyr16 peptide bond of the oxidized beta-chain of insulin. On the other hand, contrary to PRB, it had neither milk clotting activity nor esterolytic activity toward N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine ethyl ester and was stable during storage at -20 degrees C without glycerol. The enzyme also showed a lower pH optimum for hydrolysis of casein yellow than PRB.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and properties of three endopeptidases from baker's yeast. 266 27

A metal-dependent aminopeptidase (EC 3.4.11.-), designated APase Y, has been purified to homogeneity by conventional methods. The enzyme is composed of a single polypeptide chain with molecular mass of 102 kilodaltons, estimated by sodium dodecyl sulphate - polyacrylamide gel electrophoresis, with a blocked N-terminal amino acid. It possesses neither endopeptidase nor carboxypeptidase activity and is strongly inhibited by metal-chelating agents, Zn2+, and the protein inhibitor from Neurospora crassa. APase Y is insensitive to Cl anions, S--S reducing reagents, serine protease inhibitors, and the peptidase inhibitor benzamidine. Co2+, Hg2+, and p-chloromercuribenzoate can activate the enzyme up to 22, 20, and 55%, respectively. The holoenzyme is resistant to yeast endopeptidases A, B, and Y, whereas the apoenzyme (obtained after treatment with chelators) is susceptible to the serine endopeptidases B and Y. The enzyme catalyzes hydrolysis of most L peptides possessing free alpha-amino (or imino) group by stepwise removal of N-terminal residue. Peptides with L-leucine at the N terminus are cleaved preferentially. The enzyme is unable to catalyze hydrolysis of X--Pro type peptide bonds, and inefficiently hydrolyzes bonds between Asp--X and Glu--X. L-leucine p-nitroanilide hydrolyzes optimally at pH 8.2 with a Km value of 1 mM. The purified enzyme is stable during storage in 0.05 M phosphate buffer, pH 6.7, containing 40-50% glycerol, at -20 degrees C.
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PMID:The yeast aminopeptidase Y. 304 13

Rat liver cytosol has low hydrolytic activity against [3H]methylcasein at neutrality, but activity increases greatly on addition of various compounds such as poly-L-lysine, N-ethylmaleimide, and sodium dodecyl sulfate, suggesting that it contains latent proteolytic activity. The latent enzyme was found to be stabilized in the presence of 20% glycerol and to be activated by addition of poly-L-lysine. The latent enzyme was purified from a crude extract of rat liver to apparent homogeneity in the presence of 20% glycerol by conventional chromatographic techniques. The purified enzyme showed endoproteolytic activity toward various proteins when it was activated by the compounds listed above. It preferentially degraded N-substituted tripeptide substrates with a basic amino acid at the carboxyl terminus, as well as peptides containing neutral hydrophobic amino acids. It did not require activation for these peptidase activities, in contrast to its activity toward large proteins. Interestingly, a proteinase and a trypsin-like and a chymotrypsin-like peptidase activity could not be separated by customary chromatographic methods but were distinguishable by their sensitivities to various inhibitors, activators, and covalent modifiers, suggesting that the enzyme has three distinct active sites within a single protein. The enzyme seems to be a seryl endopeptidase showing maximal activity at neutral and weakly alkaline pH values. Thus, the enzyme is a unique protease with latent multifunctional catalytic sites. The distribution of the protease in soluble extracts of various rat tissues and cells was examined quantitatively by an enzyme immunoassay. The enzyme level was highest in liver and also in spleen, stomach, lung, small intestine, and kidney, but was low in heart, diaphragm, skeletal muscle, brain, and skin. The concentrations of enzyme in some established cell lines including hepatoma and rat kidney cells were comparable to that in normal liver hepatocytes. The enzyme was found mainly in the cytosol fraction, although a small amount was associated with microsomal membranes, suggesting that it is an extralysosomal protease. Immunohistochemical staining of the liver and skeletal muscles showed that the protease is distributed diffusely in panlobular hepatocytes with slight centrilobar predominance and is present in Kupffer cells, vascular endothelial cells, and bile duct epithelial cells in the liver and also diffusely in the intermyofibrillar spaces and vascular endothelial cells in skeletal muscle. The quantitative data obtained in the present study indicate the presence of the protease in the cytosol fraction of all rat tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A high molecular weight protease in the cytosol of rat liver. I. Purification, enzymological properties, and tissue distribution. 309 25

Prolipoprotein signal peptidase, a unique endopeptidase which recognizes glycyl glyceride cysteine as a cleavage site, was characterized in an in vitro assay system using purified prolipoprotein as the substrate. This enzyme did not require phospholipids for its catalytic activity and was found to be localized in the inner cytoplasmic membrane of the Escherichia coli cell envelope. Globomycin inhibited this enzyme activity in vitro with a half-maximal inhibiting concentration of 0.76 nM. Nonionic detergent, such as Nikkol or Triton X-100, was required for the in vitro activity. The optimum pH and reaction temperature of prolipoprotein signal peptidase were pH 7.9 and 37-45 degrees C, respectively. Phosphatidylglycerol:prolipoprotein glyceryl transferase (glyceryl transferase) activity was measured using [2-3H]glycerol-labeled JE5505 cell envelope and [35S]cysteine-labeled MM18 cell envelope as the donor and acceptor of glyceryl moiety, respectively. 3H and 35S dual-labeled glyceryl cysteine was identified in the product of this enzymatic reaction. The optimal pH and reaction temperature for glyceryl transferase were pH 7.8 and 37 degrees C, respectively.
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PMID:Prolipoprotein modification and processing enzymes in Escherichia coli. 636 52

Cucumisin (EC 3.4.21.25), a serine endopeptidase, was isolated by a simple purification procedure from the prince melon (Cucumis melo ssp. melo, cv. 'Prince Melon'). The enzyme is stable over a wide pH range (4-11) and to heat, 80% of its initial activity remaining even at pH 11.1 and at 60 degrees C for 20 min. The enzyme was inactive at 72 degrees C and pH 8.0, but 38% of the activity remained in the presence of 10% (w/v) glycerol. Caseinolysis by cucumisin indicated full activity in 8 M urea at pH 9.1 and 50 degrees C. Cucumisin was inactivated by treatment with trypsin at 37 degrees C for 24 h, but was not affected by alpha-chymotrypsin. The synthetic substrates benzyloxycarbonyltyrosine nitrophenyl ester (Z-Tyr-ONp) and benzoyltyrosine ethyl ester (Bz-Tyr-OEt) were cleaved, but Z-Lys-ONp and tosylarginine methyl ester (Tos-Arg-OMe) were not cleaved by cucumisin. Oxidized insulin B-chain was hydrolysed by cucumisin at 37 degrees C for 24 h, 21 cleavage sites being detected. Cucumisin could not cleave the C-termini of all the valine residues in the oxidized insulin B-chain molecule.
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PMID:Improved isolation, stability and substrate specificity of cucumisin, a plant serine endopeptidase. 757 59

Neutral endopeptidase 24.11, a membrane-bound metallopeptidase, cleaves, and degrades vasoactive peptides such as atrial natriuretic peptide, endothelin, angiotensin I, substance P, and bradykinin. Therefore, the presence of this metallopeptidase may contribute to the regulation of vascular tone and local inflammatory responses in the vascular endothelium and elsewhere. We determined neutral endopeptidase in cultured human endothelial cells from different vascular beds and studied its regulation by protein kinase C. Neutral endopeptidase was detected in all cultured endothelial cell types. Lowest concentrations were measured in human endothelial cells from umbilical veins (360 +/- 14 pg/mg protein), followed by pulmonary and coronary arteries; higher concentrations were found in endothelial cells from the cardiac microcirculation (1099 +/- 73 pg/mg protein). Neutral endopeptidase content increased during cell growth but was not affected by endothelial cell growth factor or modifications of the growth medium. Stimulation of protein kinase C with 1-oleoyl-2-acetyl-rac-glycerol (0.1 to 1 mumol/L) and phorbol 12-myristate 13-acetate (0.01 to 0.1 mumol/L) induced a time- and concentration-dependent increase of endothelial cells that was inhibited by cycloheximide (5 mumol/L), an inhibitor of protein synthesis. Incubation with phospholipase C (1 mumol/L) and thrombin (10 IU/mL) induced upregulation of neutral endopeptidase, resulting in 158 +/- 26% and 150 +/- 22% increases, respectively, compared with controls. The thrombin effect was inhibited by calphostin C (1 mumol/L), an inhibitor of protein kinase C. Endothelial neutral endopeptidase is constitutively expressed in endothelial cells from different origins and is inducible by thrombin via activation of the protein kinase C pathway.
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PMID:Regulation and differential expression of neutral endopeptidase 24.11 in human endothelial cells. 763 30

Meprin A is a zinc-dependent metallo-endopeptidase that is present in the brush border membrane of renal proximal tubular epithelial cells. Although this enzyme has been detected in the kidney of mammalian species and its biochemical structure and action have been characterized, the role of this endopeptidase in normal renal physiology and in disease states is unknown. Therefore, we assessed the susceptibility of inbred strains of mice with normal and low meprin A activity to ischemic and nephrotoxic acute renal failure. We report that two normal meprin A strains of mice, C57BL/6 and DBA/2, developed more severe renal functional and structural injury following either bilateral renal artery clamping or the injection of hypertonic glycerol, compared to two low meprin A strains, C3H/He and CBA. These findings suggest that meprin A plays a role in the pathophysiology of acute renal failure following a variety of insults to the kidney. We propose that redistribution of this metalloendopeptidase to the basolateral membrane domain during acute renal failure results in degradation of the extracellular matrix and damage to adjacent peritubular structures.
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PMID:The role of meprin A in the pathogenesis of acute renal failure. 769 99

Recent data support the view that neuropeptide mediators, in particular opioid peptides, participate in the control of hematopoiesis. The main arguments are: neuropeptides modulate the functions of lymphoid cells, macrophages and mature granulocytes; they control cell proliferation and differentiation in many tissues, particularly during embryogenesis; lymphoid cells, macrophages, polymorphonuclear granulocytes and bone marrow stromal elements express neuropeptide receptors; bone marrow cells produce opioid-like neuropeptides; the CD10/CALLA marker of lymphoid, myeloid and marrow stromal cells is an enzyme, endopeptidase, which cleaves- and thus activates/inactivates-opioid and other neuropeptides. We have shown that opioid peptides enkephalins, opioid antagonist naloxone, and the inhibitor of enkephalin-degrading endopeptidase, thiorphan, modulate the proliferation and differentiation of hematopoietic cells in clonal and long-term cultures of mouse bone marrow. The effects partly depended on the presence of the accessory hematopoietic elements, and followed a circadian pattern. The dose-responses were irregular, showed strain-dependent and individual variations, and apparently reflected the state of the activity of target cells, cellular interactions and simultaneous signals by other mediators. The enkephalins were shown to bind to specific (opioid) receptors on the target cells, and their signals to be transmitted to the cell interior by a cascade of secondary messengers including diacyl-glycerol (DAG), protein-kinase C (PKC) and Ca++ ions. Neuropeptide regulation of hematopoiesis might belong to a complex immuno-neuroendocrine network including melatonin.
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PMID:Enkephalins in hematopoiesis. 908 27

Glycerol, employed to mimic biological media with restricted water activity, has been shown to modify the activity of subtilisin BPN', an endopeptidase, towards the oxidized insulin B-chain, a well-studied substrate (FEBS Lett., 279 (1991) 123-131). Without minimizing the role of the microenvironment on the enzyme, we have studied the effect of glycerol addition on the structure of the enzyme substrate by homonuclear NMR spectroscopy and simulated annealing. Our results show that, in water, the oxidized insulin B-chain tertiary structure loses its central helix (residues B9-B19) and presents a folded structure with a flexible turn (residues B18-B24) in the beta-turn region of the insulin B-chain; whereas, in glycerol, the peptide is more rigid and is not folded. Moreover, in our experimental conditions, glycerol favors beta-strand secondary structure formation. Following these results, hypotheses about the differences observed in enzymatic activity on this substrate in glycerol have been postulated.
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PMID:Glycerol's influence on the oxidized insulin B-chain conformation in relation to the selectivity variation of subtilisin: an nuclear magnetic resonance and simulated annealing study. 998 30

Prolyl endopeptidase is the only endopeptidase that specifically cleaves peptides at proline residues. Although this unique specificity is advantageous for application in protein chemistry, the stability of the enzyme is lower than those of commonly used peptidases such as subtilisin and trypsin. Therefore, we attempted to apply a directed evolution system to improve the thermostability of the enzyme. First, an efficient expression system for the enzyme in Escherichia coli was established using the prolyl endopeptidase gene from Flavobacterium meningosepticum. Then, a method for screening thermostable variants was developed by combining heat treatment with active staining on membrane filters. Random mutagenesis by error-prone PCR and screening was repeated three times, and as a result the thermostability of the enzyme was increased step by step as the amino acid substitutions accumulated. The most thermostable mutant obtained after the third cycle, PEP-407, showed a half-life of 42 min at 60 degrees C, which was 60 times longer than that of the wild-type enzyme. The thermostable mutant was also more stable with a high concentration of glycerol, which is a necessary condition for in vitro amidation.
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PMID:Directed evolution to improve the thermostability of prolyl endopeptidase. 1096 43


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