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 maltose-regulated mlr-2 gene from the hyperthermophilic archaeon Pyrococcus furiosus having homology to bacterial and eukaryal prolyl endopeptidase (PEPase) was cloned and overexpressed in Escherichia coli. Extracts from recombinant cells were capable of hydrolyzing the PEPase substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide (ZGPpNA) with a temperature optimum between 85 and 90 degrees C. Denaturing gel electrophoresis of purified PEPase showed that enzyme activity was associated with a 70-kDa protein, which is consistent with that predicted from the mlr-2 sequence. However, an apparent molecular mass of 59 kDa was obtained from gel permeation studies. In addition to ZGPpNA (K(Mapp) of 53 microM), PEPase was capable of hydrolyzing azocasein, although at a low rate. No activity was detected when ZGPpNA was replaced by substrates for carboxypeptidase A and B, chymotrypsin, subtilisin, and neutral endopeptidase. N-[N-(L-3-trans-Carboxirane-2-carbonyl)-L-Leu]-agmatine (E-64) and tosyl-L-Lys chloromethyl ketone did not inhibit PEPase activity. Both phenylmethylsulfonyl fluoride and diprotin A inhibited ZGPpNA cleavage, the latter doing so competitively (K(lapp) of 343 microM). At 100 degrees C, the enzyme displayed some tolerance to sodium dodecyl sulfate treatment. Stability of PEPase over time was dependent on protein concentration; at temperatures above 65 degrees C, dilute samples retained most of their activity after 24 h while the activity of concentrated preparations diminished significantly. This decrease was found to be due, in part, to autoproteolysis. Partially purified PEPase from P. furiosus exhibited the same temperature optimum, molecular weight, and kinetic characteristics as the enzyme overexpressed in E. coli. Extracts from P. furiosus cultures grown in the presence of maltose were approximately sevenfold greater in PEPase activity than those grown without maltose. Activity could not be detected in clarified medium obtained from maltose-grown cultures. We conclude that mlr-2, now called prpA, encodes PEPase; the physiological role of this protease is presently unknown.
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PMID:Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus. 917 7

We have developed an algorithm (MassDynSearch) for identifying proteins using a combination of peptide masses with small associated sequences (tags). Unlike the approach developed by Matthias Mann, 'Tag searching', in which the sequence tags are generated by gas phase fragmentation of peptides in a mass spectrometer, 'Rag Tag' searching uses peptide tags which are generated enzymatically or chemically. The protein is digested either chemically or with an endopeptidase and the resultant mixture is then subjected to partial exopeptidase degradation. The mixture is analyzed by matrix assisted laser desorption and ionization time of flight mass spectrometry and a list of intact peptide masses is generated, each associated with a set of degradation product masses which serve as unique tags. These 'tagged masses' are used as the input to an algorithm we have written, MassDynSearch, which searches protein and DNA databases for proteins which contain similar tagged motifs. The method is simple, rapid and can be fully automated. The main advantage of this approach is that the specificity of the initial digestion is unimportant since multiple peptides with tags are used to search the database. This is especially useful for proteins like membrane, cytoskeletal, and other proteins where specific endopeptidases are less efficient and lower specificity proteases such as chymotrypsin, pepsin, and elastase must be used.
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PMID:An algorithm for the identification of proteins using peptides with ragged N- or C-termini generated by sequential endo- and exopeptidase digestions. 974 53

Vitamin B2 and flavin cofactors are transported tightly bound to immunoglobulin in human serum. We reasoned that anti-mitochondrial flavoprotein autoantibodies (alpha Fp-AB) present in the serum of patients with myocarditis and cardiomyopathy of unknown aetiology may form immunoglobulin aggregates with these serum proteins. However, immunodiffusion and Western blot assays demonstrated that the flavin-carrying proteins were not recognized by alpha Fp-AB. Apparently the flavin moiety in the native protein conformation was inaccessible to alpha Fp-AB. This conclusion was supported by the absence of an immunoreaction between the riboflavin-binding protein from egg white and alpha FP-AB. Intravenous application of vitamin B2 to rabbits immunized with 6-hydroxy-D-nicotine oxidase, a bacterial protein carrying covalently attached FAD, did not neutralize alpha Fp-AB which had been raised in the serum of the animals. FAD-carrying peptides generated from 6-hydroxy-D-nicotine oxidase by trypsin and chymotrypsin treatment were not recognized by the alpha Fp-AB, but those generated by endopeptidase Lys were. This demonstrates that the epitope recognized by alpha Fp-AB comprises, besides the flavin moiety, protein secondary structure elements.
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PMID:Anti-mitochondrial flavoprotein autoantibodies of patients with myocarditis and dilated cardiomyopathy (anti-M7): interaction with flavin-carrying proteins, effect of vitamin B2 and epitope mapping. 1019 10

To clarify the neuroendocrine differentiation and CD10 expression in solid-pseudopapillary tumors (SPTs) of the pancreas, we performed immunohistochemical analysis in 19 such tumors, including one solid-pseudopapillary carcinoma (SPC), along with 20 pancreatic neuroendocrine tumors (PNTs), six acinar cell carcinomas (ACCs), and one pancreatoblastoma (PB). We used antisera directed against CD56, synaptophysin, protein gene product 9.5, the alpha-subunit of Go protein, chromogranin A, CD10, trypsin, chymotrypsin, various cytokeratins (CKs), CA19-9, vimentin, and alpha-1-antitrypsin (AAT). All SPTs exhibited immunoreactivity for CD56 and CD10, and 15 expressed other neuroendocrine markers focally with the exception of chromogranin A. Frequent clustering of synaptophysin-positive cells was noted. Two cases contained a peculiar nodule that cytomorphologically and immunohistochemically resembled PNT. CD10-positive cells were scarce in one SPC. PNTs were CD56-positive, but often with faint intensity, and staining for other neuroendocrine markers, including chromogranin A, was diffusely positive. CD10 was detected, mostly in a focal pattern, in five PNTs. Pan-CK, CK8, CK18, and CK19 were more frequently demonstrated in PNT than SPT. Vimentin and AAT were often identified in PNT as well and were not specific for SPT. ACCs were CD56-negative, with the exception of one case designated as a mixed acinar-endocrine carcinoma. PB was focally positive for CD56 at the periphery of the tumor nests. Four ACCs and one PB exhibited focal CD10 reactivity. This study demonstrated the unique immunohistochemical features of SPT. Our results also suggest that SPT exhibits, at least focally, neuroendocrine differentiation, and that these neuroendocrine markers and CD10 are diagnostically useful.
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PMID:Solid-pseudopapillary tumor of the pancreas: immunohistochemical localization of neuroendocrine markers and CD10. 1102 97

The peptidolytic activity of fresh and frozen mucosal homogenates from five regions (duodenum, jejunum, ileum, caecum and colon) of possum intestine from Trichosurus vulpecula towards human Luteinizing Hormone Releasing Hormone (LHRH) was investigated. The rank of order of specific peptidolytic activity of the mucosal homogenates was jejunum > ileum > caecum> duodenum = colon, with a 3 to 4 fold difference between the least and the most active segment in both frozen and fresh samples. The formation of peptides LHRH (1-3), LHRH (1-4) and LHRH (1-5) suggest endopepetidase-24.18, endopeptidase-24.15 and angiotensin converting enzyme (ACE) might be responsible for the peptide degradation in mucosal homogenates. The inhibition of LHRH degradation by mucosal homogenates was evaluated in four regions (jejunum, ileum, caecum and colon) of possum intestine. Ethylenediaminetetraacetic acid (EDTA, 5 mM), sodium deoxycholate (SDA, 10 mM) and bacitracin (3.5 or 9 mM) inhibited the degradation of LHRH in mucosal homogenates from small intestine and hindgut. However, the serine protease inhibitor, soybean trypsin-chymotrypsin inhibitor (SBTI), did not prevent degradation of LHRH. It is concluded that combining peptides with inhibitors may enhance oral delivery of bioactive peptides or proteins to possums.
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PMID:Enzymatic degradation of luteinizing hormone releasing hormone (LHRH) by mucosal homogenates from the intestine of the common brushtail possum (Trichosurus vulpecula). 1238 85

The cDNA of a cystein peptidase inhibitor was isolated from sugarcane and expressed in Escherichia coli. The protein, named canecystatin, has previously been shown to exert antifungal activity on the filamentous fungus Trichoderma reesei. Herein, the inhibitory specificity of canecystatin was further characterized. It inhibits the cysteine peptidases from plant source papain (Ki =3.3nM) and baupain (Ki=2.1x10(-8)M), but no inhibitory effect was observed on ficin or bromelain. Canecystatin also inhibits lysosomal cysteine peptidases such as human cathepsin B (Ki=125nM), cathepsin K (Ki=0.76nM), cathepsin L (Ki=0.6nM), and cathepsin V (Ki=1.0nM), but not the aspartyl peptidase cathepsin D. The activity of serine peptidases such as trypsin, chymotrypsin, pancreatic, and neutrophil elastases, and human plasma kallikrein is not affected by the inhibitor, nor is the activity of the metallopeptidases angiotensin converting enzyme and neutral endopeptidase. This is the first report of inhibitory activity of a sugarcane cystatin on cysteine peptidases.
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PMID:Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane. 1524

Beauveria bassiana GK2016 grown in a medium with gelatin as the sole carbon and nitrogen source produced an extracellular protease. The protease production was highest when the fungus was grown on a semiliquid medium and was purified about 18-fold, with a recovery of 21%. The protease molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 35,000. It had an optimum activity at pH 8.5 and 37 degrees C and was rapidly inactivated at 50 degrees C. Its enzymatic activity was that of an endopeptidase which hydrolyzed elastin, casein, and gelatin but was much less active on bovine serum albumin and collagen. No trypsinlike activity was detected on N-alpha-benzoyl-dl-arginine-p-nitroanilide. It was, however, inhibited by phenylmethylsulfonyl fluoride, indicating that a serine residue is present in the active site. The protease was unaffected by metal-chelating agents, sulfhydryl reagents, trypsin inhibitor, and chymotrypsin inhibitor.
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PMID:Purification and Properties of an Extracellular Protease Produced by the Entomopathogenic Fungus Beauveria bassiana. 1634 95

Three isoinhibitors have been isolated to homogeneity from the C-serum of the latex of the rubber tree, Hevea brasiliensis clone RRIM 600, and named HPI-1, HPI-2a and HPI-2b. The three inhibitors share the same amino acid sequence (69 residues) but the masses of the three forms were determined to be 14,893+/-10, 7757+/-5, and 7565+/-5, respectively, indicating that post-translational modifications of the protein have occurred during latex collection. One adduct could be removed by reducing agents, and was determined to be glutathione, while the other adduct could not be removed by reducing agents and has not been identified. The N-termini of the inhibitor proteins were blocked by an acetylated Ala, but the complete amino acid sequence analysis of the deblocked inhibitors by Edman degradation of fragments from endopeptidase C digestion and mass spectrometry confirmed that the three isoinhibitors were derived from a single protein. The amino acid sequence of the protein differed at two positions from the sequence deduced from a cDNA reported in GenBank. The gene coding for the inhibitor is wound-inducible and is a member of the potato inhibitor I family of protease inhibitors. The inhibitor strongly inhibited subtilisin A, weakly inhibited trypsin, and did not inhibit chymotrypsin. The amino acid residues at the reactive site P(1) and P(1)(') were determined to be Gln45 and Asp46, respectively, residues rarely reported at the reactive site in potato inhibitor I family members. Comparison of amino acid sequences revealed that the HPI isoinhibitors shared from 33% to 55% identity (50-74% similarity) to inhibitors of the potato inhibitor I family. The properties of the isoinhibitors suggest that they may play a defensive role in the latex against pathogens and/or herbivores.
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PMID:Isolation and characterization of isoinhibitors of the potato protease inhibitor I family from the latex of the rubber trees, Hevea brasiliensis. 1643 95

Trypsin inhibitor was purified to homogeneity from seeds of the mung bean (Vigna radiata [L.] Wilczek). The protease inhibitor has the following properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel filtration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptidase in the cotyledons of mung bean seedlings.The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of beta-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts.The intracellular localization of trypsin inhibitor was determined by fractionation of crude extracts on isopycnic sucrose gradients and by cytochemistry with fluorescent antibodies. Both methods indicate that trypsin inhibitor is associated with the cytoplasm and not with the protein bodies where reserve protein hydrolysis occurs. No convincing evidence was obtained which indicates that the catabolism of trypsin inhibitor during germination and seedling growth is causally related to the onset of reserve protein breakdown.
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PMID:Trypsin inhibitor in mung bean cotyledons: purification, characteristics, subcellular localization, and metabolism. 1666 Mar 48

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination.
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PMID:Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis). 1666 4


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