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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A full-length cDNA of 794 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Pagrus major was cloned by the PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (53-91%) with the sequences of Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-47, 49, 64, and 121) and zinc (His-64, 72, 81, and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, were well conserved among all reported Cu/Zn-SOD sequences. To further characterize the Pagrus major Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native-gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. Dimer was the major form of the enzyme in equilibrium. The dimerization of the enzyme was inhibited under acidic pH (below 4.0 or higher than 10.0). The half-life was 8.6 min and the inactivation rate constant (k(d)) was 9.69 x 10(-2) min(-1) at 70 degrees C. The enzyme activity was not significantly affected under 4% SDS or 0.5 M imidazole. The enzyme was resistant to proteolysis by both trypsin and chymotrypsin.
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PMID:Characterization of copper/zinc-superoxide dismutase from Pagrus major cDNA and enzyme stability. 1182 45

RNA-binding proteins in round spermatids have previously been assigned to the coding sequence of Prm1- and Prm2-mRNA. To further characterize this protein-RNA interaction, prior to cDNA synthesis, microdissected cell profiles were digested with different proteases exhibiting a specific cleavage site followed by both conventional and real-time quantitative PCR. Best results were obtained with proteinase K and A followed by factor Xa protease, genenase I, and proteases V8. While enterokinase revealed PCR signals solely for Prm2, no amplification signal was obtained using chymotrypsin. These data suggest a protein segment rich in basic amino acids to be important for the binding to Prm1- and Prm2-mRNA. The fact that phenanthroline treatment instead of protease digestion also resulted in amplification signals suggests the involvement of zinc-finger-like protein-RNA interactions. Employing different primer pairs, RNA-binding proteins were shown to be localized at the 5' end of Prm1- and Prm2-mRNA. Since protein-RNA interactions are a common principle of posttranscriptional regulation of gene expression, the combination of microdissection, protease digestion, and real-time quantitative PCR provides a suitable tool for its investigation in a cell type-specific manner. Furthermore, the presence of RNA-binding proteins within the coding sequence of mRNAs demands proteinase K treatment prior to cDNA synthesis, a compelling necessity for the study of gene expression.
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PMID:Protamine-1 and -2 mRNA in round spermatids is associated with RNA-binding proteins. 1191 20

Four accessions of the lesser-known legume, Cassia obtusifolia L. (Sickle pod), collected from four different agroclimatic regions of Western Ghats, were evaluated for agrobotanical traits and chemical composition. Among the four accessions, the Keriparai accession had the highest values for plant height (cm), number of flowers per cluster, number of pods per cluster, pod length (cm), seeds per pod, seed weight (g) per pod and seed recovery percentage. Crude protein ranged from 18.56-22.93%, crude lipid was between 5.35-7.40%, crude fiber ranged from 6.83-9.45%, ash content ranged from 5.14-5.83% and carbohydrate varied from 57.00-60.69%. Globulins constituted the bulk of the seed protein as in most legumes. Mineral profiles, viz., sodium, potassium, calcium, magnesium, phosphorus, iron, copper, zinc and manganese ranged from 42.92-84.83, 758.05-1555.79, 559.92-791.72, 456.36-709.47, 629.13-947.79, 8.42-12.35, 0.93-2.06, 10.60-30.04 and 2.12-4.12 mg/100 g seeds flour, respectively. Seed proteins of all accessions exhibited relatively high levels of non-essential and essential amino acids, with the exception of threonine. The in vitro protein digestibility of the legume ranged from 74.66 to 81.44%. Antinutritional substances such as total free phenolics ranged from 0.34-0.66%; tannins were between 0.47-0.60%; L-DOPA content ranged from 0.98-1.34%; trypsin inhibitor activity varied from 11.4-13.5 TIU/mg protein and chymotrypsin inhibitor activity ranged from 10.8-12.3 CIU/mg protein. Phytohemagglutinating activity also was assayed. In conclusion, the accessions of C. obtusifolia, collected from Western Ghats, South India, could serve as a low-cost source of some important nutrients for humans. The antinutritional factors might have little nutritional significance, if the seeds are processed properly.
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PMID:Agrobotanical traits and chemical composition of Cassia obtusifolia L.: a lesser-known legume of the Western Ghats region of South India. 1204 47

A full-length cDNA clone of 744 bp encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from lemon (Citrus limon) was cloned by PCR approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprised an open reading frame coding for 152 amino acid residues. The deduced amino acid sequences showed high identity (65-84%) with the sequences of the Cu/Zn-SODs from other plant species. Computer analysis of the residues required for coordinating copper (His-45, -47, -62, and -119) and zinc (His-62, -70, and -79 and Asp-82), as well as the two cysteines (56 and 145) that form a single disulfide bond, showed they were well-conserved among all reported Cu/Zn-SOD sequences in the present study. To further characterize the lemon Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+), and transformed into Escherichia coliBL21(DE3). Expression of the Cu/Zn-SOD was confirmed by enzyme activity staining on a native gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose superflow. The purified enzyme showed two active forms (70% monomer and 30% dimer) in equilibrium, and the specific activity was 7 456 units/mg. The activity of the dimer was 65% higher than that of the monomer. The thermal inactivation rate constant K(d) value calculated for the dimer at 90 degrees C was -7.0 x 10(-3) min(-1), and the half-life for inactivation was 99 min. Both activity and forms of the enzyme were affected very little by acidic pH, basic pH, or 4% SDS. The dimeric structure was more resistant to heat and proteolytic attack with trypsin or chymotrypsin compared to the monomeric structure. Imidazole caused the dimer to dissociate into monomers. These studies suggested subunit interaction might be important for enzyme stability.
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PMID:Copper/zinc-superoxide dismutase from lemon cDNA and enzyme stability. 1245 42

Human chymase is a chymotryptic serine peptidase stored and secreted by mast cells. Compared with other chymotryptic enzymes, such as cathepsin G and chymotrypsin, it is much more slowly inhibited by serum serpins. Although chymase hydrolyzes several peptides and proteins in vitro, its target repertoire is limited compared with chymotrypsin because of selective interactions in an extended substrate-binding site. The best-known natural substrate, angiotensin I, is cleaved to generate vasoactive angiotensin II. Selectivity of angiotensin cleavage depends in major part on interactions involving substrate residues on the carboxyl-terminal (P1'-P2') side of the cleaved bond. To identify new targets based on interactions with residues on the aminoterminal (P4-P1) side of the site of hydrolysis, we profiled substrate preferences of recombinant human chymase using a combinatorial, fluorogenic peptide substrate library. Data base queries using the peptide (Arg-Glu-Thr-Tyr-X) generated from the most preferred amino acid at each subsite identify albumin as the sole, soluble, human extracellular protein containing this sequence. We validate the prediction that this site is chymase-susceptible by showing that chymase hydrolyzes albumin uniquely at the predicted location, with the resulting fragments remaining disulfide-linked. The site of hydrolysis is highly conserved in vertebrate albumins and is near predicted sites of metal cation binding, but nicking by chymase does not alter binding of Cu2+ or Zn2+. A synthetic peptidic inhibitor, diphenyl N alpha-benzoxycarbonyl-l-Arg-Glu-Thr-PheP-phosphonate, was designed from the preferred P4-P1 substrate sequence. This inhibitor is highly potent (IC50 3.8 nM) and 2,700- and 1,300-fold selective for chymase over cathepsin G and chymotrypsin, respectively. In summary, these findings reveal albumin to be a substrate for chymase and identify a potentially useful new chymase inhibitor.
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PMID:Albumin is a substrate of human chymase. Prediction by combinatorial peptide screening and development of a selective inhibitor based on the albumin cleavage site. 1281 38

The tannins are natural polyphenols, able to precipitate water-soluble alkaloids and possess an inhibitory action on the angiotensin converting enzyme (ACE). We identified 18 polyphenolic compounds (tannins) from Chinese herbs and examined the in vitro effects of these tannins on ACE activity, including determination of the 50% inhibitory concentrations (IC50), specificity and mode of inhibition. We also assessed the in vivo inhibitory effect of the tannins on angiotensin I-induced blood pressure elevation in spontaneously hypertensive rats (SHR). Nine tannins with an IC50 <200 microM for ACE inhibitors were identified belonging to three tannin classes: caffeoylquinates, flavan-3-ols and gallotannins. In vitro, we found caffeoylquinates chelate the ACE zinc cofactor. Two of the flavan-3-ols: epigallocatechin-3-O-gallate and epigallocatechin-3-O-methylgallate, and one of gallotannin: 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucose were non-specific inhibitors because also reduced the activity of trypsin and chymotrypsin. The ACE inhibition of 1, 2, 3, 4, 6-penta-O-galloyl-beta-D-glucose was also reduced after addition of bovine serum albumin, suggesting a non-specific mode of action. In vivo, 1,2,3,6-tetra-O-galloyl-beta-D-glucose and epigallocatechin-3-O-methylgallate had a strong dose-dependent hypotensive effect reducing the blood pressure significantly in the SHR with infusion of the angiotensin I. These findings indicate that some of the tannins isolated from herbs inhibit ACE activity non-specifically. The ACE inhibitory effect of these tannins may explain the hypotensive effects of some traditional Chinese herbs.
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PMID:Antihypertensive effects of tannins isolated from traditional Chinese herbs as non-specific inhibitors of angiontensin converting enzyme. 1286 94

We demonstrated that amyloid-forming peptides could be selected from phage-displayed library via proteolysis-based selection protocol. The library of 28-residue peptides based on a sequence of the second zinc finger domain of Zif268, and computationally designed betabetaalpha peptide, FSD-1, was presented monovalently on the surface of M13 phage. The library coupled the infectivity of phage particles to proteolytic stability of a peptide introduced into the coat protein III linker. It was designed to include variants with a strong potential to fold into betabetaalpha motif of zinc finger domains, as expected from secondary structure propensities, but with no structure stabilization via zinc ion coordination. As our primary goal was to find novel monomeric betabetaalpha peptides, the library was selected for stable domains with the assumption that folded proteins are resistant to proteolysis. After less than four rounds of proteolytic selection with trypsin, chymotrypsin, or proteinase K, we obtained a number of proteolysis-resistant phage clones containing several potential sites for proteolytic attack with the proteinases. Eight peptides showing the highest proteolysis resistance were expressed and purified in a phage-free form. When characterized, the peptides possessed proteolytic resistance largely exceeding that of the second zinc finger domain of Zif268 and FSD-1. Six of the characterized peptides formed fibrils when solubilized at high concentrations. Three of them assembled into amyloids as determined through CD measurements, Congo red and thioflavin T binding, and transmission electron microscopy.
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PMID:Amyloid-forming peptides selected proteolytically from phage display library. 1287 17

A full-length cDNA of 803 base pairs encoding a putative copper/zinc-superoxide dismutase (Cu/Zn-SOD) from Epinephelus malabaricus was cloned by the polymerase chain reaction approach. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 154 amino acid residues. The deduced amino acid sequence showed high similarity (65-91%) with the sequences of the Cu/Zn-SOD from other species. Computer analysis of the residues required for coordinating copper (His-49, -64, and -121) and zinc (His-64, -72, and -81 and Asp-84), as well as the two cysteines (58 and 147) that form a single disulfide bond, was well-conserved among all reported Cu/Zn-SOD sequences. To further characterize the E. malabaricus Cu/Zn-SOD, the coding region was subcloned into an expression vector, pET-20b(+) and transformed into Escherichia coli BL21(DE3)pLysS. The expression of the Cu/Zn-SOD was confirmed by enzyme activity stained on a native gel and purified by Ni(2+)-nitrilotriacetic acid Sepharose. The enzyme activity was inhibited under basic pH (higher than 10.0). The enzyme retained 65% activity after heating at 60 degrees C for 10 min. The inactivation rate constant (k(d)) was 6.64 x 10(-2) min(-1) at 60 degrees C. The enzyme activity was only some decrease under 3% sodium dodecyl sulfate. The enzyme was resistant to proteolysis by trypsin and chymotrypsin. The finding of Cu/Zn-SOD cDNA could be used as a probe to detect the transcription level of this enzyme, which can be used as an early biomarker of environmental pollution. The property of this enzyme could provide a reference as compared to the oxidized forms or new isoforms, which could be induced under the experiments of pollution.
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PMID:Copper/zinc-superoxide dismutase from Epinephelus malabaricus cDNA and enzyme property. 1295 20

Peptidase-catalyzed formation of macrocyclic lactams on solid phase identifies ring systems that are favorably bound in the enzyme active site. We evaluated several cyclic peptide motifs linked by ester bonds between the P2 and P1' or the P1 and P2' side chains. The depsipeptide represented by structure 5 was readily generated by a variety of peptidases from precursor omega-amino acids or omega-amino esters. This strategy for identifying ring systems for potential macrocyclic transition state analogues was demonstrated with the serine peptidases trypsin and chymotrypsin, with the aspartic peptidase pepsin, and with the zinc peptidase thermolysin.
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PMID:Identification of novel macrocyclic peptidase substrates via on-bead enzymatic cyclization. 1457 71

An inhibitor of the metallo-ectoenzyme, pyroglutamyl aminopeptidase II (PPII), a thyrotropin releasing hormone-specific peptidase, was identified by screening extracts from marine species of the Cuban coast-line belonging to the phylla Chordata, Echinodermata, Annelida, Mollusca, Cnidaria, Porifera, Chlorophyta and Magnoliophyta. Isolation of the inhibitor (HcPI), from the marine annelide Hermodice carunculata, was achieved by trichloroacetic acid treatment of the aqueous extract, followed by ion-exchange chromatography on DEAE Sephacel, gel filtration on Sephadex G-25 and reverse phase-HPLC. HcPI had a small apparent molecular weight (below 1000 Da) and was not a peptide. It inhibited rat PPII (a membrane preparation with 8.5mg protein/ml) with an apparent K(i) of 51 nM. HcPI did not inhibit serine (trypsin, chymotrypsin, elastase and dipeptidyl aminopeptidase IV), cysteine (papain, bromelain and pyroglutamyl aminopeptidase I), aspartic (pepsin and recombinant human immunodeficiency virus 1 protease (HIV1-PR)) nor other metallo proteinases (collagenase, gelatinase, angiotensin converting enzyme, aminopeptidase N and carboxypeptidase A). HcPI was non-toxic and active in vivo. Intraperitoneal injection of HcPI reduced mouse pituitary and brain PPII activity. Potency of the effect was higher in hypophysis and hypothalamus than in other brain regions. Intrathecal administration to male rats reduced PPII activity in the spinal cord. In conclusion we have identified a specific inhibitor of PPII that is the first M1 family zinc metallo-peptidase inhibitor isolated from marine invertebrates. It may be useful for elucidating the in vivo role of PPII in the pituitary and central nervous system.
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PMID:Purification of a specific inhibitor of pyroglutamyl aminopeptidase II from the marine annelide Hermodice carunculata. in vivo effects in rodent brain. 1459 39


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