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)

Using purified bacterially expressed herpes simplex virus type 1 ribonucleotide reductase large subunit (R1) and the proteolytic enzymes chymotrypsin and trypsin, we have generated stable N-terminal truncations. Chymotrypsin removes 246 amino acids from the amino terminus to produce a fragment (dN246R1) which retains full enzymic activity and affinity for the small subunit (R2). Treatment of R1 with trypsin produces a 120K protein and a cleavage at amino acid residue 305 to produce a fragment (dN305R1) which remains associated with a 33K N-terminal polypeptide. Although this 33K-dN305R1 complex retains full binding affinity for R2 its reductase activity is reduced by approximately 50%. Increasing the concentration of trypsin removes the 33K N-terminal polypeptide resulting in dN305R1 which, when bound to R2, has full ribonucleotide reductase activity. Like R1, dN246R1 and dN305R1 each exist as dimers showing that the first 305 amino acids of R1 are not necessary for dimer formation. These results indicate that, in structural studies of subunit interaction, dN246R1 or dN305R1 can be considered as suitable replacements for intact R1.
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PMID:The unique N terminus of the herpes simplex virus type 1 large subunit is not required for ribonucleotide reductase activity. 130 56

The substrate specificities of alpha-chymotrypsin and subtilisins for peptide synthesis in hydrophilic organic solvents were investigated. Chymotrypsin exhibited high specificity to aromatic amino acids as acyl donors, while subtilisin Carlsberg and subtilisin BPN' were specific to aromatic and neutral aliphatic amino acids, in accordance with the S1 specificities of the enzymes for peptide hydrolysis in aqueous solutions. On the contrary, chymotrypsin exhibited higher specificities to hydrophilic amino acid amides as acyl acceptors (nucleophiles) for peptide synthesis with N-acetyl-L-tyrosine ethyl ester, in contrast to the S1' specificity for peptide hydrolysis and peptide synthesis in aqueous solutions. Furthermore, nucleophile specificity changed with the change in water-organic solvent composition; the increase in water content led to increase in relative reactivity of leucinamide to that of alaninamide. It was also found that protection of the carboxyl group of alanine by amidation is much preferable to protection by esterification in terms of reactivity as nucleophiles.
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PMID:Peptide synthesis by proteases in organic solvents: medium effect on substrate specificity. 136 70

Chymotrypsin preparations are contaminated by autolysis products and other post-translational products derived from the zymogen. Some prevalent contaminants are difficult to detect with activity assays and molecular weight separation. However, our differential scanning calorimetry (DSC) studies of alpha-chymotrypsin have revealed that gamma-chymotrypsin is a common contaminant in commercial preparations, and the alpha- and gamma-species differ significantly in thermal stability. Thus, DSC analysis provides a sensitive and rapid means to assess the homogeneity of preparations. Moreover, free metal ion binding studies conducted indicate that the alpha- and gamma-species differ substantially in the number of metal binding sites and metal affinity. Therefore, to attempt to repurify a commercial preparation of alpha-chymotrypsin, a resolubilized sample of alpha-chymotrypsin was subjected to immobilized metal (Cu+2) affinity chromatography with pH elution and the fractions were subjected to DSC analysis. The process successfully removed the majority of the contaminating gamma-chymotrypsin.
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PMID:Metal-based affinity separation of alpha- and gamma-chymotrypsin and thermal stability analysis of isolates. 136 28

The effects of human red cell glycophorin A (GPA) on the expression of the human erythrocyte anion transporter (band 3, AE1) has been examined in Xenopus oocytes. The coexpression of GPA with band 3 increased stilbene disulfonate-sensitive chloride transport into the oocytes. The effect of GPA was particularly noticeable at low band 3 concentrations and less marked at high band 3 cRNA concentrations. The enhancement of chloride transport was specific to GPA and was not observed when either glycophorin B or glycophorin C was coexpressed with band 3. Immunoprecipitations of whole oocyte homogenates showed the amount of band 3 synthesized was not affected by GPA at subsaturating cRNA concentrations. More band 3 was detected at the oocyte surface by immunoprecipitation when GPA was also expressed. Chymotrypsin treatment of intact oocytes was also used to assess surface band 3 and greater cleavage of band 3 by chymotrypsin was observed when GPA was present. Band 3 synthesis and assembly into canine pancreatic microsomes in the reticulocyte cell-free translation system was not altered by cotranslation of GPA. We suggest that GPA facilitates the translocation of band 3 to the plasma membrane at some point during band 3 biosynthesis in Xenopus oocytes. However, GPA is not essential for the expression of band 3 in red cells, since GPA-deficient individuals have apparently normal levels of band 3. Other GPA-independent mechanisms must also allow translocation of band 3 to the surface membrane in erythroid cells and oocytes. GPA may affect the rate of accumulation of band 3 at the cell surface, rather than the final level in the plasma membrane.
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PMID:Glycophorin A facilitates the expression of human band 3-mediated anion transport in Xenopus oocytes. 138 95

delta-Chymotrypsin has been alkylated by 1-13C- and 2-13C-enriched tosylphenylalanylchloromethane. In the intact inhibitor derivative, signals due to the 1-13C- and 2-13C-enriched carbon atoms have chemical shifts which titrate from 55.10 to 59.50 p.p.m. and from 99.10 to 103.66 p.p.m. respectively with similar pKa values of 8.99 and 8.85 respectively. These signals are assigned to a tetrahedral adduct formed between the hydroxy group of serine-195 and the inhibitor. An additional signal at 58.09 p.p.m. and at 204.85 p.p.m. in the 1-13C- and 2-13C-enzyme-inhibitor derivatives respectively does not titrate when the pH is changed and it is assigned to alkylated methionine-192. On denaturation/autolysis of the 1-13C-enriched enzyme-inhibitor derivative these signals associated with the intact inhibitor derivative are no longer detected, and a new signal, which titrates from 56.28 to 54.84 p.p.m. with a pKa of 5.26, is detected. The titration shift of this signal is assigned to the deprotonation of the imidazolium cation of alkylated histidine-57 in the denatured/autolysed enzyme-inhibitor derivative. Model compounds which form stable hydrates and hemiketals in aqueous solutions have been synthesized. By comparing the 13C titration shifts of these model compounds with those of the 13C enriched trypsin- and delta-chymotrypsin-inhibitor derivatives, we deduce that, in both of the intact enzyme-inhibitor derivatives, the zwitterionic tetrahedral adduct containing the imidazolium cation of histidine-57 and the hemiketal oxyanion predominates at alkaline pH values. It is estimated that in both the trypsin and delta-chymotrypsin-inhibitor derivatives the concentration of this zwitterionic tetrahedral adduct is 10,000-fold greater than it would be in water. We conclude that the pKa of the oxyanion of the hemiketal in the presence of the imidazolium cation of histidine-57 is 7.9 and 8.9 in the trypsin and delta-chymotrypsin-inhibitor derivatives respectively and that the pKa of the imidazolium cation of histidine-57 is greater than 7.9 and greater than 8.9 when the oxyanion is present as its conjugate acid, whereas, when the oxyanion is present, the pKa of the imidazolium cation is greater than 11 in both enzyme-inhibitor derivatives. We discuss how these enzymes preferentially stabilize zwitterionic tetrahedral adducts in the intact enzyme-inhibitor derivatives and how they could stabilize similar tetrahedral intermediates during catalysis. It is suggested that substrate binding could raise the pKa of the imidazolium cation of histidine-57 before tetrahedral-intermediate formation which would explain the enhanced nucleophilicity of the hydroxy group of serine-195.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A study of the stabilization of tetrahedral adducts by trypsin and delta-chymotrypsin. 141 49

Two fluorogenic derivatives of amino acids are proposed as substrates for the purpose of enzymatic assay: N-benzyloxycarbonyl-phenylalanine-4-methyl umbelliferyl ester (substrate-1) and tert-butyloxycarbonyl-alanine-4-methyl-umbelliferyl ester (substrate-II). Chymotrypsin-like (hydrolysis of substrate-1), elastase-like (hydrolysis of substrate-II) esterase activity of bovine pancreatic chymotrypsin, activities of cathepsin G and elastase from human, porcine and rat neutrophils and esterase activity of human, porcine and rat serum were assayed. Differences in the level of chymotrypsin-like and elastase-like activities of human, porcine and rat serum were established. Activities of purified elastase and cathepsin G from human and animal neutrophils were shown to have no significant distinctions.
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PMID:[Determination of esterase activity of human and animal serine proteinases using fluorogenic esters of amino acids as substrates]. 144 26

Frequency of the labral brush movements of first, second, and fourth instars of Aedes aegypti (L.) and Aedes albopictus (Skuse) was studied comparatively in the laboratory. A frequency of 197 strokes per min for the first and second instars was observed in the former species compared to 118 strokes per min in the latter species. A faster ingestion rate of algal cells also was observed in first and second instars of Ae. aegypti (mean 57.5 cells per s) compared with first and second instars of Ae. albopictus (mean 22.4 cells per s). The digestive enzymes chymotrypsin (EC 3.4.21.1) and trypsin (EC 3.4.21.4) were more active in the peritrophic membrane (including food contents) than in the midgut epithelium of both species. Chymotrypsin activity in 11-d-old third and fourth instars of Ae. albopictus was 28 times higher than in the corresponding stadia of Ae. aegypti, indicating that the former species may have a superior enzymatic process for digesting food proteins.
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PMID:Food ingestion and digestive enzymes in larval Aedes aegypti and Ae. albopictus (Diptera: Culicidae). 146 Jun 35

Physicochemical characteristics of previously suggested surface-modified polymeric nanogranules (SMPN) and catalytic and stability properties of alpha-chymotrypsin entrapped into such nanogranules in a nonpolar solvent were investigated in more details. SMPN were obtained by polymerization of an acrylamide/N,N'-methylene-bisacrylamide mixture in a mixed reversed micellar system composed of Aerosol OT [sodium di(2-ethylhexyl)sulfosuccinate] and the polymeric surfactant Pluronic F-108 modified with polymerizable groups, followed by the chromatographic removal of the auxiliary surfactant, Aerosol OT. An optimal solvent system was found providing the required orientation of the polymeric surfactant in starting mixed micelles, i.e. with polar fragments immersed into the micellar interior and apolar fragments protruding into organic solvent. The hydrodynamic diameter of SMPN in benzene solution was estimated by means of quasi-elastic light scattering to be 84 +/- 1 nm. Catalytic and stability properties of alpha-chymotrypsin entrapped into SMPN strongly depended on conditions of preparation of SMPN. The optimal concentration of acrylamide monomers in the micellar interior and hydration degree of starting reversed micelles were found to be 20% by mass and wo = 15, respectively. alpha-Chymotrypsin-containing SMPN were used as a catalyst in the synthesis of N-acetyl-L-tyrosine ethyl ester from N-acetyl-L-tyrosine and ethanol, performed in a membrane reactor.
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PMID:Catalysis by alpha-chymotrypsin entrapped into surface-modified polymeric nanogranules in organic solvent. 148 59

1. Two chymotrypsins, called chymotrypsin I and II, were purified from the pyloric caeca of rainbow trout, by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). 2. The approximate molecular weights of chymotrypsin I and II were 28,200 (+/- 1200) and 28,800 (+/- 900), respectively, as determined by SDS-PAGE and their isoelectric points were about 5. 3. The pH optima of the enzymes were centered around nine, when assayed for succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-NA) as substrate and both enzymes were unstable at pH values below 5. 4. The amidase activity of both enzymes increased with temperature up to about 55 degrees C. Chymotrypsin I was found to be more heat stable than chymotrypsin II, an effect most likely explained by stronger calcium binding of the former. 5. The trout chymotrypsins were significantly more active than bovine alpha-chymotrypsin when assayed against Suc-AAPF-NA at 25 degrees C and casein at low temperatures (10-20 degrees C), indicating an adaptation of the activities of the trout chymotrypsins to the habitation temperatures of the fish.
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PMID:Purification and characterization of two chymotrypsin-like proteases from the pyloric caeca of rainbow trout (Oncorhynchus mykiss). 149 72

alpha-Chymotrypsin serves as a sole carbon source, sole nitrogen source, and as sole carbon plus nitrogen source for wild-type Escherichia coli in a totally defined medium. Hence, a mammalian host for E. coli may supply the necessary carbon and nitrogen nutrients for the microorganism. Growth is most rapid when chymotrypsin is a sole nitrogen source and least rapid with chymotrypsin as a carbon source. The approximate doubling times for E. coli utilizing chymotrypsin as a nitrogen source, carbon plus nitrogen source, and carbon source are 1.6, 4.6, and 11.3 h, respectively. The activity of the residual enzyme in the culture supernates falls off asymptotically over the source of time, as followed by cleavage of glutaryl-L-phenylalanine-p-nitroanilide. Chymotrypsin hydrolyzes succinyl-L-ala-L-ala-p-nitroanilide, the elastase substrate, to some extent. Peptidases do not appear to be secreted that hydrolyze such model substrates as benzoyl-DL-arginine-p-nitroanilide, the tryptic and cathepsin B substrate, L-leucine-p-nitroanilide, the leucine amino-peptidase substrate, or L-lysine-p-nitroanilide, the aminopeptidase B substrate. Growth of E. coli is generally directly related to the loss of chymotryptic activity in the medium. Hence, autolysis of chymotrypsin, i.e., self-degradation, is an important factor for the availability of degradation products of the enzyme to the bacterium for growth purposes. Accordingly, the degradation of a host protein by autolysis presents an opportunity for E. coli to survive during periods of host nutritional crisis by utilization of the degradation peptides that are produced during autolysis.
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PMID:Utilization of chymotrypsin as a sole carbon and (or) nitrogen source by Escherichia coli. 161 54


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