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)

The rates of cis to trans interconversion of Glt-(Ala)n-Pro-Phe-4-nitroanilides (n = 1-3) were estimated by means of a two-step process with chymotrypsin as the trans-substrate cleaving activity. By the aid of this system, pig kidney and several other tissues contained demonstrable catalytic activity against the cis to trans interconversion of the proline containing peptides. The active protein fraction was purified 38-fold from pig kidney cortex by ammonium sulfate precipitation and a series of column chromatographic techniques. Activity was detected against the cis to trans interconversion of Glt-Ala-Ala-Pro-Phe-4-nitroanilide to a different extent. No activity was found with Phe-Pro-4-nitroanilide. With respect to the substrate specificity, this enzyme must be classified as a peptidyl-prolyl cis-trans-isomerase. The enzyme was strongly inactivated by p-chloromercuribenzoate, sodium dodecylsulfate, Hg2+- and Cu2+-ions, but was not inhibited by metal chelators, diisopropylphosphorofluoridate and chlorotosylamidophenylbutane. The activity is abolished by incubation with trypsin. The enzyme is heat sensitive at 50 degrees C. The results presented in this paper suggest a new type of enzymes, characterized by catalytic activity against conformational interconversions. The possibility of the location of the enzyme on ribosomal particles is discussed.
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PMID:[Determination of enzymatic catalysis for the cis-trans-isomerization of peptide binding in proline-containing peptides]. 639 66

Band 3, the anion transport protein of human erythrocyte membranes, can be transferred from cells to liposomes and from liposomes back to cell membranes, retaining function and native orientation. After incubation with cells, sonicated phosphatidylcholine vesicles bind a transmembrane protein that comigrates with band 3 on sodium dodecyl sulfate-polyacrylamide gels. Like native red cell band 3, the vesicle-bound protein is cleaved by chymotrypsin into 65- and 30-kdalton fragments and is not cleaved by trypsin. The protein can be cross-linked by copper-phenanthroline oxidation either before or after transfer to vesicles; in either case, the vesicle fractions contain high molecular weight material that is dissociated into 95-kdalton species by mercaptoethanol. Band 3-vesicle complexes contain no detectable cell lipid and are specifically permeable to anions. Greater than 99% of their anion uptake can be blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). Red cells whose band 3 function has been blocked irreversibly by DIDS or eosin maleimide regain part of their anion permeability upon incubation with band 3-vesicle complexes. Under the conditions employed, an average of one copy of functional band 3 is delivered to half of the cells, increasing by 2.3-fold the number of cells containing functional anion transporters. Incubation of pure lipid vesicles or red cell membrane buds with either normal red cells or eosin maleimide inhibited cells has no detectable effect on the cells' anion permeability.
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PMID:Transfer of band 3, the erythrocyte anion transporter, between phospholipid vesicles and cells. 666 30

A histidine-rich fragment, Cp F5, with a molecular weight of 18,650 was isolated from human ceruloplasmin. It consists of 159 amino acids and contains a possible copper-binding site. The sequence of the first 18 NH2-terminal residues of Cp F5 was determined by automated Edman degradation. Cp F5 was cleaved by cyanogen bromide to produce nine fragments of from 2 to 63 residues. The amino acid sequence of all of the cyanogen bromide fragments was investigated using automated and manual Edman degradation, the fragments being digested with trypsin, chymotrypsin, thermolysin, staphylococcal protease, and pepsin as appropriate. The results, in conjunction with the data on the tryptic peptides reported in the accompanying paper (Kingston, I.B., Kingston, B.L., and Putnam, F.L. (1980) J. Biol. Chem. 255, 2886-2896), establish the complete amino acid sequence of Cp F5.
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PMID:Primary structure of a histidine-rich proteolytic fragment of human ceruloplasmin. I. Amino acid sequence of the cyanogen bromide peptides. 698 29

The effect of AG+, Cu2+, Cd2+, Co2+ and Ni2+ on the activity of alkaline mesentericopeptidase (EC 3.4.21.-) has been studied. Ag+, Cu2+ and Cd2+ were found to be reversible non-competitive inhibitors of the enzyme. The pH-dependence of Ki for Ag+-inhibition is sigmoidal with a pKa near 6. The Kilim values, calculated for the pH-independent region of the metal-enzyme inhibition, are close to the corresponding dissociation constants of metal-imidazole complexes, thus implying that the inhibitory effect of metal ions on enzyme activity is due to complex formation with the imidazole group of the active site histidine. The method of the two-component inhibition showed that Cu2+ and Ag+ bind to the same ligand of the enzyme molecule. The addition of Cu2+ decreases the rate of deacylation of the hydrolysis of p-nitrophenyl valerate, catalyzed by alkaline mesentericopeptidase in contrast to alpha-chymotrypsin where the acylation step is affected.
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PMID:Inhibitory effect of metal ions on alkaline mesentericopeptidase. 701 96

In the green alga Chlamydomonas reinhardtii, the copper-dependent accumulation of plastocyanin is effected via the altered stability of the protein in copper-deficient versus copper-sufficient medium (t1/2) < 20 min versus several hours). To understand the mechanism of plastocyanin degradation in vivo, the purified apoprotein was characterized relative to the holoprotein with respect to conformation and protease susceptibility. Circular dichroism spectroscopy revealed that the apoprotein in solution did not display the characteristic secondary structure displayed by the native or reconstituted holoprotein. The apoprotein was also susceptible to digestion in vitro by chymotrypsin whereas the holoprotein was resistant. High ionic conditions, which stabilize the folded structure of apoplastocyanin, also inhibit its degradation by chymotrypsin. These results suggest that one explanation for plastocyanin degradation in copper-deficient cells in vivo might be the increased susceptibility of the apo form to a lumenal protease. Since apoplastocyanin is a normal biosynthetic intermediate for the formation of holoplastocyanin, the increased susceptibility of apoplastocyanin to proteolysis implies that degradative and biosynthetic activities would compete for the same substrate. However, characterization of an apoplastocyanin-accumulating mutant suggests that a plastocyanin-degrading protease is active only in copper-deficient cells. Thus, apoplastocyanin is rapidly degraded in copper-deficient cells, whereas its major fate in copper-supplemented cells is holoplastocyanin formation.
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PMID:Degradation of plastocyanin in copper-deficient Chlamydomonas reinhardtii. Evidence for a protease-susceptible conformation of the apoprotein and regulated proteolysis. 755 14

The coding region of copper/zinc-superoxide dismutase (Cu/Zn-SOD) cDNA from sweet potato, Ipomoea batatas (L.) Lam. cv. Tainong 57, was introduced into an expression vector, pET-20b(+). The Cu/Zn-SOD purified by His-tagged technique showed two active forms (dimer and monomer). The amount of proteins of dimer and monomer appeared to be equal, but the activity of dimeric form was seven times higher than that of monomeric form. The enzyme was dissociated into monomer by imidazole buffer above 1.0 M, acidic pH (below 3.0), or SDS (above 1%). The enzyme is quite stable. The enzyme activity is not affected at 85 degrees C for 20 min, in alkali pH 11.2, or in 0.1 M EDTA and also quite resistant to proteolytic attack. Dimer is more stable than monomer. The thermal inactivation rate constant kd calculated for the monomer at 85 degrees C was 0.029 min-1 and the half-life for inactivation was about 28 min. In contrast, there is no significant change of dimer activity after 40 min at 85 degrees C. The enzyme dimer and monomer retained 83% and 58% of original activity, respectively, after 3 h incubation with trypsin at 37 degrees C, while those retained 100% and 31% of original activity with chymotrypsin under the same condition. These results suggest subunit interaction might change the enzyme conformation and greatly improve the catalytic activity and stability of the enzyme. It is also possible that the intersubunit contacts stabilize a particular optimal conformation of the protein or the dimeric structure enhances catalytic activity by increasing the electrostatic steering of substrate into the active site.
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PMID:Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method. 759 15

Studies have shown that the dust mites Dermatophagoides pteronyssinus and D. farinae contain several serine proteases, two of which have been shown to be allergenic, and to include trypsin and chymotrypsin, corresponding to the groups III and VI mite allergens. However, mites also contain other serine proteases, and the data reported in this study show that an elastase-like enzyme is present in both species. This enzyme was differentiated from the other serine proteases, particularly chymotrypsin, on the basis of charge, substrate specificity, and inhibition by copper and mercury cations. Its apparent mol. mass, as judged by gel filtration, was similar to those previously described for trypsin and chymotrypsin, i.e., 30 kDa. Several isoforms were detected by isoelectric focusing, but the isoelectric points of the major forms in both D. pteronyssinus and D. farinae were 10.5 and 9.8, respectively, contrasting with the acidic mite chymotrypsins. All three serine proteases were detected in whole mite and faecally enriched extracts, but the activities of trypsin and the elastase-like enzyme were greater in the latter type of extract. These data were similar to those obtained by quantitative immunochemical analysis of the D. farinae group III allergen in appropriate extracts, suggesting that culture conditions may modulate protease production. A monoclonal antibody affinity matrix specific for the group III allergen from D. farinae was shown to bind mite trypsin. However, a small amount of mite chymotrypsin also bound, suggesting limited immunologic cross-reactivity, a finding consistent with known sequence data.
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PMID:A comparative study of three serine proteases from Dermatophagoides pteronyssinus and D. farinae. 782 23

An affinity capillary electrophoresis method has been developed that employs small ligands covalently bound to a replaceable soluble polymer matrix. The metal chelate iminodiacetate-Cu(II) coupled to polyethylene glycol was used as a model system and the interactions of different model proteins, namely ribonucleases, cytochromes c, chymotrypsin, and kallikrein, were investigated. The method allowed for easy determination of dissociation constants using a modified Langmuir adsorption isotherm equation which is applicable to interactions with fast on/off kinetics. It is shown that the general rules for protein interaction with matrix-bound metal chelates established for immobilized metal ion affinity chromatography (IMAC) are maintained in immobilized metal ion affinity capillary electrophoresis (IMACE). In contrast to gel electrophoresis, IMACE allowed for using similar conditions as in IMAC, especially regarding the high salt concentrations, usually employed with the latter technique. The usefulness of the method for quantification of low-affinity interactions and studying protein surface structure and structure/function relationship using the metal affinity is demonstrated.
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PMID:Immobilized metal ion affinity capillary electrophoresis of proteins--a model for affinity capillary electrophoresis using soluble polymer-supported ligands. 871 92

Crystalline alpha-chymotrypsin preparations are contaminated by the post translational variant, gamma-chymotrypsin. The contaminant can account for 5-50 weight percent of the preparation based on thermal analysis. Such contamination can be problematic because this serine protease has both commercial and deactivation model system utility, and the presence of the contaminant may not be detectable by activity assays. Prior work has shown that simple pH gradient elution can separate the two chymotrypsins when loaded to a Cu(2+)-IMAC column; gamma-chymotrypsin eluted first indicating that its interaction with immobilized Cu2+ is weaker. The molecular features that endow these serine proteases with metal affinity has been investigated further by performing differential scanning calorimetry (DSC) studies in the presence and absence of Cu2+, and at different pH values. The dependence of thermostability on pH for fixed metal concentration reveals an interplay between stabilizing and destabilizing metal binding events. The results are consistent with Cu(2+)-chymotrypsin interaction occurring, in part, through binding to a glutamate- or aspartate-containing chelation site. The strength of this site may differ in the two chymotrypsins.
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PMID:Using differential scanning calorimetry to elucidate metal-protein binding sites in alpha- and gamma-chymotrypsin. 872 Aug 50

Two 15-d nutrient balance trials were conducted using a total of 32 weanling barrows (averaging 6.8 kg, 26 d). The effect of the addition of 15 or 250 ppm Cu (as CuSO4.5H2O) to diets containing 0 or 5% added animal fat on nutrient utilization, digestive enzyme activities, and tissue mineral levels in weanling pigs was investigated. In each trial, four groups of four littermate barrows were randomly assigned to one of four treatments in a 2 x 2 factorial arrangement. The addition of 250 ppm Cu improved apparent fat digestibility and apparent nitrogen retention (P < .02). The addition of 5% fat increased apparent fat digestibility (P < .01). There were no Cu x fat interactions (P > .10) for any of the digestibility indices measured. The addition of 250 ppm of Cu stimulated small intestinal lipase (P < .01) and phospholipase A (P < .05) activities but had no effect (P > .10) on pancreatic lipase or phospholipase activities and no effect on trypsin, chymotrypsin, or amylase activities in the small intestine or the pancreas. The addition of 250 ppm Cu to the diet increased Cu (P < .001) in plasma, liver, and kidney and decreased Fe in plasma (P < .05) and liver (P < .02). The addition of 5% fat increased Fe in kidney (P < .05) and heart (P < .08). Copper x fat interactions were observed for spleen Ca (P < .01), Mg (P < .08), Na (P < .05), and K (P < .08) and spleen weight (P < .05). In additional in vitro assays, increased Cu concentrations tended to consistently stimulate purified porcine pancreatic lipase activity (linear, P < .01) but not purified porcine pancreatic phospholipase A activity (P > .10). The results from this study indicate that 250 ppm Cu stimulated intestinal lipase and phospholipase A activities, leading to an improvement of dietary fat digestibility in weanling pigs.
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PMID:Effect of dietary copper and fat on nutrient utilization, digestive enzyme activities, and tissue mineral levels in weanling pigs. 885 43


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