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)

We have altered the amino acid at the center of the reactive site (methionine 73) of Streptomyces subtilisin inhibitor (SSI) by site-directed and cassette mutagenesis. Replacement by lysine or arginine resulted in trypsin inhibitory activity, replacement only by lysine gave inhibition of lysyl endopeptidase, and replacement by tyrosine or tryptophan resulted in inhibition of alpha-chymotrypsin. The four mutant SSIs retained their native activity against subtilisin BPN'. Thus by altering only one amino acid residue at the reactive site of SSI to the substrate specificity of the respective protease we could successfully change its inhibitory profile.
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PMID:Alteration of the specificity of the Streptomyces subtilisin inhibitor by gene engineering. 136 38

The channel-forming protein aerolysin is secreted as a protoxin which can be activated by proteolytic removal of a C-terminal peptide. The activation and subsequent oligomerization of aerolysin were studied using a variety of spectroscopic techniques. Mass spectrometric determination of the molecular weights of proaerolysin and aerolysin permitted identification of the sites at which the protoxin is processed by trypsin and chymotrypsin. The results of far- and near-UV circular dichroism measurements indicated that processing with trypsin does not lead to major changes in secondary or tertiary structure of the protein. An increase in tryptophan fluorescence intensity and a small red shift in the maximum emission wavelength of tryptophans could be observed, suggesting that there is a change in the environment of some of the tryptophans. There was also a dramatic increase in the binding of the hydrophobic fluorescent probe 1-anilino-8-naphthalenesulfonate during activation, leading us to conclude that a hydrophobic region in the protein is exposed by trypsin treatment. Using measurements of light scattering, various parameters influencing oligomerisation of trypsin-activated aerolysin were determined. Oligomerization rates were found to increase with the concentration of aerolysin, whereas they decreased with increasing ionic strength.
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PMID:Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation. 138 79

We have developed and validated a new radioimmunoassay for cholecystokinin. In order to establish that the antiserum binds large and small forms of CCK to an equal extent, we used the microbial enzyme clostripain, which cleaves large forms of CCK yielding CCK 8. Cleavage by clostripain of synthetic and purified forms of CCK, and CCK extracted at from human jejunum and CCK in human plasma was found not to affect immunoactivity, indicating that the antiserum reacts similarly with all forms of CCK. There is controversy over whether intraduodenal trypsin inhibits release of CCK in man. We used our radioimmunoassay to investigate whether chymotrypsin, rather than trypsin, could be the major mediator of negative feedback control of CCK release. Six normal subjects received an intraduodenal infusion of L-phenylalanine and L-tryptophan on two occasions, with the addition of either 1 g/l bovine chymotrypsin or 1 g/l albumin. Plasma CCK concentrations rose in response to the amino acid infusion, but were not affected by the addition of chymotrypsin, indicating that this enzyme is not a mediator of CCK feedback regulation in man.
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PMID:Effect of chymotrypsin on human cholecystokinin release: use of clostripain in the validation of a new radioimmunoassay. 143 74

At the aim of investigating whether the early rapid phase of enzyme turnover is different in reverse micelles compared with bulk water, the kinetic properties of alpha-chymotrypsin have been studied in reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. Pre-steady state and steady-state kinetic constants, in water and in reverse micelles, have been determined by stopped-flow spectrophotometry for the hydrolysis of two substrates, namely acetyl-L-tryptophan-p-nitrophenyl ester and p-nitrophenyl acetate. It has been shown that, for both substrates, the acylation rate constant (k2) is very much lower in reverse micelles than in water. However, the deacylation rate constant (k3) and the turnover number (kcat) are not significantly changed in reverse micelles with respect to bulk water. Therefore, despite considerable rate changes in the acylation step, deacylation is rate limiting both in water as well as in reverse micelles, under the experimental conditions used.
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PMID:Kinetic behaviour of alpha-chymotrypsin in reverse micelles. A stopped-flow study. 151 84

The synthesis reaction of the peptide, N-Cbz-L-tryptophanyl-glycineamide, catalyzed by alpha-chymotrypsin was performed in a 20% water/80%, 1,4-butanediol mixture. The synthesis yield reached 90.9% at the end of the reaction and 72.3% after purification. The effects on the yield of both pH and the ratio between total initial concentrations of glycineamide and N-Cbz-L-tryptophan are examined. The high yield, specificity, simplicity and reproducibility of this method make it complementary of the chemical methods.
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PMID:Gram-scale enzymatic synthesis of a peptide bond. 152 Nov 72

Fast skeletal myosins were isolated from carp acclimated to 10 and 30 degrees C, and their structural and enzymatic properties were compared. Myosins in 0.5 M KCl were subjected to limited proteolysis by using various proteases including alpha-chymotrypsin, trypsin, and papain, and different SDS-PAGE patterns were seen for the 10- and 30 degrees C-acclimated myosins in all cases. Myosin subfragment-1 (S1) prepared from the 10 degrees C-acclimated myosin by alpha-chymotryptic digestion in 0.12 M NaCl showed higher acto-S1 Mg(2+)-ATPase activity and lower thermostability than S1 from the warm-acclimated myosin. The peptide maps and ATP-induced spectral changes of tryptophan fluorescence also showed an obvious difference between the two types of S1. Temperature acclimation further caused changes in the rod region of myosin, since the apparent sizes of light meromyosin were different from each other for the two types of myosin. Myosin from carp acclimated to 20 degrees C showed intermediate properties between those of the 10- and 30 degrees C-acclimated myosins. Myosin isoforms might be expressed in a temperature-dependent manner to compensate for the effect of seasonal environmental temperature variation on swimming ability.
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PMID:Fast skeletal myosin isoforms in thermally acclimated carp. 153 74

The classical hydrolysis of proteins with hydrochloric acid using tryptamine [3-(2-aminoethyl)indole] as additive revealed that tryptophan can be measured without destruction together with other amino acids by gas chromatography. An extensive study was made to establish the optimum conditions for protein hydrolysis (time and temperature of hydrolysis, amount of tryptamine) and for the derivatization of amino acids. The amino acid contents (including tryptophan) of standard proteins such as lysozyme, bovine and human albumin, human gamma-globulin, casein and alpha-chymotrypsin and protein matrices (meat and fish meals, sunflower) were determined, after hydrochloric acid hydrolysis (4 h, 145 degree C) in the presence of tryptamine. as N, O, (S)-trifluoroacetyl isobutyl esters with SE-30 as the stationary phase. The reproducibility of the measurements was 4.6% (relative standard deviation) or less.
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PMID:Gas chromatography of tryptophan together with other amino acids in hydrochloric acid hydrolysates. 170 86

The potent vasoconstrictor peptide endothelin-1 is proposed to arise via proteolysis of a precursor molecule, "big endothelin," at a unique cleavage site. To aid in the identification of a putative endothelin-converting enzyme, we have developed an assay that mimics the relevant cleavage reaction. The assay takes advantage of the intramolecular fluorescence energy transfer between the scissile-site tryptophan and a dansyl moiety present in the same synthetic substrate. Cleavage of the peptide chain separates the fluorophore and quencher, resulting in an increase in fluorescence. The assay has been validated using chymotrypsin as a model protease and has been employed in the identification of novel endothelin-converting enzyme activities.
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PMID:A fluorogenic assay for endothelin-converting enzyme. 180 35

Pretreatment of the purified jack bean inhibitor with enterokinase activated human pancreatic preparation for 1 hr decreased its inhibitory capacity against crystalline bovine alpha-chymotrypsin by 30% but did not affect its trypsin inhibitory activity. Preincubation of the inhibitor with bovine chymotrypsin for 60 min resulted in partial loss of the inhibitory potency. Complex formation studies by gel chromatography on Sephadex G-100 indicated that the trypsin-inhibitor and chymotrypsin-inhibitor complexes dissociated to release inactivated inhibitor and active proteinases. Gel chromatography of the inhibitor in presence of 1.5 M ammonium sulphate indicated that the inhibitor showed a tendency to aggregate without loss of biological activity. However, in 4.2 M salt medium after 3 hr, antichymotryptic activity was lost completely without any effect on antitryptic activity. Treatment with methylamine, a nucleophile, caused a greater loss of antichymotryptic activity. Trinitrobenzene sulphonate and ethylacetamidate, the amino group modifiers, affected only the antichymotryptic activity. Treatment with ninhydrin, a specific arginine modifier, at pH 9.0 abolished the antitryptic activity whereas only 50% of the antichymotryptic activity was lost. Diethylpyrocarbonate, a histidine reagent, also decreased only the antitryptic activity. Modification of tryptophan and cysteine residues of the inhibitor had no effect on its inhibitory potency. Treatment with mercaptoethanol and sodium borohydride caused nearly 50% loss of antitryptic and antichymotryptic activities. Chloramine-T, a reagent that modifies methionine residues, inactivated the inhibitor.
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PMID:Chemical modification and complex formation studies with jack bean proteinase inhibitor. 181 77

The myoglobin, cytochrome b5 and alpha-chymotrypsin hydrophobic nucleus sizes were calculated as well as sizes of theoretical spherical nucleus, radii that are equal to the lengths of phenylalanine and tryptophan lateral groups. All calculated values of sizes lie in the (0.99-1.65) nm3 interval. The quantitative estimation of analyzed proteins nucleus heterogeneous composition has been shown.
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PMID:[A comparative analysis of the structure of the myoglobin, cytochrome b5, and alpha-chymotrypsin hydrophobic nuclei]. 181 2


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