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PubMed1979

Genetic control of the immune response to mammalian chymotrypsins in mice. I. The immune response to high doses of bovine alpha-chymotrypsin.

The primary and secondary immune response to the antigen bovine pancreatic alpha-chymotrypsin was investigated in inbred mice. It was found that strain differences in the immune response only became apparent after secondary immunization. The genetic control of the immune response was investigated in twelve different strains of mice, F1, F2 and F1 backcross hybrids, following secondary immunization. A continuous distribution for the mean antibody responsiveness was obtained. High responsiveness was associated with both the H-2 haplotype and three non-H-2 loci. Furthermore the F1 hybrids produced a greater quantitative antibody response to chymotrypsin than either of the corresponding parental strains.

Animals↗

PubMed1991

Determination of faecal chymotrypsin concentration and 72-hour faecal chymotrypsin output in the detection of pancreatic steatorrhoea.

In 96 consecutive patients who underwent a 72-h faecal fat determination because of suspected nutrient malassimilation (maldigestion and/or malabsorption) faecal chymotrypsin (F-Chym) was estimated with a commercial photometric test (Monotest Chymotrypsin), comparing F-Chym concentrations in the first 24-h stool with the total 72-h F-Chym output. In the first 24-h faeces, the F-Chym concentration, calculated as a mean of three random samples, did not significantly differ from a single value obtained after homogenization. In known pancreatic disease, a F-Chym concentration less than 3.0 U/g wet faeces distinguished well between steatorrhoic patients (n = 12) and nonsteatorrhoic (n = 13) (positive predictive value (PV), 91%; negative PV, 86%) but was less suitable as a screening test for pancreatic steatorrhoea in the unselected patient group (positive PV, 61%; negative PV, 98%). Although the estimation of 72-h F-Chym output could differentiate between various subgroups of patients to a certain extent, the positive PV for discovery of pancreatic steatorrhoea in a single patient was low. Four patients had excessively high F-Chym output and increased bile acid excretion after ileal resection (n = 3) and radiation ileitis (n = 1), respectively, possibly indicating the removal of an inhibitory mechanism of pancreatic and biliary secretion in these conditions.

Adult↗

PubMed1982

Separation of free and chymotrypsin-bound alpha 2-macroglobulin by affinity chromatography. Its use to demonstrate that the two chymotrypsin-binding sites of alpha 2-macroglobulin are equivalent and independent.

Binary and ternary alpha 2-macroglobulin-chymotrypsin complexes may be quantitatively adsorbed on BH-Sepharose-D-tryptophan methyl ester at pH 8.0 and quantitatively eluted either with acetic acid or with 40% glycerol, pH 8.0. This is the first report of a preparative separation of free and proteinase-bound alpha 2-macroglobulin. Using this affinity chromatographic system, we were able to demonstrate that the two chymotrypsin binding sites of alpha-2-macroglobulin are equivalent and independent.

Animals↗

PubMed1998

Acetaldehyde inhibits chymotrypsin and serum anti-chymotrypsin activity.

BACKGROUND: Chymotrypsin (CT) and CT-like enzymes contribute to the dynamics of metabolism by their participation in digestion, peptide hormone generation and catabolism, fertilization of ova and inhibition of thrombin-induced platelet aggregation, among other processes. The frequency of pancreatitis is observably higher in alcoholics, and pancreatic enzymes have been associated with localized vascular damage, thrombosis and pancreatic necrosis. METHODS: Since CT is a major pancreatic enzyme and may serve as a link between pancreatitis, coagulopathy, and alcoholism, the affect of acetaldehyde (AcH) the primary metabolite of ethanol, upon the enzyme and upon the influence of human serum thereon was studied. RESULTS: It was observed that CT activity upon glutaryl-L-phenylalanine-b-naphthylamide was inhibited to the extent of 23.7%, 52.5%, and 96.7% by 44.7, 89.4, and 447 mmol/L AcH in a fluorometric assay whereby the enzyme was dialyzed to remove excess AcH prior to assay. The p values were < 0.04. Aliquots of human serum (10 microL, 20 microL, 30 microL, 40 microL, 50 microL, and 100 microL) inhibited 40 micrograms of CT by 13%, 37.7%, 65.3%, 89.8%, and 92.8%, respectively (n = 6; p = < 0.05). The serum did not hydrolyze the fluorogenic substrate. On the other hand, AcH added to serum at 447, 224, 112, or 56 mmol/L resulted in 42.6%, 42.6%, 52.9%, and 60.3% inhibition of CT relative to a 69.1% inhibition of the enzyme by serum alone (n = 6; = p < 0.01). CONCLUSIONS: These data show that AcH clearly decreases the antichymotryptic activity of serum (consisting of alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin). The incomplete inactivation of chymotrypsin by serum and partial inactivation of CT inhibitor(s) by AcH suggest the possibility that CT leaked into the circulation, (in alcoholic pancreatitis) may be available in blood to lower the clotting potential induced by thrombin-activated platelets, and that a greater amount of CT might be available in the blood of alcoholics, thereby contributing, in part, to the prolongation of clotting times.

Acetaldehyde↗

PubMed1991

Structure of gamma-chymotrypsin in the range pH 2.0 to pH 10.5 suggests that gamma-chymotrypsin is a covalent acyl-enzyme adduct at low pH.

Crystals of gamma-chymotrypsin (gamma-CHT) grown at pH 7.0 are stable from pH 2.0 to 11.0. Crystalline gamma-CHT therefore provides an unusually favourable system to observe the structure of a protein and its bound solvent over a broad range of pH. In this report we describe the high-resolution refined structure of gamma-CHT at pH values of 2.0, 7.0 and 10.5. The apparent tetrapeptide seen bound in the active site of gamma-CHT at pH 7.0 is also present at pH 2.0 and 10.5 although it is better defined at low pH. A comparison of the respective structures shows that there is additional electron density in the low pH structure at the point where the side-chain of Ser 195 approaches most closely to the presumptive inhibitor. This suggests that the adduct is most likely to be covalently linked to the enzyme at low pH and to be non-covalent at higher pH. As the pH is lowered from 7.0 to 2.0, the side-chain of His 40 rotates approximately 120 degrees about its C alpha-C beta bond and, in concert, the side-chain of Gln 34 also rotates approximately 140 degrees about its C alpha-C beta bond. Apart from these localized rearrangements in the vicinity of His 40, the structure of gamma-CHT at pH 2.0 is very similar to that at neutral pH. The structure of gamma-CHT at pH 10.5 is also seen to be almost identical with that at neutral pH. There is no indication that the internal salt bridge between Asp 194 and the alpha-amino group of lle 16 begins to dissociate at pH 10.5. With the exception of the vicinity of His 40, the structure of the bound solvent in the crystal structures at low, neutral and high pH is very similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Chymotrypsin↗

PubMed1994

S'-subsite mapping of polyethylene glycol-modified alpha-chymotrypsin and alpha-chymotrypsin: a comparative study.

Nucleophile specificities of polyethylene glycol-modified alpha-chymotrypsin and the native enzyme were investigated via acyl transfer reactions using Ac-Tyr-OEt as acyl donor and a large series of peptides and amino-acid amides as nucleophiles. In acyl transfer reactions with amino-acid amides both enzymes prefer basic and bulky amino-acid residues. However, peptides with bulky aliphatic or aromatic residues in P1' position were very poor nucleophiles for both enzymes. Surprisingly, peptides having bulky aliphatic or aromatic residues in P2' were preferred by the modified enzyme and were apparently more efficient nucleophiles for both enzymes than those with such residues in P1'. Generally, peptides with a longer chain were weaker nucleophiles in the reactions catalyzed by polyethylene glycol-modified enzyme. In the series of peptides containing a positively charged amino-acid residue in various locations, the order of nucleophilic efficiency is with this location being: P1' > P3' > P2'; this is valid for both enzymes.

Amino Acid Sequence↗

PubMed2003

A study of the effect on nucleophilic hydrolytic activity of pancreatic elastase, trypsin, chymotrypsin, and leucine aminopeptidase by boronic acids in the presence of arabinogalactan: a subsequent study on the hydrolytic activity of chymotrypsin by boronic acids in the presence of mono-, di-, and trisaccharides.

The hydrolytic activity of trypsin, chymotrypsin, elastase, and leucine aminopeptidase, is inhibited by different boronic acids. However, all the enzymes are inhibited by the compound CbzAla(boro)Gly(OH)(2). Therefore, these additives can control the nucleophilic hydrolytic activity of these enzymes.

Boronic Acids↗

PubMed1990

Recombinant chymotrypsin inhibitor 2: expression, kinetic analysis of inhibition with alpha-chymotrypsin and wild-type and mutant subtilisin BPN', and protein engineering to investigate inhibitory specificity and mechanism.

The serine protease inhibitor chymotrypsin inhibitor 2 (CI2 or BSPI2) has been expressed in Escherichia coli with the pINIIIompA3 expression vector to produce 20-40 mg/L of culture. Recombinant CI2 purified from this system has been characterized and found to be identical with CI2 from barley. Slow-binding kinetics were observed for the interaction between CI2 and subtilisin BPN', with Ki = 2.9 x 10(-12) M. Analysis of slow-binding data indicates that binding of the inhibitor follows the simplest model of E + I = EI with no kinetically detectable intermediate steps or proteolytic cleavage of the reactive site bond in CI2 (Met-59-Glu-60). This, in agreement with crystallographic data, indicates that the enzyme-inhibitor adduct is the Michaelis complex, which is not chemically processed by the enzyme. Three mutant CI2 molecules with new P1 residues have also been examined with a range of serine proteases, including a mutant subtilisin. In agreement with earlier studies, we find the P1 amino acid an important determinant of specificity. CI2 Met----Lys-59 was found to be a temporary inhibitor of subtilisin BPN' but an effective inhibitor of subtilisin Carlsberg and subtilisin BPN'(Glu----Ser-156). The structural reasons for this are discussed in relation to mechanisms of inhibition of serine proteases.

Base Sequence↗

PubMed1977

Thermodynamics of binding to native alpha-chymotrypsin and to forms of alpha-chymotrypsin in which catalytically essential residues are modified; a study of "productive" and "nonproductive" associations.

The standard free energy (deltaG degrees), enthalpy (deltaH degrees), and entropy (deltaS degrees) of association for proflavin and D- and L-N-AcTrp have been obtained at pH 7.8 for native alpha-chymotrypsin (Cht) and for forms of Cht in which essential catalytic residues of the active site are modified. The modified Cht forms studied are dehydroalaninyl-195-alpha-Cht and N-methylhistidinyl-57-alpha-Cht. Associations to native Cht (pH 7.8) are characterized by negative deltaH degrees and deltaS degrees values (i.e., for L-AcTrp deltaH degrees = -9.1 kcal/mol and deltaS degrees = -21 eu at T = 25 degreesC). In contrast, we found associations to modified Chts to be characterized by an enthalpy near zero and a positive entropy of association, the values of the deltaH degrees and deltaS degrees for association to the modified Cht forms being similar to those expected for transfer of small aromatic molecules from water to a nonpolar solvent phase. Differences in deltaH degrees and deltaS degrees observed for binding of substrate analogues and inhibitors to modified and native Cht (pH 7.8) are approximately + 10 kcal/mol and +30 eu, respectively. Data from D. D. F. Shiao ((1970), Biochemistry 9, 1083) similarly show differences of comparable magnitude between binding of substrate analogues to active alpha-Cht (pH 7.8) and the His-57 protonated form of alpha-Cht (pH 5.6). The negative deltaH degrees and deltaS degrees values of associations for binding to active alpha-Cht indicate that a substrate-induced conformational change occurs on substrate association with the primary binding site (S1), which does not occur in Ser-195 and His-57 modified Cht. From these differences we infer a linkage between binding of substrate into S1 and the catalytic residues in the nucleophilic subsite (S1-S1'). Our data also show that associations of substrate analogues into potentially productive Michaelis complexes S1 cannot be easily differentiated from associations that are nonproductive (i.e., nonactivated) from their deltaG degrees obsd, but may be differentiated by their respective deltaH degrees obsd and deltaS degrees obsd for association. Accordingly, it is indicated that the probable substrate association-activation process, characterized thermodynamically in this work, occurs in the substrate binding step and leads to lowered free energies of activation in catalytic steps succeeding binding however, the process does not influence the observed strength of substrate binding.

Binding Sites↗

PubMed1989

A 13C-n.m.r. investigation of the ionizations within an inhibitor--alpha-chymotrypsin complex. Evidence that both alpha-chymotrypsin and trypsin stabilize a hemiketal oxyanion by similar mechanisms.

13C-n.m.r. was used to investigate the structure of the inhibitor enzyme complex formed when alpha-chymotrypsin is alkylated by L-1-chloro-4-phenyl-3-tosylamido-[2-13C]butan-2-one. Two signals are detected. The one at 204.82 +/- 0.11 p.p.m. does not titrate from pH 3 to 9 and is assigned to alkylated methionine-192. The second signal titrates from 99.08 p.p.m. to 103.44 p.p.m. with pKa 8.67. This signal is assigned to a tetrahedral adduct formed between the hydroxy group of serine-195 and the inhibitor. The titration shift of the tetrahedral adduct is ascribed to the ionization of the hemiketal hydroxy group. It is proposed that the resulting oxyanion is stabilized by interaction with the imidazolium ion of histidine-57. It is argued that this interaction must raise the pKa of at least 70% of histidine-57 to greater than 11. On denaturation/autolysis of the inhibitor-enzyme complex neither of the signals associated with the intact complex is detected, but a new signal is observed that titrates from 203.52 p.p.m. to 206.08 p.p.m. with pKa = 5.27. This titration shift is assigned to the ionization of the imidazolium ion of alkylated histidine, confirming that the inhibitor has alkylated histidine-57. The significance of these results for the catalytic mechanism of the serine proteinases is discussed.

Alkylation↗

PubMed1982

Specific and non-specific effects of inert anions and cations on the dimerization of alpha-chymotrypsin and the catalytic activities of monomeric and dimeric alpha-chymotrypsins.

The effect of salts on the dimerization and the catalytic activity of dimeric alpha-chymotrypsin (alpha-CT) was investigated. The observed effect was mainly related to the anionic constituent of the salt, and its order depended on the salt concentration. The order of effect of anions on several parameters at [salt] less than 0.02 M was SO4 2-, ClO4-, NO3-, Br-, Cl-, which reflected the electrostatic interaction between the anion and the positively charged surface of alpha-CT. On the other hand, the anion dependence at moderate salt concentrations (0.1-0.2 M) followed the Hofmeister series, SO4 2-, Cl-, Br-, NO3-, ClO4-, which was related to the direct and indirect interactions between the anion and non-charged groups of the protein. The anion order for the dimerization constant of this enzyme, however, was the complete reverse of that generally observed in the aggregation of proteins at high salt concentration (greater than 1 M). This result was accountable for in terms of a specific interaction in the formation of the dimeric enzyme (probably that between the active site of one monomer and Tyr-146 of the other).

Anions↗

PubMed1979

Genetic control of the immune response to mammalian chymotrypsins in mice. II. The immune response to low doses of bovine alpha-chymotrypsin.

The immune response to the antigen bovine pancreatic alpha-chymotrypsin was investigated in ten recombinant strains of mice. Using a fixed antigen-percentage bound isotope technique, it was found that the quantity of antibody produced was related to the H-2 haplotype of the responding animal. A continuous distribution for the mean antibody responses was obtained for the ten strains of mice. High responsiveness was associated with the H-2 haplotype gamma2. The genetic control of the immune response to this immunogen was found to be both quantitative and qualitative.

Animals↗

PubMed1988

Is chymotrypsin output a better diagnostic index than the measurement of chymotrypsin in random stool?

This study compares the diagnostic utility of fecal chymotrypsin (CT) output in timed stool collections and random stools using a new photometric enzyme assay. The CT output (mean +/- SD, U/24 h) was 1,487 +/- 1,980 in 127 children with normal fat absorption and negative sweat-chloride test (mean age 45 months), and 1,804 +/- 1,452 in 27 cases with fat malabsorption due to nonpancreatic disease (mean age 41 months). 66 cases of cystic fibrosis (CF) were examined (mean age 119 months). Stool output in 19 newly diagnosed patients before therapy was 85 +/- 94, in 42 patients receiving enzyme replacement therapy was 3,462 +/- 2,841, and in 5 patients with pancreatic sufficiency 1,754 +/- 1,482. Using nonparametric statistics, 120 U/24 h was defined as the lower limit of the 95-percentile for stool CT output. Only 5 of the 127 patients with normal fat absorption had output below that limit. None of the patients with nonpancreatic malabsorption and only 1 treated CF patient had lower values. Sixteen of the newly diagnosed CF patients had stool CT less than 120 U/24 h. The sensitivity of the test is therefore 84% and its specificity 97% at this decision threshold. However, no diagnostic advantage is gained from measuring CT output in timed stool collections as compared to random stools.

Age Factors↗

PubMed1965