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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)

Three proteolytic enzymes, trypsin, chymotrypsin, and aminopeptidase-N (APN), were purified from laboratory-reared western spruce budworm, Choristoneura occidentalis [Freeman], larvae. Budworm trypsin exhibited a high degree of substrate specificity, was inactivated by DFP and TLCK, and was inhibited by trypsin inhibitors. The western spruce budworm chymotrypsin hydrolyzed SAAPFpNA and SAAPLpNA, but not SFpNA, SGGFpNA, SGGLpNA or BTpNA. The chymotrypsin was inactivated by DFP, and was inhibited by chymostatin and the chymotrypsin inhibitor, POT-1. Purified budworm chymotrypsin exhibited little BTEE esterolytic activity and was insensitive to inhibition with TPCK. The N-terminal sequence of budworm trypsin, chymotrypsin, and APN were obtained. Similar levels of trypsin and APN gut activities were found in laboratory-reared and field-collected larvae. However, in comparison to laboratory-reared insects, considerably less chymotrypsin activity, and a much higher level of gut carboxypeptidase activity were found in field-collected western spruce budworm larvae.
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PMID:Purification and characterization of the western spruce budworm larval midgut proteinases and comparison of gut activities of laboratory-reared and field-collected insects. 1038 Jun 52

Reactive phosphonate diesters were designed and prepared as inhibitors of serine proteases and esterases. Inactivation of trypsin, chymotrypsin, and butyrylcholinesterase was determined by residual enzymatic activity as well as by the release of a chromogenic or fluorogenic product of the inhibition reaction. Second-order rate constants were determined from rates of nitrophenol formation. Application of the reaction for active-site titration of enzyme preparations is demonstrated. A basic functional group present in the nitrophenyl tropane phosphonate diester was shown to confer selectivity for inactivation of trypsin and chymotrypsin. Biotinylated derivatives of the phosphonate diesters were prepared to permit analysis of proteins modified in the inhibition reaction. Labeled polypeptides were resolved by SDS-PAGE, electroblotted, and detected by streptavidin-peroxidase staining. A detection limit of less than 4 ng, corresponding to 20 nM of trypsin, was demonstrated. Pretreatment of enzymes with DFP or nonbiotinylated phosphonates specifically blocks the labeling. This technique permits identification of serine proteases in complex mixtures with good sensitivity and specificity.
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PMID:Inhibition and labeling of enzymes and abzymes by phosphonate diesters. 1082 63

Prostate specific antigen (PSA, also known as human kallikrein 3) is an important diagnostic indicator of prostatic disease. PSA exhibits low protease activity (>10(4)-fold less than chymotrypsin) under the usual in vitro assay conditions. In addition, PSA does not react readily with prototypical serine protease inactivators. We expressed human PSA (rh-PSA) in Escherichia coli and have demonstrated that rh-PSA has properties similar to those of native PSA isolated from human seminal fluid. Both PSA and rh-PSA are >10(3)-fold more active in the presence of 1.3 M Na(2)SO(4). This activation is anion-dependent, following the Hofmeister series when normality is considered: SO(4)(2)(-) approximately citrate > Ac(-) > Cl(-) > Br(-) > I(-). The nature of the cation has little effect on salt activation. The rate of inactivation of rh-PSA by DFP is 30-fold faster in the presence of 0.9 M Na(2)SO(4), and the rate of inactivation by Suc-Ala-Ala-Pro-Phe-CK is >20-fold faster under these conditions. Azapeptides containing Phe or Tyr at position P(1) also inactivate rh-PSA in the presence of high salt concentrations. These compounds represent the first described inhibitors designed to utilize the substrate binding subsites of PSA. CD spectroscopy demonstrates that the conformation of rh-PSA changes in the presence of high salt concentrations. Analytical ultracentifugation and dynamic light scattering indicate that PSA remains monomeric under high-salt conditions. Interestingly, human prostatic fluid contains as much as 150 micro mol citrate/g wet weight, which suggests that salt concentrations may regulate PSA activity in vivo.
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PMID:Modulation of recombinant human prostate-specific antigen: activation by Hofmeister salts and inhibition by azapeptides. Appendix: thermodynamic interpretation of the activation by concentrated salts. 1157 Aug 74

Oil-induced mononuclear phagocytes (MN) were quantitatively assayed for various hydrolases as unfractionated suspensions of frozen and thawed cells. They apparently contain two proteases. The first, measured with urea- or acid-denatured hemoglobin, was similar to purified Proteinase I of lung with respect to pH optimum (pH 4), stability, hydrolytic and polymerizing activities, and reactions to various inhibitors. The second protease resembled chymotrypsin in its hydrolysis of glycyl-L-phenylalanine amide, acetyl-L-tyrosine ethyl ester and N-benzoyl-DL-phenylalanine-beta-naphthol ester (BPN). With the latter, its pH optimum was between 5.0 and 5.8, and its action was inhibited by diisopropylphosphorofluoridate (DFP) and p-chloromercuribenzoate. When assayed under the above conditions, polymorphonuclear exudate cells (PMN) and red blood corpuscles (RBC) manifested little or no hydrolysis of either hemoglobin or BPN. MN also contained esterases that split methyl butyrate and beta-naphthyl acetate. The pH optimum with the latter was 7.4, and its hydrolysis was partially inhibited by DFP, fluoride, taurocholate, and eserine. PMN had low esterase activity; RBC had little or none. MN, but not PMN or RBC, contained a stable lipase with a pH optimum of 6.1 in maleate buffer. Protamine, NaCl, heat, p-chloromercuribenzoate, ethylenediamine tetraacetate, taurocholate, and DFP were inhibitory, but no appreciable activation occurred in the presence of heparin or serum. Thus it possessed some of the characteristics of Korn's lipoprotein lipase, but not others.
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PMID:HYDROLYTIC ENZYMES OF RABBIT MONONUCLEAR EXUDATE CELLS. I. QUANTITATIVE ASSAY AND PROPERTIES OF CERTAIN PROTEASES, NON-SPECIFIC ESTERASES, AND LIPASES OF MONONUCLEAR AND POLYMORPHONUCLEAR CELLS AND ERYTHROCYTES. 1415 92

In the present study, we applied for the first time (31)P diffusion NMR to resolve different species obtained by the addition of organophosphorus compounds (OP) such as diisopropyl phosphorofluoridate (DFP) or 1-pyrenebutyl phosphorodichloridate (PBPDC) to alpha-chymotrypsin (Cht). (31)P diffusion NMR was used since the products of these reactions constitute a mixture of OP-covalent conjugates of the enzyme and OP-containing hydrolysis products that have noninformative (1)H NMR spectra. It was shown that the peak, attributed to the covalent native diisopropylphosphoryl-Cht (DIP-Cht) conjugate by chemical shift considerations, has a greater diffusion coefficient (D = (0.65 +/- 0.01) x 10(-5) cm(2) s(-1)) than expected from its molecular mass (approximately 25 kDa). This peak was therefore suggested to consist of at least two superimposed signals of diisopropyl phosphoryl (DIP) pools of high and low molecular weights that happen to have the same chemical shift. This conclusion was substantiated by the use of DMSO-d(6) that separated the overlapping signals. Diffusion measurements performed on the extensively dialyzed and unfolded DIP-Cht conjugate still resulted in a high diffusion coefficient ((0.30 +/- 0.05) x 10(-5) cm(2) s(-1)) relative to the assumed molecular mass. This observation was attributed to a dynamic dealkylation at the OP moiety (i.e., aging) that occurred during the relatively long diffusion measurements, where DIP-Cht was converted to the corresponding monoisopropyl phosphoryl Cht (MIP-Cht) conjugate. Homogeneous aged forms of OP-Cht were obtained by use of DFP and heat-induced dealkylation of DIP-Cht, and by PBPDC that provided the aged form via the hydrolysis of a P-Cl bond (PBP-Cht). The thermally stable aged conjugates enabled a reliable determination of the diffusion coefficients over several days of data acquisition, and the values found were (0.052 +/- 0.002) x 10(-5) cm(2) s(-1) and (0.054 +/-0.004) x 10(-5) cm(2) s(-1) for the MIP-Cht and the PBP-Cht adducts, respectively, values in the range expected for a species with a molecular weight of 25 kDa. The advantages and limitations of (31)P diffusion NMR in corroborating the type of species that prevail in such systems are briefly discussed.
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PMID:Probing the molecular interaction of chymotrypsin with organophosphorus compounds by 31P diffusion NMR in aqueous solutions. 1562 38

The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase.
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PMID:Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph. 1693 80


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