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 synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (approximately 4.9 x 10(3) M-1.min-1) and reagent 2 (approximately 1.0 x 10(5) M-1.min-1) respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatible with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase.
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PMID:The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases. 157 91

Cleavage of yeast invertase by alpha-chymotrypsin produced a number of small glycopeptides that were highly active as elicitors of ethylene biosynthesis and phenylalanine ammonia-lyase in suspension-cultured tomato cells. Five of these elicitors were purified and their amino acid sequence determined. They all had sequences corresponding to known sequences of yeast invertase, and all contained an asparagine known to carry a N-linked small high mannose glycan. The most active glycopeptide elicitor induced ethylene biosynthesis and phenylalanine ammonia-lyase half-maximally at a concentration of 5-10 nM. Structure-activity relationships of the peptide part were analyzed by further cleavage of a defined glycopeptide elicitor with various proteolytic enzymes. Removal of the C-terminal phenylalanine enhanced the elicitor activity, whereas removal of N-terminal arginine impaired it. A glycopeptide with the peptide part trimmed to the dipeptide arginine-asparagine was still fully active as elicitor. Glycopeptides with identical amino acid sequences were further separated into fractions differing in the oligosaccharide side chain. A given peptide had high elicitor activity when carrying a glycan with 10-12 mannosyl residues (Man10-12GlcNAc2), a 3-fold lower activity when carrying Man9GlcNAc2 and a 100-fold lower activity when carrying Man8GlcNAc2. The oligosaccharides, released by endo-beta-N-acetylglucosaminidase H from the pure glycopeptide elicitors, acted as suppressors of elicitor-induced ethylene biosynthesis and phenylalanine ammonia-lyase activity. A series of such oligosaccharides in the size range of Man8-13GlcNAc was purified. The structure and composition of the purified oligosaccharides corresponded to the known small high mannose glycans of yeast invertase as verified by 1H NMR spectroscopy at 600 MHz. The highest suppressor activities were obtained with the oligosaccharides containing 10-12 mannosyl residues (Man10-12GlcNAc). The oligosaccharide Man8 GlcNAc was ineffective as a suppressor. Thus, the structural requirements for the free oligosaccharides to act as efficient suppressors were the same as for the oligosaccharide side chains of the glycopeptides for high elicitor activity. We propose that the glycan suppressors bind to the same recognition site as the glycopeptide elicitors without inducing a response.
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PMID:Elicitors and suppressors of the defense response in tomato cells. Purification and characterization of glycopeptide elicitors and glycan suppressors generated by enzymatic cleavage of yeast invertase. 158 15

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

We have studied an indirect role of serine and thiol proteases in the activation of human neutrophils in vitro. Stimulation was evaluated using a chemiluminescence (CL) generation system. Receptor-dependent and receptor-independent stimuli were studied, e.g. opsonized zymosan, formyl-methionyl-leucyl-phenylalanine, platelet activating factor, phorbol myristate acetate, and calcium ionophore A23187. The serine protease inhibitors TPCK and TLCK, and thiol protease inhibitor PHMB, diminished the CL with different potencies and in a dose-dependent manner after treatment of cells with the various stimuli. Non-specific serine protease inhibitor, PMSF, and trypsin substrate TAME, showed a low inhibitory potency with respect to CL generation. Synthetic substrates for chymotrypsin (BTEE, ATEE) significantly inhibited CL with the various stimuli used with some differences in susceptibility to their inhibition. Specific chymotrypsin inhibitors diminished both the resting and activator-induced CL. We suggest that cell-bound chymotrypsin-like protease(s) is involved in the activation of signal transduction in human neutrophils after both receptor-dependent and receptor-independent stimulation.
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PMID:The effect of serine and thiol protease inhibitors on the chemiluminescence of human neutrophils in investigations in vitro. 164 40

The midgut chymotrypsins (EC 3.4.4.5) of three species of shrimps, Penaeus monodon, Penaeus japonicus and Penaeus penicillatus were purified and studied in detail to clarify previous ambiguity in their identification. In each of the species there are two major forms of chymotrypsin, both single-chained with three disulfide bonds. One has a pI of 3.2 and Mr 27,000 or 28,000, while the other has a pI of 3.0 and Mr 25,000 or 26,000. The N-terminal amino acid sequences of the P. monodon enzymes are homologous to those of the crab (Uca pugilator) collagenase and to the other chymotrypsins. However, the active sites of the shrimp chymotrypsins are different from that of the well studied bovine alpha-chymotrypsin in some respects: (1) in spite of showing the typical specificity of chymotrypsin, the shrimp enzymes are more stringently selective for substrates with extended polypeptide chain; (2) some titration agents of alpha-chymotrypsin, including t-cinnamoylimidazole, 4-nitrophenyl guanidinobenzoate and its fluorescent derivative, do not react with the shrimp enzymes, neither do some of the alpha-chymotrypsin inhibitors: Tosyl-PheCH2Cl, methyl-4-nitrobenzenesulfonate and benzeneboronic acid; (3) the shrimp chymotrypsins are more reactive than the bovine enzyme toward native protein substrates including collagen; (4) the kinetic-salt-effects of the shrimp enzyme toward N-succinyl- and acetyl-Ala-Ala-Pro-Phe-4-nitroanilide mainly reflect electrostatic rather than hydrophobic interactions between the substrates and the enzyme. The shrimp enzymes are acid-labile but resistent to autolysis. Our results suggest that most Crustacea decapods contain chymotrypsins as one of the major digestive endopeptidases.
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PMID:The midgut chymotrypsins of shrimps (Penaeus monodon, Penaeus japonicus and Penaeus penicillatus). 165 78

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.
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PMID:A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein". 169 76

Nucleolin, also called protein C23, is a RNA-associated protein implicated in the early stages of ribosome assembly. To study the general conformation and map the nucleic acid binding regions, rat nucleolin was subjected to limited proteolysis using trypsin and chymotrypsin in the presence or absence of poly(G). The cleavage sites were classified according to their locations in the three putative domains: the highly polar amino-terminal domain, the central nucleic acid binding domain, which contains four 90-residue repeats, and the carboxyl-terminal domain, which is rich is glycine, dimethylarginine, and phenylalanine. The most labile sites were found in basic segments of the amino-terminal domain. This region was stabilized by Mg2+. At low enzyme concentrations, cleavage by trypsin or chymotrypsin in the amino-terminal domain was enhanced by poly(G). Trypsin produced a relatively stable 48-kDa fragment containing the central and carboxyl-terminal domains. The enhanced cleavage suggests that binding of nucleic acid by the central domain alters the conformation of the amino-terminal domain, exposing sites to proteolytic cleavage. At moderate enzyme concentrations, the 48-kDa fragment was protected by poly(G) against tryptic digestion. At the highest enzyme concentrations, both enzymes cleaved near the boundaries between repeats 2, 3, and 4 with some sites protected by poly(G), suggesting that the repeats themselves form compact units. The carboxyl-terminal domain was resistant to trypsin but was cleaved by chymotrypsin either in the presence or in the absence of poly(G), indicating exposure of some phenylalanines in this region. These studies provide a general picture of the topology of nucleolin and suggest that the nucleic acid binding region communicates with the amino-terminal domain.
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PMID:Limited proteolysis as a probe of the conformation and nucleic acid binding regions of nucleolin. 169

The authors suggest a highly sensitive rapid and simple method for measuring esterase activities of bovine pancreatic chymotrypsin, human neutrophilic cathepsin D, and elastase, and of human blood serum chymotrypsin-like esterase and elastase-like esterase activities, with fluorogenic synthetic ethers, amino acid derivatives, employed as substrates: N-benzoxycarbonylphenylalanine 4-methylumbelliferyl ester (Z-Phe-OMC) and tret-butyloxycarbonyl-1-alanine 4-methylumbelliferyl ester (BOC-Ala-OMC).
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PMID:[Determination of the esterase activity of serine proteinases using synthetic substrates]. 171 86

2E7 is a human monoclonal IgM autoantibody that binds to a site on the heavy chain of the human platelet integrin alpha subunit glycoprotein IIb. The epitope recognized by 2E7 is stable to denaturation with sodium dodecyl sulfate and reduction of disulfide bonds but is destroyed by proteolysis with papain, chymotrypsin or elastase. By evaluating the reaction of 2E7 with a number of protein sequences from the IIb heavy chain, we have determined that the epitope is located in the octapeptide Phe-Asp-Gly-Tyr-Trp-Gly-Tyr-Ser (FDGYWGYS), corresponding to residues 231-238, and that substitution of the Trp at position 235 completely destroys the epitope. This represents the first precise localization of an epitope on the human platelet integrin IIb-IIIa or on any platelet membrane glycoprotein that is recognized by a human autoantibody.
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PMID:Human monoclonal autoantibody 2E7 is specific for a peptide sequence of platelet glycoprotein IIb. Localization of the epitope to IIb231-238 with an immunodominant Trp235. 171 97

During the formation of an inhibitory complex with neutrophil elastase, alpha 1 antitrypsin (alpha 1 AT) undergoes a structural rearrangement and the resulting alpha 1 AT-elastase complex becomes endowed with chemoattractant activities, mediates an increase in synthesis of alpha 1 AT, and is rapidly cleared from the circulation. In previous studies we have provided evidence that these biological activities involve the recognition of a conformation-specific domain in the alpha 1 AT molecule by a cell surface receptor on human hepatoma HepG2 cells and human monocytes. The receptor has been termed the serpin-enzyme complex (SEC) receptor because it also recognizes complex of serpins antithrombin III, alpha 1 anti-chymotrypsin, and C1 inhibitor with their cognate enzymes. Because a pentapeptide domain of alpha 1 AT (amino acids 370-374, Phe-Val-Phe-Leu-Met) is sufficient for binding to the SEC receptor and the sequence of this domain is remarkably similar to those of substance P, several other tachykinins, bombesin, and the amyloid-beta peptide, we have examined the possibility that these other ligands bind to the SEC receptor. The results indicate that substance P, several other tachykinins, and bombesin compete for binding to, and cross-linking of, the SEC receptor. The SEC receptor is distinct from the substance P receptor by several criteria. There is no substance P receptor mRNA in HepG2 cells; the SEC receptor is present in much higher density on receptor-bearing cells and binds its ligands at lower affinity than the substance P receptor; the SEC receptor is much less restricted in the specificity with which it recognizes ligand; ligands for the SEC receptor including peptide 105Y (based on alpha 1 AT sequence 359-374), alpha 1 AT-protease complexes, and bombesin do not compete for binding of substance P to a stable transfected cell line expressing the substance P receptor. Finally, we show here that the amyloid-beta peptide competes for binding to the SEC receptor but does not bind to the substance P receptor, therein raising the possibility that the SEC receptor is involved in certain biological activities, including the recently described neurotrophic and neurotoxic effects ascribed to the amyloid-beta peptide.
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PMID:Amyloid-beta peptide, substance P, and bombesin bind to the serpin-enzyme complex receptor. 171 86


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