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)

A new method for cross-linking of protein was proposed in our previous work. The method is based on the spontaneous chelate formation process involving three components, salicylaldehyde moiety, alpha-amino acid residue and copper(II). In this paper versatility of the method as a purpose of immobilization of enzyme was described. Chymotrypsin-salicylaldehyde conjugate was immobilized to the agarose gel attached alpha-amino acid residue in the presence of copper(II) ion The enzyme was not eluted from the gel by washing with a copper free buffer though it was exclusively eluted by a medium containing EDTA. Catalytic activity of the chymotrypsin salicylaldehyde conjugate was not changed upon the immobilization. The method was proposed as a new tool for reversible immobilization of enzyme.
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PMID:Immobilized chymotrypsin by means of Schiff base copper(II) chelate. 275 59

The steroid-binding subunit of the glucocorticoid receptor is known to be a approximately 100-kDa phosphoprotein composed of an immunogenic, DNA-binding, and steroid-binding domain. When isolated from WEHI-7 cells, this protein contains between two and three phosphoryl groups per steroid-binding site (Mendel WEHI-7 cells, this protein contains between two and three phosphoryl groups per steroid-binding site (Mendel et al., 1987). To identify the domains that contain these phosphorylated sites, we have analyzed the phosphate content of selected proteolytic fragments of the approximately 100-kDa steroid-binding protein from nonactivated and activated receptors. The approximately 100-kDa steroid-binding protein from WEHI-7 cells grown in the presence of [32P]orthophosphate was covalently labeled with [3H]dexamethasone 21-mesylate, purified with the BuGR2 monoclonal antibody, digested with chymotrypsin or trypsin, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Chymotrypsin digestion of this protein yields a approximately 45-kDa fragment containing both the steroid-binding and DNA-binding domains, which contained both 32P and 3H. Trypsin digestion of the protein yields a approximately 29-kDa fragment encompassing the steroid-binding domain but not the DNA-binding domain of the approximately 100-kDa protein, which also contained both 32P and 3H. The 32P/3H ratio of each fragment provides a measure of phosphate content per steroid-binding site and indicated that each fragment has approximately 30% of the phosphate content of the intact protein. This is sufficient to account for one of the three receptor phosphoryl groups.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylated sites within the functional domains of the approximately 100-kDa steroid-binding subunit of glucocorticoid receptors. 276 97

Transcription factor IIIC2 is required for in vitro transcription of the adenovirus 2 VA1 gene and binds with high affinity to its B-box promoter element which is an 18 bp perfect inverted repeat. Partial proteolysis of TFIIIC2 with chymotrypsin and Staphylococcus aureus V8 protease yielded a species which produced a discrete band in a gel shift assay with about twice the mobility of the undigested complex. Chymotrypsin-digested TFIIIC2 produced a DNase I footprint virtually identical to that of the undigested protein, but the stability of the protein-VA1 DNA complex was drastically reduced and the in vitro transcriptional activity was eliminated. These results indicate that a chymotrypsin-resistant domain of TFIIIC2 binds to the B-box sequence. We speculate that stable binding requires protease sensitive cooperative interactions between TFIIIC2 DNA-binding domains.
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PMID:A DNA-binding domain of human transcription factor IIIC2. 279 26

The glycoprotein thrombospondin is distributed between the extracellular matrix and the platelet-sequestered pool in the resting state and it undergoes redistribution upon platelet stimulation. It is believed to play a role in matrix structure and in coagulation. We have studied the structural domains of endothelial cell (EC) thrombospondin by use of the serine proteases thrombin, trypsin and chymotrypsin and have characterized the heparin-binding domains of this molecule. For this purpose we used purified thrombospondin synthesized and secreted by bovine aortic endothelial cells grown in the presence of radiolabeled methionine. We find that the susceptibility of EC thrombospondin to proteolysis is five-fold smaller than that of platelet thrombospondin. In the presence of 2 mM Ca ions the molecule is cleaved by 20 U/ml thrombin at a single locus, to yield fragments of 160 kDa and 35 kDa. Trypsin digestion for 5 min at room temperature at an enzyme-to-substrate ratio of 1:20 produces a stable fragment of 140 kDa but not the 30-kDa fragment observed in platelet thrombospondin. Chymotrypsin, under identical conditions to those used for trypsin, cleaves EC thrombospondin into four stable fragments of 160 kDa, 140 kDa, 27 kDa and 18 kDa. Chelation of Ca by EDTA increases susceptibility of the molecule to proteolysis. Under the conditions used a cryptic thrombin-cleavage site, not hitherto observed in platelet thrombospondin, was observed in EC thrombospondin. The location of this site is near a chymotrypsin-susceptible site, which has been observed in the long connecting arm, which is particularly Ca-stabilized. Heparin-binding capacity of EC thrombospondin was observed in at least two separate loci. Both thrombin and chymotrypsin produced small fragments (35 kDa and 27 kDa respectively) which bound to heparin with high affinity, and large fragments (160 kDa for thrombin and 140 kDa for chymotrypsin) which had low affinity. Chelation of Ca substantially decreased the low-affinity binding of the large fragments but not the high-affinity binding of the small fragments. Two-dimensional gel electrophoresis of the chymotryptic heparin-binding fragments shows that each molecule gave rise to a heterogeneous array of fragments of high molecular mass bound by disulfide bonds, indicating that there is a difference in the rate of cleavage between the three subunits of EC thrombospondin. Trypsin, despite its limited degradation, completely eliminated the heparin-binding capacity of both high and low-affinity loci, in contrast to platelet thrombospondin where the high affinity remains intact.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The structure of endothelial cell thrombospondin. Characterization of the heparin-binding domains. 282 10

Chymotrypsin, trypsin, carboxypeptidase A and B, elastase and enterokinase activities were measured in buffer solutions and in human duodenal juice after incubation with wheat bran, cellulose, guar gum, pectin, psyllium and lignin. The different types of dietary fiber led to inhibition of enzymatic activity in most experiments, e.g., lignin could totally ablish the activity of isolated trypsin and chymotrypsin. Only in enterokinase was there no influence. Inhibition depended on incubation time; the effect was proportional to fiber concentration and inversely related to enzyme level. Treatment of fiber with hydrochloric acid (pH 1.5) and heat (95 degrees C) destroyed inhibitory activity in some experiments. The effect of lignin on one enzyme (trypsin) was reduced by the addition of another enzyme (chymotrypsin). It is concluded that dietary fiber could affect digestion by inhibiting proteolytic pancreatic enzymes.
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PMID:Effect of dietary fiber on proteolytic pancreatic enzymes in vitro. 282 29

Di-isopropyl fluorophosphate (DFP) and other organophosphorus inhibitors recruit the catalytic power of their target enzymes: the enzyme catalyzes its own irreversible phosphorylation. The magnitude of the catalytic acceleration can approach the factor by which the enzyme catalyzes its own acylation by natural substrates. The reaction of DFP with five serine proteases [chymotrypsin, elastase, dipeptidyl peptidase IV (DP IV), subtilisin and thermitase] exhibits in all cases the same pH dependence as does enzyme acylation by natural substrates. Chymotrypsin and elastase form a "fast" class of enzymes which react about ten-fold faster than the other three "slow" enzymes. All enzymes show k(HOH)/k(DOD) of about 2 but the proton inventory indicates one-proton character for "slow" enzymes and multiproton character for "fast" enzymes. Enthalpies of activation are about 33 kJ/mol (subtilisin, "slow") and 10 kJ/mol (elastase, "fast"). Entropies of activation are about -120 J.T-1.mol-1 (subtilisin, "slow") and -175J.T-1.s-1 (elastase, "fast"; T = temperature in K).
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PMID:Molecular interactions in intermediate and transition states in the self-stimulated inhibition of enzymes. 284 12

The influence of alpha-chymotrypsin and diazepam on the phasic (mainly direct) and tonic (indirect, probably substance P-mediated) components of intestinal cholinergic contractions, induced by the GABA-A receptor agonist 3-aminopropane sulphonic acid (3-APS), was investigated in the guinea-pig ileum. alpha-Chymotrypsin, at a concentration (20 U/ml) not affecting submaximal Ach (0.1 microM) contractions, preferentially depressed the tonic component of the 3-APS (30 microM)-induced response. A brief exposure (10 or 60 sec) to diazepam (0.1 microM) potentiated both the phasic and the tonic contractions evoked by low (10, 30 microM) 3-APS concentrations. This potentiation was prevented by bicuculline (30 microM), hyoscine (1 microM) and flumazenil (1, 3 microM). These results provide further support for an involvement of a peptide neurotransmitter on GABA-A receptor-mediated cholinergic response in the ileum. The modulation of this response by diazepam is probably exerted through recognition sites resembling the "central type" benzodiazepine receptors.
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PMID:Cholinergic contractions induced by GABA-A receptor activation in the guinea-pig ileum are inhibited by alpha-chymotrypsin and potentiated by diazepam. 285 13

Parathyroid hormone (PTH) -degrading activity was studied using osteoblast-like UMR-106 cells. PTH-degrading activity was assessed by the amount of PTH fragments produced in the medium after exposure of intact human PTH-(1-84) to UMR-106 cells. PTH immunoreactivity recovered in trichloroacetic acid-soluble products of the medium and in fractions eluted from reverse-phase high-performance liquid chromatography (HPLC) was measured by radioimmunoassay using an antibody specific for the mid-region and C-terminus of PTH. In this study, intact UMR-106 cells but not extracellular enzymes cleaved human PTH(1-84) into fragments which were released into the medium (in a time- and temperature-dependent fashion). HPLC analysis of the PTH fragments depicted three immunoreactive peaks (peaks 1, 2 and 3) besides intact PTH, indicating a limited PTH-hydrolyzing activity of the cells. Furthermore, a 1000-fold molar excess of either hPTH-(3-34) or [Nle8,Nle18,Tyr34]hPTH-(3-34)amide inhibited PTH-degrading activity by 63% and 80% of control, respectively, whereas neither calcitonin, vasopressin nor growth hormone suppressed it. Additionally, HPLC analysis of the samples treated with [Nle8,Nle18,Tyr34]hPTH-(3-34)amide showed a reduction of the three peaks, suggesting an involvement of PTH receptor in the production of PTH fragments. This PTH-degrading activity was strongly inhibited by phenylmethylsulfonyl fluoride and chymostatin, but not by soybean trypsin inhibitor, elastatinal or inhibitors of cysteine, aspartic or metalloproteinases, indicating that it is due to a seryl chymotrypsin-like endopeptidase. Chymotrypsin-like activity seems to be solely responsible for PTH-degrading activity in intact UMR-106 cells, since all three PTH fragments were predominantly suppressed in the presence of chymostatin. Further analysis of chymotrypsin-digested products of hPTH-(1-84) eluted from HPLC exhibited five fragments detected by ultraviolet absorbance at 210 nm, three of which were measurable by PTH radioimmunoassay, each corresponding to the three PTH fragments produced by UMR-106 cells. To explore the cleavage sites of PTH further, amino acid analysis of chymotrypsin-cleaved products was performed. The results strongly support the view that the chymotrypsin-like enzyme in UMR-106 cells cleaved the hormone between residues 23-24 and 34-35, to produce, at least, hPTH-(24-84) and -(35-84). Our present study indicates that a chymotrypsin-like endopeptidase is solely responsible for limited hydrolysis of PTH by intact UMR-106 cells.
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PMID:Parathyroid hormone degradation by chymotrypsin-like endopeptidase in the clonal osteogenic UMR-106 cell. 291 1

Tissue plasminogen activator was treated with Sepharose-bound trypsin or chymotrypsin. Trypsin rapidly converted the one-chain activator to the two-chain form. This caused a marked increase in the amidolytic activity, while plasminogen activation initially increased but then decreased again. SDS/polyacrylamide gel electrophoresis in combination with [3H]diisopropylfluorophosphate active-site labeling revealed that after the conversion to the two-chain activator a minor cleavage occurred in the B chain, while the A chain was substantially degraded. Chymotrypsin caused a marked decrease in both amidolytic activity and plasminogen activation. SDS/polyacrylamide gel electrophoresis under reducing conditions revealed that two pairs of new bands had appeared, with Mr or about 50,000/52,000 and 17,000/20,000 respectively. N-terminal sequence analysis identified cleavage sites at peptide bonds 420-421 and 423-424. These bonds are located in a region of the activator which is homologues to the segments of trypsin and chymotrypsin, where autocatalytic cleavages occur during their activations. However, treatment of two-chain activator with chymotrypsin had markedly less effect on plasminogen activation and amidolytic activity. By treatment of samples of chymotrypsin-digested one-chain activator with plasmin, amidolytic activity could be largely restored. Thus, chymotrypsin may, by cleaving bonds 420-421 and 423-424, convert the active one-chain activator into an 'inactive' zymogen, which is again 'activated' by plasmin cleavage.
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PMID:Proteolytically induced variations in the enzymatic properties of tissue plasminogen activator. Activations, inactivations and reactivations. 294 92

Molecular composition of Tetrahymena ciliary dynein has been examined by electron microscopy and gel electrophoresis. SDS-urea gel electrophoresis revealed that Tetrahymena 22S dynein contains three (A alpha, A beta, and A gamma) heavy chains whereas 14S dynein contains only one. The molecular masses of 22S and 14S dynein heavy chains were estimated to be approximately 490 and 460 kD, respectively. Electron microscopy of negatively stained specimens showed 22S dynein has three globular heads and thin stalks, whereas 14S dynein consists of a single head. Chymotrypsin digested each of the three 22S dynein heavy chains into large fragments with different time courses. Sucrose density gradient centrifugation separated the digestion products as two peaks. The one with a larger sedimentation coefficient mainly consisted of two-headed particles having binding ability to doublet microtubules, whereas the other with a smaller sedimentation coefficient consisted of only isolated globular particles. Both fractions had ATPase activities. Thus, one active head of 22S dynein can be isolated by chymotrypsin digestion.
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PMID:Chymotryptic digestion of Tetrahymena 22S dynein. I. Decomposition of three-headed 22S dynein to one- and two-headed particles. 295 81


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