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)

An inhibitory protein for the 20S proteasome (also known as macropain, the multicatalytic proteinase complex and 20S proteinase) has been purified from bovine red blood cells. The inhibitor has an apparent molecular weight of 31,000 on SDS-PAGE and appears to form multimers under nondenaturing conditions. This protein inhibited all three of the putatively distinct catalytic activities of proteasome A (the active form of the proteinase) characterized by the hydrolysis of synthetic peptides such as Z-VLR-MNA, Z-GGL-AMC or Suc-LLVY-AMC and Z-LLE-beta NA. The inhibitor also prevented the hydrolysis of large protein substrates such as casein, lysozyme and bovine serum albumin. Proteasome L (the latent form of the proteinase) does not degrade these large protein substrates, but does hydrolyze the three synthetic peptides at rates similar to those by proteasome A. The inhibitor inhibited only two of these peptidase activities of proteasome L (hydrolysis of Z-GGL-AMC and of Z-LLE-beta NA or Suc-LLVY-AMC); it had no effect on the hydrolysis of Z-VLR-MNA. The inhibitor was specific for inhibition of the proteasome and had no effect on the activity of any other proteinase tested including trypsin, chymotrypsin, papain, subtilisin and both isoforms of calpain. Kinetic analysis indicates that the inhibitor interacted with the proteasome by a mechanism involving tight-binding. Because the proteasome appears to be a key component of the ATP/ubiquitin-dependent pathway of intracellular protein degradation, the inhibitor may represent an important regulatory protein of this pathway.
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PMID:Purification and characterization of a protein inhibitor of the 20S proteasome (macropain). 131 59

The egg jelly-induced acrosome reaction of the sea urchin, Strongylocentrotus intermedius, was inhibited by succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide (Suc-Leu-Leu-Val-Tyr-MCA), but not by Suc-Ala-Ala-Pro-Phe-MCA. The proteases with hydrolytic activity toward the former were purified from sperm extract by DEAE-Sephacel and hydroxylapatite chromatographies, Sephacryl S-300 gel filtration, and heparin-Sepharose CL-6B chromatography. Two types of protease were separated, and the molecular weights were estimated to be 65 and 700 kDa, respectively, by gel filtration. The former was accompanied by hydrolytic activity toward Suc-Ala-Ala-Pro-Phe-MCA, which was not hydrolyzed by the latter. Polyacrylamide gel electrophoresis of 700 kDa protease gave a single protein band under nondenaturing conditions and at least eight bands in the range of 22-33 kDa in the presence of sodium dodecyl sulfate (SDS). The substrate specificity and the inhibitor sensitivity of 700 kDa protease indicate that it contains two types of the activity, one is chymotrypsin-type and the other trypsin-type. The former activity was enhanced by poly-L-lysine or SDS. These properties of 700 kDa protease are similar to those of proteasomes (multicatalytic proteinases) isolated from various eukaryotic sources. We had previously shown that inhibitors of chymotrypsin-like proteases inhibit the increase of intracellular Ca2+ concentration by egg jelly, resulting in the inhibition of the acrosome reaction of St. intermedius (Matsumura and Aketa, Gamete Res 23:255-266, 1989). Bringing these findings together, we suggest that the chymotrypsin-like activity of sperm proteasome participates in the onset of the acrosome reaction of St. intermedius.
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PMID:Proteasome (multicatalytic proteinase) of sea urchin sperm and its possible participation in the acrosome reaction. 187 26

Proteasome, a high molecular weight multicatalytic protease, was purified from the cytosolic fraction of human platelets for the first time. The biochemical properties of the enzyme including substrate specificity, optimal pH and effects of various inhibitors were almost identical with those of other cells. During the purification with a Heparin-Sepharose chromatography, a novel endogenous activator of the protease was identified and was partially purified. The activator enhanced both chymotrypsin or trypsin like activities of the proteasome in a dose related manner and was inactivated by heating at 56 degrees C for 30 min. This newly identified activator may serve as an important regulator or cofactor of intracellular activities of the proteasome.
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PMID:Proteasome and its novel endogeneous activator in human platelets. 206 66

We previously identified a benzyloxycarbonyl(Z)-Leu-Leu-Leu-4-methylcoumaryl-7-amide (ZLLL-MCA) degrading activity in proteasome as a candidate for the regulator of neurite outgrowth. As its counterpart, we purified a protein from bovine brain that specifically inhibits the ZLLL-MCA degrading activity in proteasome. This protein is heat stable and has no effect on the other catalytic activities in proteasome, or on the activities of trypsin, chymotrypsin, or m- and mu-calpains either. The molar ratio of inhibitor-to-proteasome that inhibits 50% of the ZLLL-MCA degrading activity of proteasome is 1:1. The inhibitory mechanism of the inhibitor against proteasome is non-competitive. Finally, the inhibitor was identified as heat-shock protein 90 (HSP90) by partial amino acid sequencing and immunodetection. The results suggest that HSP90 initiates neurite outgrowth through the inhibition of the ZLLL-MCA degrading activity in proteasome.
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PMID:Purification and characterization of an endogenous inhibitor specific to the Z-Leu-Leu-Leu-MCA degrading activity in proteasome and its identification as heat-shock protein 90. 818 90

We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hslVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome beta subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coli expressing heat-shock proteins constitutively and 100-fold in cells expressing HslV and HslU from a high copy plasmid. Although HslV and HslU could be coimmunoprecipitated from cell extracts of both strains with an anti-HslV antibody, these two components were readily separated by various types of chromatography. ATP stimulated peptidase activity up to 150-fold, whereas other nucleoside triphosphates, a nonhydrolyzable ATP analog, ADP, or AMP had no effect. Peptidase activity was blocked by the anti-HslV antibody and by several types of inhibitors of the eukaryotic proteasome (a threonine protease) but not by inhibitors of other classes of proteases. Unlike eukaryotic proteasomes, the HslVU protease lacked tryptic-like and peptidyl-glutamyl-peptidase activities. Electron micrographs reveal ring-shaped particles similar to en face images of the 20S proteasome or the ClpAP protease. Thus, HslV and HslU appear to form a complex in which ATP hydrolysis by HslU is essential for peptide hydrolysis by the proteasome-like component HslV.
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PMID:HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. 865 Jan 74

The antitumor drug aclacinomycin A was previously shown to inhibit the degradation of ubiquitinated proteins in rabbit reticulocyte lysates with an IC50 of 52 microM (Isoe, T., Naito, M., Shirai, A., Hirai, R., and Tsuruo, T.(1992) Biochim. Biophys. Acta 1117, 131-135). We report here that from all the catalytic activities of the 20 S proteasome tested, the chymotrypsin-like activity was the only one affected by the antitumor drug. An important requirement for inhibition of the chymotrypsin-like activity seemed to be the presence of hydrophobic nonpolar residues in positions P1 to P3. Degradation of Z-E(OtBu)AL-pNA and Z-LLL-AMC at pH 7.5 was dramatically (87-98%) inhibited by 50 microM of the drug, while that of Z-GGL-pNA (containing uncharged polar residues in positions P2 and P3) and succinyl-LLVY-AMC (containing an uncharged polar residue in the P1 position) was inhibited only 11 and 24%, respectively. Aclacinomycin A had no effect on cathepsin B, stimulated trypsin, and inhibited chymotrypsin and, to a lesser extent, calpain. The aglycone and sugar moieties of the cytotoxic drug are essential for inhibition. The results presented here support a major role for the chymotrypsin-like activity in the degradation of ubiquitinated proteins. Aclacinomycin A is the first described non-peptidic inhibitor showing discrete selectivity for the chymotrypsin-like activity of the 20 S proteasome.
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PMID:The antitumor drug aclacinomycin A, which inhibits the degradation of ubiquitinated proteins, shows selectivity for the chymotrypsin-like activity of the bovine pituitary 20 S proteasome. 866 10

We purified by fractionation on 10-40% glycerol gradients, 26S proteasomes from normal human spermatozoa. These proteasomes, which participate in the ATP-dependent degradation of ubiquitinated proteins, share a similar sedimentation coefficient to those purified from other human tissues. Fluorogenic peptide assays reveal they have chymotrypsin, trypsin and peptidyl-glutamyl-like peptide hydrolysing activities; the chymotrypsin activity is ablated by the specific 26S proteasome inhibitor MG132. Confirmation that these large proteases are 26S proteasomes is provided by detection of the 20S proteasome subunits HC2, XAPC7, RN3 and Z and regulatory ATPases MSS1, TBP1, SUG1 and SUG2 by Western analyses with monoclonal antisera. These antigens are found only in the gradient fractions enriched in proteolytic activities. We have also shown that, although mature spermatozoa from mice have considerably reduced amounts of a ubiquitin-conjugating enzyme (E2) and ubiquitin-protein conjugates in comparison with less mature germ cells, they retain relatively high values of 26S proteasome activity. This suggests that proteasomes may have further roles to play in normal sperm physiology.
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PMID:Purification and characterization of 26S proteasomes from human and mouse spermatozoa. 946 50

For an effective CD8+ cytotoxic T cell response to occur during infection, MHC class I molecules must be loaded with antigenic peptides in the endoplasmic reticulum. The cytosolic factor responsible for peptide generation is believed to be the proteasome, with the TAP heterodimer mediating peptide transport into the endoplasmic reticulum. However, the rate-determining step(s) in this intracellular pathway of Ag presentation is currently unresolved. The availability of a specific and irreversible proteasome inhibitor called lactacystin has enabled us to determine the amount of proteasomes required for the peptide loading of MHC class I molecules in four cell types. In the absence of the IFN-gamma-inducible proteasome subunits LMP2 and LMP7, the trypsin-like (but not the chymotrypsin-like) activity of the proteasome is directly related to MHC class I peptide loading. However, IFN-gamma stimulation or assimilation of catalytic LMP2 and LMP7 subunits into proteasomes causes both chymotrypsin- and trypsin-like activities of the proteasome to become limiting for the loading of class I molecules. Our data suggest that upon full IFN-gamma stimulation, peptide supply by the proteasome is the limiting step in the assembly of MHC class I polypeptides. This mechanism may enable the cell to prevent competition between novel Ags and the pool of endogenous proteins for binding to MHC class I molecules.
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PMID:Proteasome activity limits the assembly of MHC class I molecules after IFN-gamma stimulation. 955 Mar 86

The induction of apoptosis in thymocytes by the glucocorticoid dexamethasone was used as a model system to investigate whether there are changes in 20 S and 26 S proteasome activities during apoptosis. We observed that thymocytes contain high concentrations of proteasomes and that following treatment with dexamethasone, cell extracts showed a decrease in proteasome chymotrypsin-like activity which correlated with the degree of apoptosis observed. The decrease in chymotrypsin-like activity of 20 S and 26S proteasomes was still apparent after these complexes had been partially purified from apoptotic thymocyte extracts and was therefore not due to competition resulting from a general increase in protein turnover. The trypsin-like and peptidylglutamylpeptide hydrolase activities of proteasome complexes were also observed to decrease during apoptosis, but these decreases were reversed by the inhibition of apoptosis by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone. However, the chymotrypsin-like activity of proteasomes decreased further in the presence of the apoptosis inhibitor. Val-Ala-Asp-fluoromethylketone was found to inhibit the chymotrypsin- and trypsin-like activity of 26 S proteasomes in vitro. The decrease in proteasome activities in apoptosis did not appear to be due to a decrease in the concentration of total cellular proteasomes. Thus, the early decreases in 20 S and 26 S proteasome activities during apoptosis appear to be due to a down-regulation of their proteolytic activities and not to a decrease in their protein concentration. These data suggest that proteasomes may be responsible, in thymocytes, for the turnover of a protein that functions as a positive regulator of apoptosis.
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PMID:Proteasome activities decrease during dexamethasone-induced apoptosis of thymocytes. 960 Oct 58

The 20S proteasome purified from animal cells has various latent peptidase activities. Fatty acids such as linoleic, linolenic and oleic acids strongly activate both the chymotrypsin-type and peptidylglutamylpeptide (PGP) hydrolase-type activities, but have been reported to have little activation or inhibition of the trypsin-type activity. We show here that an increase of the fatty acid concentration produces activation of chymotrypsin-type and PGP hydrolase-type in a biphasic fashion: no effect until the threshold concentration and then a sharp activation. In contrast, the trypsin-type activity was markedly inhibited at low concentrations of fatty acid, slightly activated at higher concentrations, and inhibited again at even higher concentrations. The inhibition was removed when the concentration of fatty acid was reduced by dilution after pre-incubation with the fatty acid. As a result, the activation pattern became biphasic, which was identical to that of chymotrypsin-type and PGP hydrolase-type activities. These results suggest that in the chymotrypsin-type and PGP hydrolase-type peptidase fatty acids bind first to a class of sites without direct effect on the peptidase activity, but after saturation of this class it permits more fatty acid to bind to another class of sites involved in the activation. In the trypsin-type peptidase an additional class of fatty acid binding sites is uniquely present, which is involved in the enzyme inhibition. The dilution procedure described above removes the fatty acid molecules bound to the inhibition sites, but not the fatty acid molecules bound to the activation sites; this results in the fatty acid activation profile indistinguishable from that of the chymotrypsin- and PGP hydrolase-type peptidases.
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PMID:Irreversible potent activation and reversible inhibition of trypsin-like activity of 20S proteasome purified from Xenopus oocytes by fatty acid. 961 17


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