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:3.4.21.4 (trypsin)
42,187 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 multicatalytic proteinase complex (MPC), also referred to as proteasome, is a large molecular mass intracellular particle (approximately 700 kDa), which exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPH), all sensitive to inhibition by 3,4-dichloroisocoumarin (DCI). The presence of a component resistant to inhibition by DCI with an apparent preference toward bonds on the carboxyl side of branched-chain amino acids has also been recently established. Peptide aldehydes and peptide alpha-keto esters containing a hydrophobic residue in the P1 position have been tested as potential inhibitors of the chymotrypsin-like activity. Three peptide aldehydes (benzyloxycarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-CHO), N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO), and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO) were found to be slow-binding reversible inhibitors with Ki values of 0.46, 5.7, and 33 microM, respectively. The simplest kinetic model for inhibition is consistent with a mechanism involving a slow and reversible association of the enzyme with the inhibitor to form a EI complex. The aldehyde inhibitors also inhibited the trypsin-like and PGPH activities of the complex albeit with much higher Ki values than those for chymotrypsin-like activity. Z-LLF-CHO, the most selective of the three aldehydes, did not inhibit the PGPH activity at concentrations of up to 200 microM and inhibited the trypsin-like activity with a Ki approximately 2 orders of magnitude higher than that for the chymotrypsin-like activity. The activity of the DCI-resistant component was not affected by Z-LLF-CHO.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of the chymotrypsin-like activity of the pituitary multicatalytic proteinase complex. 135 35

The proteasome (multicatalytic protease complex), a high molecular weight protein complex, has been purified from spinach leaves by successive chromatography on DEAE-cellulose, Bio-Gel A-1.5m, DEAE-TOYOPEARL 650C, and DEAE-5PW. The molecular mass was estimated to be 850 kDa by gel filtration. Polyacrylamide gel electrophoresis of the proteasome gave a single protein band under nondenaturing conditions and at least 10 bands in the range of 21-32 kDa in the presence of sodium dodecyl sulfate. By electron microscopy after negative staining with uranyl acetate, the proteasome from spinach appeared as symmetrical ring-shaped particles. The substrate specificity of proteasomes indicates that they contain at least three types of activity, namely, chymotrypsin-like, Staphylococcus aureus V8 protease-like, and trypsin-like activities. The former two activities were enhanced by poly-L-lysine or sodium dodecyl sulfate. Moreover, we examined the immunological reactivities of proteasomes from various eukaryotes. As a result, cross-immunoreactivities of some subunits were observed. These properties of the proteasome are similar to those of proteasomes isolated from various other eukaryotic sources.
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PMID:Purification and initial characterization of the proteasome from the higher plant Spinacia oleracea. 140 Apr 79

Chemical modification of the proteasome with N-ethylmaleimide (NEM) was performed for the purpose of identifying amino acid residues that play a role in the enzyme's proteolytic function. Modification of the proteasome with NEM specifically and irreversibly suppressed one of the three peptidase activities of the enzyme, viz., the "trypsin-like" activity. Leupeptin, a reversible competitive inhibitor of this activity, protected the activity from NEM inactivation, suggesting that NEM modifies a residue in the leupeptin binding site. Comparisons of enzyme samples labeled with [14C]NEM either in the presence or in the absence of leupeptin allowed the identification of a proteasome subunit containing an NEM-modified, leupeptin-protected cysteinyl residue. The leupeptin protection experiments suggest that residues of this subunit contribute to the active site responsible for the proteasome's trypsin-like activity. This subunit was purified by reverse-phase high-performance liquid chromatography. Peptide mapping and N-terminal amino acid sequencing were employed to acquire information about the primary structure of the subunit, including the sequence surrounding the leupeptin-protected cysteinyl residue. The sequencing data suggest that this proteasome subunit is evolutionarily related to other proteasome subunits that have been sequenced, which show no homology to other known proteases. The assignment of a catalytic function to a member of the proteasome family supports the hypothesis that proteasome subunits represent a structurally and possibly mechanistically novel group of proteases.
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PMID:Identification and localization of a cysteinyl residue critical for the trypsin-like catalytic activity of the proteasome. 151 Sep 24

A protease involved in oocyte maturation of a starfish, Asterina pectinifera, was explored. Trypsin-like and chymotrypsin-like activities of the 650-kDa protease in oocyte extract were revealed to increase more than twice under the influence of 1-methyladenine before germinal vesicle breakdown (GVBD) during maturation. The inhibitory potencies of leupeptin and its five analogs against the chymotrypsin-like activity, but not the trypsin-like activity, of this protease was well in accord with those against GVBD (Takagi Sawada et al. (1989). Dev. Biol. 133, 609-612). These results indicate that the chymotrypsin-like activity of the 650-kDa protease (most probably 20 S proteasome) plays a key role in starfish oocyte maturation.
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PMID:Participation of 650-kDa protease (20 S proteasome) in starfish oocyte maturation. 155 83

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

Subunit HsN3 of the human proteasome is a beta-type subunit homologous to PRE4 from yeast, X1 beta from Xenopus and RN3 from the rat. Using electron microscopy, the binding sites of a monoclonal antibody with specificity for subunit HsN3 have been located in the two juxtaposed inner rings of the human proteasome. Subunit HsN3 was present in two copies, one in each ring, in accordance with our concept of two identical halves making up the complete human proteasome. The subunit is involved in the trypsin-like as well as the peptidylglutamyl-peptide cleavage activities.
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PMID:The human proteasome subunit HsN3 is located in the inner rings of the complex dimer. 753 29

The multicatalytic proteinase complex (MPC), also called the proteasome, is a ubiquitous particle (19S) that is required for life. It is found in the cytoplasm and nucleus of all eukaryotic cells where it degrades selected cytosolic and nuclear proteins. It forms the proteolytic core of the 26S complex that represents the final step in the ubiquitin-dependent pathway of proteolysis. The MPC expresses at least five distinct proteolytic activities. Three activities preferring cleavages on the carboxyl side of neutral amino acids were described: an activity cleaving after branched chain residues, termed branched chain amino acid preferring, that is a major factor in the degradation of proteins, an activity preferring cleavages after bulky hydrophobic residues designated chymotrypsin-like, and an activity cleaving between small neutral amino acids. Activities cleaving after basic (trypsin-like) and acidic residues (peptidylglutamyl peptide-hydrolyzing) have also been described. The expression of multiple proteolytic activities with diverse specificities may provide a functional advantage that allows efficient hydrolysis of target proteins.
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PMID:Catalytic components of the bovine pituitary multicatalytic proteinase complex (proteasome). 769 27

We have isolated and characterized a cDNA encoding the mouse proteasome subunit MC3 and identified four proteasome subtypes which differ in their peptide-hydrolyzing and polypeptide-cleavage properties. Immunoblotting data show that the 25-kD MC3 subunit is a constitutive proteasome subunit which exists in several isoforms. In addition, by immunoprecipitation of proteasomes with AbMC3, a subset of enzyme complexes could be recognized which differ in their relative subunit composition from the bulk of proteasomes. Using DEAE-column chromatography we identified three different proteasome subtypes in sol-80 mouse liver extracts and, by Trition X-100 extraction, a distinct membrane-bound subtype. The four proteasome subtypes are shown to differ in their trypsin- and chymotrypsin-like hydrolyzing activities as well as in their ability to cleave a 25mer polypeptide substrate derived from the MCMV IE pp89. Our data indicate that the enzymatic properties observed for the total proteasome population may be the summary of cleavage properties of different types of proteasome complexes.
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PMID:Characterization of mouse proteasome subunit MC3 and identification of proteasome subtypes with different cleavage characteristics. Proteasome subunits, proteasome subpopulations. 769 31


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