Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.22.6 (chymopapain)
 407 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A rabbit model system is described. It allows accurate measurement of the dose-dependent loss of glycosaminoglycan from the nucleus pulposus of lumbar intervertebral discs after injection of a proteinase. At the dose equivalent to that of chymopapain used in human chemonucleolysis, two human serine proteinases, cathepsin G and chymotrypsin, were as effective as chymopapain in removing up to 80% of the glycosaminoglycan from the disc. A cysteine proteinase, cathepsin B released no more than 45% of glycosaminoglycan. X-ray films clearly showed narrowing of the disc space when 30-40% of glycosaminoglycan was removed. The degradation of the nucleus pulposus was seen histologically as loss of toluidine blue metachromasia.
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PMID:Quantitative assessment of human proteinases as agents for chemonucleolysis. 304 17

1. 2-(N'-Acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide [compound (III)] and 2-(acetamido)ethyl 2'-pyridyl disulphide [compound (IV)] were synthesized by acylation of the common intermediate, 2-aminoethyl 2'-pyridyl disulphide, to provide examples of chromogenic thiol-specific substrate-derived two-protonic-state electrophilic probe reagents. These two reagents, together with n-propyl 2-pyridyl disulphide [compound (II)], provide structural variation in the non-pyridyl part of the molecule from a simple hydrocarbon side chain in compound (II) to a P1-P2 amide bond in compound (IV) and further to both a P1-P2 amide bond and a hydrophobic side chain (of phenylalanine) at P2 as a potential occupant of S2 subsites. 2. These disulphides were used as reactivity probes to investigate specificity and binding-site-catalytic-site signalling in a number of cysteine proteinases by determining (a) the reactivity at pH 6.0 at 25 degrees C at I 0.1 of compound (III) (a close analogue of a good papain substrate) towards 2-mercaptoethanol, benzimidazol-2-ylmethanethiol [compound (V), as a minimal catalytic-site model], chymopapains B1-B3, chymopapain A, papaya proteinase omega, actinidin, cathepsin B and papain, (b) the effect of changing the structure of the probe as indicated above on the reactivities of compound (V) and of the last five of these enzymes, and (c) the forms of pH-dependence of the reactivities of papain and actinidin towards compound (III). 3. The kinetic data suggest that reagents of the type investigated may be sensitive probes of molecular recognition features in this family of enzymes and are capable not only of detecting differences in binding ability of the various enzymes but also of identifying enzyme-ligand contacts that provide for binding-site-catalytic-site signalling mechanisms. 4. The particular value of this class of probe appears to derive from the possibility of activating the 2-mercaptopyridine leaving group not only by formal protonation, as was recognized previously [see Brocklehurst (1982) Methods Enzymol. 87C, 427-469], but also by hydrogen-bonding to the pyridyl nitrogen atom when the appropriate geometry in the catalytic site is provided by enzyme-ligand contacts involving the non-pyridyl part of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Substrate-derived two-protonic-state electrophiles as sensitive kinetic specificity probes for cysteine proteinases. Activation of 2-pyridyl disulphides by hydrogen-bonding. 366 11

The release of 5-fluorouracil from polymer-based conjugates can be influenced by the type of linkages used to bind the drug to the polymer carrier. The use of specific oligopeptide sequences designed to be biorecognizable by intracellular enzymes is a promising approach for increasing the site-specific release of 5-fluorouracil from polymer-based conjugates. In this study, we investigated the biorecognizability of specific oligopeptide sequences linking 5-fluorouracil to a water-soluble copolymer carrier based on N-(2-hydroxypropyl) methacrylamide by human cathepsin B (EC 3.4.22.1), cathepsin H(EC 3.4.22.6), and a homogenate of the human colon adenocarcinoma cell line SW 480. The cathepsins were chosen based on the hypothesis that they were two principal lysosomal enzymes responsible for the release of 5-fluorouracil from these conjugates. Our results support this hypothesis; however, these two enzymes may not be the only lysosomal enzymes responsible for the release kinetics observed. While the results for cathepsin B corresponded well to our hypothesis, the cleavage via cathepsin H was lower than predicted, suggesting the presence of additional lysosomal enzymes with catalytic activity toward these 5-fluorouracil derivatives.
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PMID:Intracellularly biorecognizable derivatives of 5-fluorouracil. Implications for site-specific delivery in the human condition. 878 16

Cysteine proteinases are widely distributed among living organisms. According to the most recent classifications (Rawlings and Barrett, 1993, 1994), they can be subdivided on the basis of sequence homology into 14 or even 20 different families, the most important being the papain and the calpain families. The papain-like cysteine proteinases are the most abundant among the cysteine proteinases. The family consists of papain and related plant proteinases such as chymopapain, caricain, bromelain, actinidin, ficin, and aleurain, and the lysosomal cathepsins B, H, L, S, C and K. Most of these enzymes are relatively small proteins with Mr values in the range 20000-35000 (reviewed in Brocklehurst et al., 1987; Polgar, 1989; Rawlings and Barrett, 1994; Berti and Storer, 1995), with the exception of cathepsin C, which is an oligomeric enzyme with Mr approximately 200000 (Metrione et al., 1970; Dolenc et al., 1995). A number of cysteine proteinases are located within lysosomes. Four of them, cathepsins B, C, H and L, are ubiquitous in lysosomes of animals, whereas cathepsin S has a more restricted localisation (Barrett and Kirschke, 1981; Kirschke and Wiederanders, 1994). The enzymes, except cathepsin C, are endopeptidases (reviewed in Kirschke et al., 1995), although cathepsin B was found also to be a dipeptidyl carboxypeptidase (Aronson and Barrett, 1978) and cathepsin H also an aminopeptidase (Koga et al., 1992). Cathepsin C is a dipeptidyl aminopeptidase, but at higher pH it exhibits also dipeptidyl transferase activity (reviewed in Kirschke et al., 1995). Among the lysosomal cysteine proteinases, cathepsin L was found to be the most active in degradation of protein substrates, such as collagen, elastin and azocasein (Barrett and Kirschke, 1981; Maciewicz et al., 1987; Mason et al., 1989), arid cathepsin B the most abundant (Kirschke and Barrett, 1981). All the enzymes are optimally active at slightly acidic pH, although their pH optima for degradation of synthetic substrates vary from 5.5 for cathepsin L to 6.8 for cathepsin H (reviewed in Kirschke et al., 1995). Several other lysosomal cysteine proteinases, such as cathepsins N, T and K, are known, although their properties are less well characterised (reviewed in Kirschke et al., 1995). In particular cathepsin K has attracted recent interest (Bromme et al., 1996; Shi et al., 1995; Bossard et al., 1996; Drake et al., 1996) and was found to be expressed specifically in osteoclasts (Drake et al., 1996) with properties similar to cathepsin L (Bossard et al., 1996).
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PMID:Structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors. 916 64

Cystatin CsC, a cysteine proteinase inhibitor from chestnut (Castanea sativa) seeds, has been purified and characterized. Its full-length cDNA clone was isolated from an immature chestnut cotyledon library. The inhibitor was expressed in Escherichia coli and purified from bacterial extracts. Identity of both seed and recombinant cystatin was confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis, two-dimensional electrophoresis and N-terminal sequencing. CsC has a molecular mass of 11,275 Da and pI of 6.9. Its amino acid sequence includes all three motifs that are thought to be essential for inhibitory activity, and shows significant identity to other phytocystatins, especially that of cowpea (70%). Recombinant CsC inhibited papain (Ki 29 nM), ficin (Ki 65 nM), chymopapain (Ki 366 nM), and cathepsin B (Ki 473 nM). By contrast with most cystatins, it was also effective towards trypsin (Ki 3489 nM). CsC is active against digestive proteinases from the insect Tribolium castaneum and the mite Dermatophagoides farinae, two important agricultural pests. Its effects on the cysteine proteinase activity of two closely related mite species revealed the high specificity of the chestnut cystatin.
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PMID:A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests. 986 28

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X=all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXabeta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1=Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.
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PMID:High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays. 1570 70

An extracellular cysteine protease inhibitor (ECPI-2) was purified to homogeneity from the culture filtrate of Chlorella sp. 4533 by the combination of various column chromatographies. The molecular mass of the inhibitor was estimated to be 340 kDa by SDS-PAGE. The inhibitor was extremely heat-stable under acidic or neutral condition. ECPI-2 exhibited an inhibitory activity against the proteolytic activity of papain, ficin, or chymopapain, but not against stem bromelain or cathepsin B. The inhibitor showed no inhibitory activity against trypsin, alpha-chymotrypsin or thermolysin. ECPI-2 contains 33.6% carbohydrate residues by weight and inhibits papain at a molar ratio of 1:2. The proteolysis of the inhibitor by trypsin or alpha-chymotrypsin was apparent, but the inhibitory activity of ECPI-2 was unaffected by these enzymes. The alpha-chymotrypsin hydrolysis product from ECPI-2 was further separated into six fractions by gel filtration. From these results, it is suggested that ECPI-2 has several reactive sites for papain.
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PMID:Purification and characterization of extracellular cysteine protease inhibitor, ECPI-2, from Chlorella sp. 1656 14