Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.22.25 (chymopapain)
 430 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock.
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PMID:Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode, Heligmosomoides polygyrus, in vitro. 1572 70

Current control of gastrointestinal nematodes relies primarily on the use of synthetic drugs and encounters serious problems of resistance. Oral administration of plant cysteine proteinases, known to be capable of damaging nematode cuticles, has recently been recommended to overcome these problems. This prompted us to examine if plant cysteine proteinases like the four papaya proteinases papain, caricain, chymopapain, and glycine endopeptidase that have been investigated here can survive acidic pH conditions and pepsin degradation. The four papaya proteinases have been found to undergo, at low pH, a conformational transition that instantaneously converts their native forms into molten globules that are quite unstable and rapidly degraded by pepsin. As shown by activity measurements, the denatured state of these proteinases which finally results from acid treatment is completely irreversible. It is concluded that cysteine proteinases from plant origin may require to be protected against both acid denaturation and proteolysis to be effective in the gut after oral administration.
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PMID:Structural characterization of the papaya cysteine proteinases at low pH. 1643 27

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

The mechanical wounding impact on the Carica papaya latex protein pattern was investigated by analyzing three latexes. A first one commercially available, a second harvested from unripe but fully grown fruits, both obtained from regularly tapped fruits. A third one was collected from similar fruits but wounded for the first time. The results demonstrated both quantitative and qualitative changes in the protein content and in the enzymatic activity. Repeated wounding results in either, accumulation or activation (or both of them) of papain, chymopapain and caricain. Furthermore, new cysteine protease activity was found to transiently accumulate in the latex collected from newly wounded fruits. The possible implication of this enzymatic material in the papaya cysteine endopeptidases pro-forms activation is discussed.
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PMID:Effects of mechanical wounding on Carica papaya cysteine endopeptidases accumulation and activity. 1658 Jul 24

Protein crystals isolated from potato tubers were found to consist of a proteinase inhibitor active against the cysteine proteinases papain, chymopapain, and ficin. The molecular weight as determined by gel filtration at pH 4.3 or by gel electrophoresis in the presence of dodecylsulfate was 80 kilodaltons. When the inhibitor was evaluated at pH 8.4 in a linear concentration (4-30% polyacrylamide) under nondenaturing conditions, it appeared as two bands of approximately 320 to 350 kilodaltons indicating that the inhibitor forms tetrameric aggregates in neutral or weakly alkaline media, while the monomeric form predominates under acidic conditions. Gel filtration in the presence of varying amounts of papain suggested that the monomer combines with four papain molecules. The inhibitor contains no cystine.
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PMID:Naturally occurring protein crystals in the potato : inhibitor of papain, chymopapain, and ficin. 1666 31

We studied the effect of three electrolytes (LiCl, Na(2)SO(4), GuHCl) on the unfolding reaction of chymopapain, a two-domain protein belonging in the papain family of cysteine proteinases. Due to methodological reasons, these studies were carried out at pH 1.5 where the protein unfolds following biphasic kinetics. We have observed the presence of two different effects of electrolyte concentration on the unfolding reactions. At low ionic strength, the ionic atmosphere brought about an increase in reaction rates, regardless of the type of ions being present; this effect is attributed to a general "electrostatic screening" of charge-charge interactions in the macromolecule. At high ionic strength, each electrolyte exerted a distinctively different effect: both rate constants were largely increased by GuHCl (a well-known protein denaturant), but only slightly by LiCl; in contrast, Na(2)SO(4) (a good precipitant) decreased the value of both unfolding rates. These ion-specific (Hofmeister) effects were further used to estimate changes in accessible surface area (DeltaASA) upon formation of the transition states (TS) for unfolding. Results obtained with LiCl and Na(2)SO(4), which we analyzed by means of a parameterization derived from published solubility data of amino acid derivatives, are consistent with DeltaASA increments (for each phase) of about 8.0% of the total theoretical DeltaASA for complete unfolding of the chymopapain molecule. Results in the presence of GuHCl, which were analyzed by using a previous parameterization of protein unfolding data, gave larger DeltaASAs of activation, equivalent to 13 and 16% of the total unfolding DeltaASA.
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PMID:Hofmeister effects in protein unfolding kinetics: estimation of changes in surface area upon formation of the transition state. 1683 56

The Proton Inventory (PI) method has been applied in the hydrolysis of synthetic substrates by papain, chymopapain and stem bromelain, comparing also their corresponding pH-(k(cat)/K(m)) profiles, and it was found: (a) k(cat)/K(m)=k(1), and thus K(S)=k(2)/k(1) is a dynamic equilibrium constant, (b) bowed-downward PI for k(cat)/K(m) exhibiting large inverse SIE, and (c) linear PI exhibiting large normal SIE for K(S), k(2) and k(3). A novel finding of this work is that the association of substrates onto all three studied cysteine proteinases proceeds via a stepwise pathway, in contrast to purely concerted pathways found previously for both acylation and deacylation. A hydrogen bond, which seems more likely to be developed across a pK(a)-value close to 4.00, connecting [see text] (papain/chymopapain or bromelain numbering), constitutes another novelty of this work.
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PMID:The catalytic mode of cysteine proteinases of papain (C1) family. 1699 46

Latex of all Vasconcellea species analyzed to date exhibits higher proteolytic amidase activities, generally attributed to cysteine proteinases, than the latex of Carica papaya. In the present study, we show that this higher activity is correlated with a higher concentration of enzymes in the latex of Vasconcellea fruits, but in addition also results from the presence of other cysteine proteinases or isoforms. In contrast to the cysteine proteinases present in papaya latex, which have been extensively studied, very little is known about the cysteine proteinases of Vasconcellea spp. In this investigation, several cDNA sequences coding for cysteine proteinases in Vasconcellea x heilbornii and Vasconcellea stipulata were determined using primers based on conserved sequences. In silico translation showed that they hold the characteristic features of all known papain-class cysteine proteinases, and a phylogenetic analysis revealed the existence of several papain and chymopapain homologues in these species. Ion-exchange chromatography and gel filtration procedures were applied on latex of V. x heilbornii in order to characterize its cysteine proteinases at the protein level. Five major protein fractions (VXH-I-VXH-V) revealing very high amidase activities (between 7.5 and 23.3 nkat x mg protein(-1)) were isolated. After further purification, three of them were N-terminally sequenced. The observed microheterogeneity in the N-terminal and cDNA sequences reveals the presence of several distinct cysteine proteinase isoforms in the latex of Vasconcellea spp.
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PMID:Purification and characterization of the cysteine proteinases in the latex of Vasconcellea spp. 1722 50

Thiol proteases are industrially significant proteins with catalytic efficiency. The effect of low, medium and high molecular-weight poly (ethylene glycol) (PEG- 400, 6000 and 20000) on the stability of thiol proteases (papain, bromelain and chymopapain) has been studied by activity measurements using synthetic substrate. Structural studies performed on papain by far UV circular dichroism spectroscopic measurements indicate that there is loss in secondary structure of the protein in presence of increasing concentration of PEGs. Intrinsic fluorescence measurements lead us to conclude that tryptophan residues of protein encounter non-polar microenvironment in presence of PEG solvent while acrylamide quenching shows greater accessibility of tryptophan residues of papain in presence of PEGs. Extrinsic fluorescence measurements lead us to conclude that PEGs bind to the hydrophobic sites on the protein and thus destabilize it. Thermal denaturation studies show that melting temperature of papain is decreased in presence of PEGs. Possible mechanism of destabilization is discussed next. The results imply that caution must be exercised in the use of PEGs with thiol proteases or hydrophobic proteins in general, for different industrial applications, even at room temperature.
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PMID:Effect of polyethylene glycols on the function and structure of thiol proteases. 1750 90


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