Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.22.32 (bromelain)
 1,025 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel total enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives was accomplished in low-water content systems at a preparative scale. alpha-Chymotrypsin, papain, thermolysin and bromelain adsorbed on Celite were used as catalysts. Organic solvents such as acetonitrile and ethyl acetate with small amounts of buffer added or at specific water activity were used as reaction media. Simple readily available amino acid ester derivatives were used as starting building blocks. This feature allowed the possibility of using the products in one step directly as acyl-donor ester, without any chemical or enzymatic modification, in the next enzymatic coupling. The optimal strategy for the synthesis of the enkephalin derivatives was different depending on the carboxy terminal group. The preparation of the carboxy-terminal amide derivatives (R-Tyr-Gly-Gly-Phe-Leu[Met]-NH2) was achieved via 4 + 1 fragment condensation catalyzed by alpha-chymotrypsin. The carboxy-terminal ethyl ester derivatives (R-Tyr-Gly-Gly-Phe-Leu[Met]-OEt) were obtained via 2 + 3 condensation catalyzed by bromelain, a quite unusual protease for peptide synthesis but more effective than papain in this coupling. Both syntheses were carried out in four enzymatic steps and one or two chemical deprotection steps routinely used in peptide synthesis. The overall yields of pentapeptide derivatives were between 40-54% of pure product.
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PMID:Enzymatic peptide synthesis in low water content systems: preparative enzymatic synthesis of [Leu]- and [Met]-enkephalin derivatives. 760 86

The ligand-filled 32-kDa fragment of the porcine estradiol receptor extending from His267 to the C-terminal Ile595 was purified to homogeneity by adsorption to mAb 13H2. The native protein was exposed at 4 degrees C to a panel of proteases: thermolysin, subtilisin, pronase, elastase, ficin, bromelain, endopeptidase Lys-C, both in the dimer and the monomer state, and chymotrypsin at pH 8.2 only. The digests were analysed by SDS/PAGE/Western blotting for Coomassie staining and immunostaining. Peptides were sequenced from blots. The majority of cleavage sites in upper domain E (8 out of 11) amassed in the Leu296-Leu310 stretch. Cleavage at Leu319 was seen with subtilisin and at Tyr328 with chymotrypsin. Susceptability to enzymic proteolysis was also pronounced in Thr465-Glu470 at the center of domain E. Three peptides, 13 kDa with thermolysin, beginning at Leu337, 6 kDa and, in low yield, 5 kDa with endopeptidase Lys-C beginning at Asp473 resp. Cys417 were only obtained from the monomer substrate. The various digests featured either 27-23-kDa peptides or mixtures of 17-13-kDa and 12-7-kDa peptides separable by SDS/PAGE. All peptides with N-termini between Leu297 and Ser329 reacted with mAb 13H2. The digests showed high peaks of bound estradiol in the dimer position of 32-kDa fragment controls on density gradient centrifugation at pH 7.4. However, the property of proton-driven dissociation was only preserved in the pronase, elastase and chymotrypsin digests with peptides extending beyond the His547-ArgLeuHis550 motif. The preservation of the estradiol-binding niche in the tightly complexed peptides of domain E was also demonstrated by refilling after steroid removal. The sites exposed to proteolytic enzymes and the epitope for 13H2 attachment are in good agreement with surface probability plots.
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PMID:Surface mapping of the ligand-filled C-terminal half of the porcine estradiol receptor by restricted proteolysis. 763 63

Polymorphonuclear neutrophils (PMN) can be primed for enhanced release of reactive oxygen species (ROS) by exposure to cytokines and biological response modifiers. ROS are considered to possess tumoricidal activity. The polyenzyme preparation Wobenzym (WE) contains pancreatin, papain, bromelain trypsin and chymotrypsin and is used in adjuvant tumor therapy. We investigated killing of WE-exposed PMN against tumor cells and analyzed WE influence on ROS production in a chemiluminescence assay in PMN in vitro and in vivo. Depending on dose WE stimulates the cytotoxic capacity of PMN in vitro against tumor cells (50 micrograms/ml:p < 0.01). Exposure of PMN to Wobenzym caused a time-dependent significant (p < 0.02) increase in release of ROS. Similarly, oral administration of Wobenzym to healthy volunteers (n = 28) resulted in significant increases (p < 0.01) in ROS production, depending on dose (peak with 20 tablets) and time (peak 4 hours after Wobenzym administration). In contrast, ROS production was not elevated in the PMN of healthy volunteers receiving placebo (n = 8) or no treatment (n = 16). These findings point to an immunomodulatory capacity of WE in adjuvant tumor therapy.
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PMID:Stimulation of reactive oxygen species production and cytotoxicity in human neutrophils in vitro and after oral administration of a polyenzyme preparation. 766 74

Protein inhibitors of cysteine proteinases possessing unusual properties have been found in soya (Glycine max) seeds. One of the inhibitor forms has also been detected in Bowman-Birk inhibitor preparations (both commercial and purified by affinity chromatography on chymotrypsin-Sepharose ones). A peculiarity of the inhibitors is that they irreversibly lose their activity in the presence of reducing agents; therefore their effects are normally unobserved under standard conditions of cysteine proteinase inhibitor assays. Soybean inhibitors are represented by two forms with pI of 5.9 and 3.2. The molecular mass of the inhibitor whose pI is equal to 5.8 is about 14 kDa. Both inhibitors suppress the activity of papain, ficin and bromelain.
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PMID:[Cysteine proteinase inhibitors from soy seeds]. 769 28

Pigeon-pea seed extracts have been analyzed for the protease inhibitors using a new, sensitive and simple method for visualization of electrophoretically separated protease inhibitors. The visualization involves equilibrating the gel successively in the protease assay buffer, protease solution, rinsing the gel in protease assay buffer, and exposing it to an exposed, undeveloped X-ray film. Gelatin on the film in places corresponding to the inhibitor bands remains unhydrolyzed. By this method the pigeon-pea seed extract was found to contain nine trypsin and at least seven chymotrypsin inhibitors but no papain or bromelain inhibitors.
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PMID:Detection of electrophoretically separated protease inhibitors using X-ray film. 806 16

A fully active form of hydroxylamine oxidoreductase from Nitrosomonas has been purified with high recovery and shown by reverse-phase high performance liquid chromatography and N-terminal analysis to contain only a 63-kDa subunit and to lack the 11-kDa protein previously thought to be a second subunit. Based on the previously published values of molecular weight in solution, hydroxylamine oxidoreductase probably has an alpha 2 or alpha 3 oligomeric structure. The enzyme was digested separately with trypsin and chymotrypsin and peptides which contained covalently bound heme were separated by high performance liquid chromatography and their amino acid sequences determined. A total of seven heme-containing peptides of unique amino acid sequence were obtained. Six of these heme-containing peptides clearly contained a single c-heme with optical properties indistinguishable from the tryptic heme-containing peptide from horse heart cytochrome c. No noncovalently bound heme was observed. One of the seven heme-containing peptides (T7) was unusual in that it released 2 amino acid residues after each cycle of the Edman degradation due to a nondisulfide cross-link and exhibited a Soret band that was broadened in both the ferric form at neutral pH and the pyridine ferrohemochrome. Subdigestion of peptide T7 with nonspecific proteases (Pronase, bromelain, or pepsin) resulted in the isolation of two smaller heme-containing peptides of unique sequences. One of these was spectrally identical to the other c-heme containing peptides, whereas the second was still apparently cross-linked, again releasing 2 amino acid residues after each Edman cycle. This second peptide possessed a heme-like chromophore with absorption bands (Soret, alpha and beta) red-shifted about 6 nm relative to the spectrum of c-heme-containing peptides. Thus, hydroxylamine oxidoreductase contains a total of eight covalently bound hemes per subunit, seven of which are c-hemes. The eighth, which is attached to a cross-linked peptide, is probably the unusual P460 heme which is unique to hydroxylamine oxidoreductase and thought to be at the active site.
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PMID:Hydroxylamine oxidoreductase from Nitrosomonas europaea is a multimer of an octa-heme subunit. 832 41

Young adult Sprague-Dawley rats were partially hepatectomized (two-thirds organ removal) and administered a basal diet supplemented with various animal- and plant-derived enzymes (trypsin, alpha-chymotrypsin, pepsin, lipase, alpha-amylase, malt diastase, ficin and bromelain) over a post-operative period of up to 10 days. Porcine or bovine dialyzed and lyophilized crystalline trypsin products containing 2400-3200 NF u/mg in addition to enteric-coated tablets with trypsin to chymotrypsin in a ratio of 6:1, were tested at supplementary levels of up to 4980 u/g ration. With the weight of tissue regenerated or the liver increment as indicator, trypsin in excess of 1000-1200 u/g ration proved inhibitory. This effect did not extend to alpha-chymotrypsin (levels of up to 4000 u/g diet) and the remaining 6 enzyme products specified above, nor to the s.c. injection of trypsin daily at 12,860 u/rat for the 1st 7 days. The last route promoted little change in increment with soy bean trypsin inhibitor (8.0 mg/rat daily for days 1 to 9). When a portion of the group fed a trypsin supplement of 2000 u/g was injected with phenobarbital i.p. at 80 mg/kg daily on each of the last 3 days, the resulting liver increment rose to the control range. As with lysine and arginine, acids of pertinence in tryptic proteolysis, no significant change was elicited by feeding a diet supplemented with peptone from tryptic digestion of casein. The enzyme-containing diets fed to sham-operated rats over a similar interval, did not affect the wet- or dry-liver weight per 100 g body weight. Microsomal parameters as total protein, cytochrome P-450 and the enzymes, aminopyrine demethylase and benzo[a]pyrene hydroxylase of livers from the partially hepatectomized or sham-operated rats fed trypsin and the other enzyme diets, presented no significant changes in the respective levels. The possible action of dietary trypsin in conjunction with inhibitors and growth factors controlling liver regeneration is discussed.
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PMID:Liver regeneration in trypsin-fed partially hepatectomized rats. 843 34

The plasma kininogens, high (HK) and low (LK) molecular weight kininogens, are the parent proteins for bradykinin, a potent vasoactive peptide that locally influences vascular biology. Binding of both HK and LK to the endovascular wall contributes to bradykinin delivery. Recently, we found one preparation of LK (LKd) which had reduced inhibition of biotin-HK binding to endothelium. The functional defect in LKd was not merely due to bradykinin loss because two preparations of bradykinin-free LK blocked biotin-HK binding. However, using two different particular monoclonal antibodies to bradykinin, LKd, but no other preparation of LK, had its epitope to bradykinin exposed on non-reduced samples on immunoblot. These data suggested that LKd had an altered conformation which exposed the amino terminal arginine of bradykinin to antigenic detection. The altered conformation of LKd allowed it to be more susceptible to trypsin proteolysis. On circular dichroism, the percentage of alpha-helix was significantly increased, indicating an alteration in the protein. This alteration in LKd was not due to a loss of molecular mass of the protein. On laser desorption mass spectroscopy, the molecular mass of LKd was similar to the other preparations of LK. Investigations were performed to ascertain the mechanism by which LKd had altered ability to bind to cells. LKd was found to be proteolyzed by an unknown protease at the beginning of domain 2 between threonine119 and alanine120. Reduction of functional LK with dithiothreitol to expose its bradykinin epitope did not produce the LKd defect. Proteolysis of functional LK with plasma kallikrein, elastase followed by plasma kallikrein, chymotrypsin, or bromelain also did not produce the defect seen in LKd. These combined data indicated that LK maintains a particular conformation that allows the protein to orient itself such that it can bind to endothelial cells. Proteolysis in the surface exposed region between domains 1 and 2 probably allows for the protein to unfold and contributes to its lost ability to bind to endothelial cells.
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PMID:Conformational changes in low molecular weight kininogen alters its ability to bind to endothelial cells. 856 Apr 18

Bromelain inhibitor VI from pineapple stem (BI-VI) is a unique double-chain inhibitor with an 11-residue light chain and a 41-residue heavy chain by disulfide bonds and inhibits the cysteine proteinase bromelain competitively. The structure of BI-VI in aqueous solution was determined using nuclear magnetic resonance spectroscopy and simulated annealing-based calculations. Its three-dimensional structure was shown to be composed of two distinct domains, each of which is formed by a three-stranded antiparallel beta-sheet. Unexpectedly, BI-VI was found to share a similar folding and disulfide bond connectivities not with cystatin superfamily inhibitors which inhibit the same cysteine proteinases but with the Bowman-Birk trypsin/chymotrypsin inhibitor from soybean (BBI-I). BBI-I is a 71-residue inhibitor which has two independent inhibitory sites toward the serine proteinases trypsin and chymotrypsin. These structural similarities with BBI-I suggest that they have evolved from a common ancestor and differentiated in function during a course of molecular evolution.
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PMID:Solution structure of bromelain inhibitor IV from pineapple stem: structural similarity with Bowman-Birk trypsin/chymotrypsin inhibitor from soybean. 861 27

This work represents our first step toward fulfilling the need to discover a model system for experimental investigations of temporal oscillations, pattern formations, and other non-linearity related dynamic behavior in immobilized-enzyme-membrane systems. In this paper, the regions in the parameter space where self-sustained pH oscillations can be induced were determined via extensive numerical simulation for five enzyme-membrane systems involving four proteolytic enzymes: alpha-chymotrypsin, trypsin, bromelain, and ficin. From this study, we concluded that, even with current enzyme-immobilization techniques, the possibility of experimentally observing self-sustained pH oscillations in a flat membrane immobilized with alpha-chymotrypsin and using N-acetyl-L-tryptophan ethyl ester as a substrate is high. Under suitable conditions and with extra efforts, self-sustained oscillations may also occur in membrane systems immobilized with ficin, trypsin and bromelain.
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PMID:Self-sustained pH oscillations in immobilized proteolytic enzyme systems. 886 29


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