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

Ca(2+)-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial-Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or alpha(1)-casein were hydrolysed with a maximum rate at 30 degrees C, pH7.5, and with 5mm-CaCl(2), but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, alpha-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry15, 2150-2158]. The Ca(2+)-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca(2+)-activated neutral proteinase.
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PMID:Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle. 53 1

Benzofuroxan reacts with the catalytic-site thiol group of cathepsin B (EC 3.4.22.1) to produce stoichiometric amount of the chromophoric reduction product, o-benzoquinone dioxime. In a study of the pH-dependence of the kinetics of this reaction, most data were collected for the bovine spleen enzyme, but the more limited data collected for the rat liver enzyme were closely similar both in the magnitude of the values of the second-order rate constants (k) and in the shape of the pH-k profile. In acidic and weakly alkaline media, the reaction is faster than the reactions of benzofuroxan with some other cysteine proteinases. For example, in the pH region around 5-6, the reaction of cathepsin B is about 10 times faster than that of papain, 15 times faster than that of stem bromelain and 6 times faster than that of ficin. The pH-dependence of k for the reaction of cathepsin B with benzofuroxan was determined in the pH range 2.7-8.3. In marked contrast with the analogous reactions of papain, ficin and stem bromelain [reported by Shipton & Brocklehurst (1977) Biochem. J. 167, 799-810], the pH-k profile for the cathepsin B reaction contains a sigmoidal component with pKa 5.2 in which k increases with decrease in pH. This modulation of the reactivity of the catalytic-site -S-/-ImH+ ion-pair state of cathepsin B (produced by protonic dissociation from -SH/-ImH+ with pKa approx. 3) towards a small, rigid, electrophilic reagent, in a reaction that appears to involve both components of the ion-pair for efficient reaction, suggests that the state of ionization of a group associated with a molecular pKa of approx. 5 may control ion-pair geometry. This might account for the remarkable finding [reported by Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] that, although the ion-pair appears to be generated in cathepsin B as the pH is increased across pKa 3.4, catalytic competence is not generated until the pH is increased across pKa 5-6.
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PMID:Chemical evidence for the pH-dependent control of ion-pair geometry in cathepsin B. Benzofuroxan as a reactivity probe sensitive to differences in the mutual disposition of the thiolate and imidazolium components of cysteine proteinase catalytic sites. 380 Sep 26

Cysteine protease inhibitors that specifically reacted with several cysteine proteases were found in KSCN extract of human melanoma tissue. From 30 gm of the tissue, approximately 593.5 U inhibitor was obtained. The inhibitors were adsorbed on a papain-Sepharose column and could be eluted with 10 mmol/L phosphate buffer, pH 6.0, containing NaCl or KCl, or with 20 mmol/L acetate buffer, pH 4.0, containing KSCN. They revealed a strong inhibitory activity for cysteine proteases such as ficin, papain, and cathepsin B, but did not react with cysteine protease bromelain or serine protease trypsin. No immunologic relationship was confirmed between the inhibitor and other well-known plasma inhibitors such as alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, antithrombin III, C1-in-activator, and alpha 2-plasmin inhibitor. With Sephadex G-100, two main peaks of molecular weight 40,000 and 10,000 were detected in the KSCN extract of the human melanoma tissue. However, the inhibitors revealed three molecular weights of 10,000, 25,000, and 80,000 when estimated by Sephadex G-100 gel filtration after papain-Sepharose affinity chromatography. On the other hand, the molecular weights of the inhibitors changed to two peaks of 25,000 and 10,000 on rechromatography with a papain-Sepharose column.
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PMID:Cysteine protease inhibitors isolated from human malignant melanoma tissue. 393 99

A new proteinase inhibitor has recently been found in human serum or plasma which specifically inhibits cysteine proteinases such as ficin, papain, bromelain and cathepsin B. However, serum contains alpha 2-macroglobulin which also inhibits these cysteine proteinases and, consequently, interferes with the assay of the new alpha-cysteine proteinase inhibitor. Therefore, assay of the inhibitor in serum has not been established previously. In the present method, the alpha 2-macroglobulin is inactivated by preincubating the serum in methylamine solution at 55 degrees C, while the alpha-cysteine proteinase inhibitor retains its activity. The inhibitory power against cysteine proteinases is found to be due mainly to this protein in human serum. This inhibitor is also found in mammals such as cows, pigs and rats. Vitamin E deficient rats show a very high inhibitor level. Therefore, the present method will enable us to investigate the relation between diseases and the activity of the alpha-cysteine proteinase inhibitor. Also, this method is simple and inexpensive. The necessary amount of serum is only 10 microliter.
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PMID:Assay of alpha-cysteine proteinase inhibitor in serum or plasma. 617 74

The papain inhibitor from human spleen was purified by extraction in isotonic sucrose, acetone fractionation, papain-Sepharose affinity chromatography and gel filtration on Sephadex G-50. The purified inhibitor was fractionated by electrofocusing into four major isoelectric variants with pI values of 4.7, 5.0, 6.0 and 6.5. These variants can be classified into two groups: the acidic type, comprising the variants with pI 4.7 and 5.0, and the neutral type, comprising the variants with pI 6.0 and 6.5. The following properties distinguish the two types: 1. Immunological properties: antibodies raised against either of the neutral variants precipitated both of these, but not the acidic variants. The antiserum against the human epidermal cysteineproteinase inhibitor precipitated the acidic variants, but not the neutral variants. 2. Molecular size: two-dimensional electrophoresis of the purified inhibitor gave molecular weights of 11400 for the acidic variants and 12000 for the neutral variants. The pI 6.0 variant contained two compounds with molecular weights of 12000 and 12800. 3. Enzyme spectrum: human cathepsin B was inhibited by the acidic type, while the neutral type was a poor inhibitor. Both types inhibited cathepsin H, papain, ficin and bromelain, although the inhibition of bromelain did not exceed 70%. Human cathepsin D, bovine trypsin and chymotrypsin and porcine elastase were not inhibited by either type.
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PMID:Human spleen cysteineproteinase inhibitor. Purification, fractionation into isoelectric variants and some properties of the variants. 618 75

A thiol protease inhibitor (TPI) was found in culture media of human malignant melanoma cells (Bowes) at 1.5 to 2.3 units/day/flask (full sheet, 75 sq cm). This amount well exceeded that for cultured nonmalignant cells (human fetal lung fibroblasts). In the intracellular region of the melanoma cells, TPI activity was localized mainly in the cytosol fraction. The difference in specific activities between the intracellular and extracellular TPI and the TPI accumulation in the culture media indicated that cultured melanoma cells release TPI. Partial purification and characterization of the TPI by column chromatography using Sephadex G-150, papain-Sepharose, and Sephadex G-50, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed two distinct TPIs with molecular weights of 56,000 and 9,800 to 10,800. The latter (main) TPI had a high specificity for thiol proteases and was heat stable (60 degrees for 60 min), like previously reported normal human TPIs. The inhibitor, however, differed from normal human TPIs in that it had a lower molecular weight than any normal TPI, was unable to inhibit bromelain, and exhibited a mosaic pattern; namely, the low-molecular-weight TPI resembled liver-type TPI but the pH stability curve resembled serum-type TPI. The thiol protease, cathepsin B, was not detected in culture media of this human melanoma cell line.
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PMID:A thiol protease inhibitor released from cultured human malignant melanoma cells. 637 66

An acidic protein of human saliva, which we named SAP-1 previously, is now shown to be an inhibitor of several cysteine proteinases. The protein inhibited papain and ficin strongly, and stem bromelain and bovine cathepsin C partially. However, it did not inhibit either porcine cathepsin B or clostripain. The mode of the inhibition of papain was found to be non-competitive. The name cystatin S has been proposed for this salivary protein in view of the similarities in activity and structure to other cysteine proteinase inhibitors such as chicken egg-white cystatin and human cystatins A, B, and C. The cystatin S antigen was detected immunohistochemically in the serous cells of human parotid and submaxillary glands.
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PMID:Cystatin S: a cysteine proteinase inhibitor of human saliva. 639

Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated.
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PMID:Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment. 654 32

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

This study investigated whether protease treatment ameliorates the progressive course of chronic failure in the rat model of subtotal nephrectomy. Fourteen male Wistar rats underwent 5/6 nephrectomy, and were randomized into a control group (C, n = 7) given 2 ml of 0.9% NaCl intraperitoneally (i.p.) daily, and a study group (P, n = 7) treated with 12 mg Phlogenzym (combination of trypsin, bromelain, and rutosid) in 2 ml saline i.p. daily. After 6 weeks treatment, the Phlogenzym group showed lower proteinuria (C: 19.6 +/- 9.1 vs. 10.2 +/- 6.2 mg/24 h, p < 0.05). Endogenous creatinine clearance was higher (C: 192.3 +/- 99.4, P: 300.5 +/- 47.9 microliters/min per 100 g, p < 0.05), while plasma creatinine was decreased (C: 106.7 +/- 33.9, P: 76.0 +/- 6.3 mumol/l, p < 0.01). Blood urea nitrogen levels did not change, although urea clearance tended to a higher level in the protease-treated rats. Decreased renal formation of cytokines was reflected by a lower urinary excretion ratio of transforming growth factor (TGF)-beta/ creatinine (C: 0.363 +/- 0.183, P: 0.232 +/- 0.085 ng TGF-beta/mg creatinine, p < 0.05). Renal morphology revealed less infiltration of mononuclear cells and an amelioration of interstitial fibrosis as expressed by the volume index of the cortical region (C: 17.17 +/- 1.43; P: 12.3 +/- 0.5%, p < 0.001). In addition, the activities of lysosomal proteinases (cathepsin B, L + B, and H), which are decreased in the remnant kidney model of chronic renal failure, were significantly higher in the enzyme-treated group both in isolated glomeruli and proximal tubules. The body and kidney weight tended to be lower, probably due to a catabolic action of the enzymes. In summary, we provide evidence that protease treatment may be beneficial in a nonimmune mediated renal disease. Phlogenzym ameliorated the course of chronic renal failure in the rat model of subtotal nephrectomy and retarded the development of tubulointerstitial fibrosis. Decreased cytokine formation in the remnant kidney is supposed to play a key role.
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PMID:Effects of protease therapy in the remnant kidney model of progressive renal failure. 938 36


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