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PubMed1974

The structure and mechanism of stem bromelain. Evaluation of the homogeneity of purified stem bromelain, determination of the molecular weight and kinetic analysis of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-L-phenylalanyl-L-serine methyl ester.

1. Purified stem bromelain (EC 3.4.22.4) was eluted from Sephadex G-100 as a single peak. The specific activity across the elution peak was approximately constant towards p-nitrophenyl hippurate but increased with elution volume with N(2)-benzoyl-l-arginine ethyl ester as substrate. 2. The apparent molecular weight, determined by elution analysis on Sephadex G-100, is 22500+/-1500, an anomalously low value. 3. Purified stem bromelain was eluted from CM-cellulose CM-32 as a single peak and behaved as a single species during column electrophoresis on Sephadex G-100. 4. Purified stem bromelain migrates as a single band during polyacrylamide-gel electrophoresis under a wide variety of conditions. 5. The molecular weight determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate is 28500+/-1000. 6. Sedimentation-velocity and equilibrium-ultracentrifugation experiments, under a variety of conditions, indicate that bromelain is an apparently homogeneous single peptide chain of mol.wt. 28400+/-1400. 7. The N-terminal amino acid composition is 0.64+/-0.04mol of valine and 0.36+/-0.04mol of alanine per mol of enzyme of mol.wt. 28500. (The amino acid recovery of the cyanate N-terminal amino acid analysis was standardized by inclusion of carbamoyl-norleucine at the cyclization stage.) 8. The pH-dependence of the Michaelis parameters of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester was determined. 9. The magnitude and pH-dependence of the Michaelis parameters have been interpreted in terms of the mechanism of the enzyme. 10. The enzyme is able to bind N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester relatively strongly but seems unable to make use of the binding energy to promote catalysis.

Amino Acid Sequence↗
PubMed1974

Kinetics of the hydrolysis of N-benzoyl-L-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses.

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S(0)]/v(i) (initial substrate concn./initial velocity) versus [S(0)] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S(0)]/v(i) versus [S(0)] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S(0)]/v(i) versus [S(0)] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25 degrees C is characterized by K(m) (1) (the dissociation constant of ES)=1.22+/-0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57x10(-2)+/-0.32x10(-2)s(-1), K(a) (2) (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38+/-0.06m, and k' (the rate constant for the breakdown of SES to E+P+S)=0.45+/-0.04s(-1). 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester and of alpha-N-benzoyl-l-arginine amide; K(m) (1) and k for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine amide hydrolysis and K(as) and k' for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of k(cat.) and K(m) for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (K(m)=52+/-4mm, k(cat.)=2.80+/-0.1s(-1) at pH7.0, I=0.1, 25.0 degrees C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain.

Binding Sites↗
PubMed2005

Susceptibility of the interchain peptide of a bromelain inhibitor precursor to the target proteases bromelain, chymotrypsin, and trypsin.

Bromein, a cysteine proteinase inhibitor from pineapple stem, is a unique double-chain inhibitor. The 27.5-kDa precursor protein is processed by the removal of three interchain, two interdomain, and two terminal-flanking peptides, thus resulting in the release of mature isoinhibitors of approximately 6 kDa. To characterize the processing of the interchain peptide Thr15-Ser-Ser-Ser-Asp, we expressed a single-chain precursor with this peptide and monitored proteolytic cleavage by the target proteinase bromelain. By peptide sequencing and mass spectrometric analysis, the initial cleavage was found to occur in vitro between the light-chain and interchain peptides; subsequent trimming formed the terminal-ragged peptides Thr15-Lys60, Ser17-Lys60, Ser18-Lys60, and Asp19-Lys60. However, bromelain did not show any cleavage activity between the interchain and heavy-chain peptides. We also discovered that cleavage between the light-chain and interchain peptides is essential for the single-chain inhibitor to exhibit full inhibitory activity. Notably, the incompletely processed intermediates showed higher inhibitory activity than either the native bromein or the single-chain precursor. Bromein is also known to weakly inhibit the serine proteinases chymotrypsin and trypsin; however, a recombinant single-chain inhibitor with the interchain peptide was no longer able to inhibit these serine proteinases.

Bromelains↗
PubMed1983

Effects in mice of rat bromelain-treated RBC and lipopolysaccharide on autoantibody production against bromelain-treated isologous RBC.

Autoantibody production against mouse bromelain-treated (brom) red blood cells (RBC) was significantly increased in mice injected with rat brom RBC. These autoantibodies were not adsorbed by rat brom RBC in serological assays and did not lyse rat brom RBC in plaque-forming cell (PFC) assays using mixtures of rat brom RBC and mouse brom RBC as targets. These data suggest that the increased response induced by rat brom RBC is not due to the presence of common, or similar, antigens on the two types of RBC. The spleens of mice injected with lipopolysaccharide (LPS), or with both LPS and rat brom RBC, had a markedly increased number of PFC lysing mouse brom RBC. About 20% of the PFC induced by LPS and rat brom RBC also lysed rat brom RBC. The autoimmune response was not increased in mice injected twice with rat brom RBC and the secondary response induced by two injections of LPS was lower than that induced by one injection of LPS. However, injection of LPS after an initial challenge with rat brom RBC induced an autoimmune response similar in size to that induced by LPS alone. The decreased secondary response against mouse brom RBC following a second injection of rat brom RBC was associated with decreased production of antibodies of various specificities as detected in a reverse PFC assay. These results do not support the hypothesis that the poor secondary responses against mouse brom RBC following a second injection of rat brom RBC are due to the exhaustive differentiation of autoimmune B cells as part of a fail-safe mechanism to prevent autoimmunity.

Animals↗
PubMed2005

Proteinase activity and stability of natural bromelain preparations.

Bromelain is a complex mixture of proteinases typically derived from pineapple stem. Similar proteinases are also present in pineapple fruit. Beneficial therapeutic effects of bromelain have been suggested or proven in several human inflammatory diseases and animal models of inflammation, including arthritis and inflammatory bowel disease. However, it is not clear how each of the proteinases within bromelain contributes to its anti-inflammatory effects in vivo. Previous in vivo studies using bromelain have been limited by the lack of assays to control for potential differences in the composition and proteolytic activity of this naturally derived proteinase mixture. In this study, we present model substrate assays and assays for cleavage of bromelain-sensitive cell surface molecules can be used to assess the activity of constituent proteinases within bromelain without the need for biochemical separation of individual components. Commercially available chemical and nutraceutical preparations of bromelain contain predominately stem bromelain. In contrast, the proteinase activity of pineapple fruit reflects its composition of fruit bromelain>ananain approximately stem bromelain. Concentrated bromelain solutions (>50 mg/ml) are more resistant to spontaneous inactivation of their proteolytic activity than are dilute solutions, with the proteinase stability in the order of stem bromelain>fruit bromelain approximately ananain. The proteolytic activity of concentrated bromelain solutions remains relatively stable for at least 1 week at room temperature, with minimal inactivation by multiple freeze-thaw cycles or exposure to the digestive enzyme trypsin. The relative stability of concentrated versus dilute bromelain solutions to inactivation under physiologically relevant conditions suggests that delivery of bromelain as a concentrated bolus would be the preferred method to maximize its proteolytic activity in vivo.

Bromelains↗
PubMed2004

Proteolytic activity and immunogenicity of oral bromelain within the gastrointestinal tract of mice.

Bromelain is a mixture of proteinases derived from pineapple stem that is marketed by health food stores as a "digestive aid". A number of studies suggest that bromelain may also have anti-inflammatory activity in vivo, including an anecdotal report describing potential efficacy in inflammatory bowel disease. We and others have previously shown that proteolytically active bromelain removes certain cell surface molecules and affects leukocyte migration, activation, and production of cytokines and inflammatory mediators in vitro. The purpose of this study was to determine whether ingested bromelain retains proteolytic activity within the murine gastrointestinal tract in vivo. The proteolytic activity of bromelain was determined in vitro using model substrates or immunofluorescence assays after administration of various doses and formulations orally to mice. Immune responses against bromelain were detected by enzyme immunoassays. When formulated in antacid, oral bromelain retained substantial proteolytic activity throughout the gastrointestinal tract. Bromelain concentrations within the colon were dependent on both dose and formulation and were sufficient to remove bromelain-sensitive molecules from both leukocytes and colon epithelial cells. Peak activity in the stool was observed 4 h after oral dosing. Although anti-bromelain IgG was detected in both serum and stool after long-term oral therapy, these antibodies did not prevent bromelain proteolytic activity within the gastrointestinal tract. These studies demonstrate that bromelain enzymes can remain intact and proteolytically active within the murine gastrointestinal tract. They provide further support for the hypothesis that oral bromelain may potentially modify inflammation within the gastrointestinal tract via local proteolytic activity within the colonic microenvironment.

Administration, Oral↗
PubMedDate not supplied

Effects of oral bromelain administration on the impaired immunocytotoxicity of mononuclear cells from mammary tumor patients.

The protease bromelain from pineapple was suggested for adjuvant therapy of malignant diseases. We studied immunological effects of an orally applied bromelain drug on 16 breast cancer patients in comparison with healthy donors. Bromelain was applied for 10 days with a daily dose of 3000 F.I.P. units and the immunocytotoxicity of blood monocytes and lymphocytes against the leukemic K562 and MDA-MB-231 mammary carcinoma target cells was determined in vitro. In addition, the expression of the cell surface markers CD44, CD16, CD11a and CD62L on lymphocytes and the secretion of IL-2 and IL-1beta from monocytes was measured. Patients leukocytes expressed lower bMAK-, MAK-, NK- and LAK-cell activities, compared with those from healthy donors. Orally applied bromelain increased the reduced bMAK- and MAK-cell activity of patients monocytes about 2-fold. When the patients were classified on the basis of bromelain effects on the monocytic cytotoxicity into bromelain responders and nonresponders, about 40% of the patients responded to bromelain with an increase of cytotoxicity from 7.8% to 54% (bMAK-cell activity) and from 16% to 47% (MAK-cell activity). Bromelain was less effective on the higher cytotoxicity of monocytes from healthy donors, but stimulated the secretion of IL-1beta from monocytes. In contrast, patient monocytes secreted no detectable IL-1beta, before, during and after bromelain treatment. Bromelain had no effects on the impaired patients NK- and LAK-cell activity, but reduced the LAK-cell activity of healthy donors. No IL-2 was found in the supernatants of untreated and treated lymphocytes from healthy donors. Bromelain reduced the expression of CD44, but weakly increased CD11a and CD62L expression on patient lymphocytes, whereas CD16 remained unchanged. In vitro bromelain application to lymphocytes had similar effects, with greater reduction rates of CD44 and CD16 expression. As to coagulation parameters in plasma of healthy donors, the activated partial thromboplastin time was increased from 38 to 46 sec, leaving prothrombin time and plasminogen unchanged. These data suggest, that orally applied bromelain stimulates the deficient monocytic cytotoxicity of mammary tumor patients, which may partially explain its proposed antitumor activity.

Administration, Oral↗
PubMed2002

Bromelain treatment alters leukocyte expression of cell surface molecules involved in cellular adhesion and activation.

Bromelain is a natural proteinase preparation derived from pineapple stem that is marketed for oral use as a digestive aid and as an antiinflammatory agent. Bromelain treatment in vitro has been previously shown to selectively remove certain cell surface molecules that may affect lymphocyte migration and activation. This study reports the effects of bromelain on a broad range of cell surface molecules and on lymphocytes, monocytes, and granulocytes under physiologically relevant conditions. In vitro bromelain treatment of leukocytes in whole blood proteolytically altered 14 of 59 leukocyte markers studied. Constitutively expressed bromelain-sensitive molecules included CD7, CD8alpha, CD14, CD16, CD21, CD41, CD42a, CD44, CD45RA, CD48, CD57, CD62L, CD128a, and CD128b. The proteolytic effect of bromelain increased as the concentration of plasma decreased, with EC50 ranging from >1000 microg/ml for 100% plasma to approximately 1 microg/ml in the absence of plasma, indicating the presence of an inhibitor of bromelain in plasma. alpha2-macroglobulin purified from plasma mimicked the inhibitory effect of whole plasma on bromelain activity. If proteolysis is required for the antiinflammatory actions of oral bromelain, these data suggest that the required concentrations are more likely to be achieved locally in the gastrointestinal tract or in other tissue sites where the plasma concentration is low, rather than in the bloodstream. The cell surface molecules altered by bromelain are involved in leukocyte homing and cellular adhesion and activation. Thus bromelain could potentially exert an antiinflammatory effect by multiple mechanisms, including alterations in leukocyte migration and activation.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2006

Oral immunogenicity of the plant proteinase bromelain.

Bromelain is a natural mixture of proteolytic enzymes derived from pineapple stem that has been shown to have anti-inflammatory activity when administered orally. Although most proteins given orally without adjuvant (e.g., food) result in tolerance, we previously reported that long-term oral exposure to bromelain stimulated the development of high serum anti-bromelain antibody titers. The purpose of these studies was to further investigate the mechanisms responsible for the immunogenicity of oral bromelain. Results showed that repeated exposure was required for development of anti-bromelain antibodies, with strong antibody responses in all mice that received at least 12 doses of bromelain either orally or intragastrically over 3-6 weeks. Proteolytic activity was required for strong oral immunogenicity in the absence of conventional adjuvant, with strong serum antibody responses generated against proteolytically active bromelain and trypsin, but not against ovalbumin, lysozyme, or inactivated bromelain. Significantly higher anti-bromelain antibody titers were seen in IL-10-deficient versus wild-type mice, suggesting that simultaneous treatments that decrease IL-10 activity may further enhance systemic antibody responses following oral exposure. The antibodies generated did not affect the proteolytic activity of bromelain. The data demonstrate that proteolytically active antigens such as bromelain can stimulate both systemic and mucosal immune responses following repeated oral exposure. Further studies of the mechanisms involved in generation of immune responses following oral exposure to proteolytically active antigens can lead to a better understanding of mechanisms of oral tolerance and to the development of novel adjuvants for oral vaccines.

Adjuvants, Immunologic↗
PubMed2001

Bromelain modulates T cell and B cell immune responses in vitro and in vivo.

The ability to modulate immune responses is a major aim of many vaccine and immunotherapeutic development programs. Bromelain, a mixture of cysteine proteases, modulates immunological responses and has been proposed to be of clinical use. However, the identity of the immune cells affected by bromelain and the specific cellular functions that are altered remain poorly understood. To address these shortcomings in our knowledge, we have used both in vitro and in vivo immunological assays to study the effects of bromelain. We found that bromelain enhanced T cell receptor (TCR) and anti-CD28-mediated T cell proliferation in splenocyte cultures by increasing the costimulatory activity of accessory cell populations. However, despite increased T cell proliferation, bromelain concomitantly decreased IL-2 production in splenocyte cultures. Additionally, bromelain did not affect TCR and CD28-induced proliferation of highly purified CD4+ T cells, but did inhibit IL-2 production by these cells. In vivo, bromelain enhanced T-cell-dependent, Ag-specific, B cell antibody responses. Again, bromelain induced a concomitant decrease in splenic IL-2 mRNA accumulation in immunized mice. Together, these data show that bromelain can simultaneously enhance and inhibit T cell responses in vitro and in vivo via a stimulatory action on accessory cells and a direct inhibitory action on T cells. This work provides important insights into the immunomodulatory activity of bromelain and has important implications for the use of exogenous cysteine proteases as vaccine adjuvants or immunomodulatory agents.

Animals↗
PubMed1998

Bromelain protects piglets from diarrhoea caused by oral challenge with K88 positive enterotoxigenic Escherichia coli.

BACKGROUND: K88 positive enterotoxigenic Escherichia coli (K88+ ETEC) is an important cause of diarrhoea in young piglets. K88+ ETEC pathogenesis relies on attachment to specific glycoprotein receptors located on the intestinal mucosa. Proteolytic treatment of these receptors in vitro and in vivo prevents attachment of K88+ ETEC to piglet small intestines and may be of clinical use to prevent K88+ ETEC pathogenesis. AIMS: To determine whether bromelain, a proteolytic extract obtained from pineapple stems, would protect piglets against K88+ ETEC diarrhoea and to confirm and extend earlier findings on the effects of bromelain on K88+ ETEC receptors in vivo. METHODS: Bromelain (0, 12.5, or 125 mg) was orally administered to just weaned piglets for 10 days. One day following commencement of bromelain treatment, piglets were challenged with K88+ ETEC (5 x 10(10) K88ac:0149) for seven days. Intestinal contents from unchallenged piglets were obtained via an intestinal fistula, and tested for their ability to bind K88+ ETEC before and after bromelain treatment. RESULTS: Both doses of bromelain were successful in reducing the incidence of K88+ ETEC diarrhoea and protected piglets from life threatening disease. Bromelain treated pigs also had significantly increased weight gain compared with untreated pigs. Bromelain only temporarily inhibited K88+ ETEC receptor activity, with receptor activity being regenerated 30 hours following treatment, consistent with the regeneration of new enterocytes. CONCLUSION: Results show that bromelain can temporarily inactivate ETEC receptors in vivo and protect against ETEC induced diarrhoea. Bromelain may therefore be an effective prophylaxis against ETEC infection.

Animals↗
PubMedDate not supplied

Bromelain proteases reduce human platelet aggregation in vitro, adhesion to bovine endothelial cells and thrombus formation in rat vessels in vivo.

The thiol protease, bromelain, an extract from pineapple stem, was suggested to have antithrombotic and anticoagulant activities in vivo. We studied the effects of bromelain on cell size distribution of isolated human platelets in vitro by Coulter Counter measurements. Preincubation of platelets with bromelain (10 micrograms/mL) completely prevented the thrombin (0.2 U/mL) induced platelet aggregation. Papain was less active in preventing platelet aggregation. In vitro, bromelain (0.1 microgram/mL) reduced the adhesion of bound, thrombin stimulated, fluorescent labeled platelets to bovine aorta endothelial cells. In addition, preincubation of platelets with bromelain, prior to thrombin, activation, reduced the platelet adhesion to the endothelial cells to the low binding value of unstimulated platelets. On the basis of mass concentrations, the proteases papain and trypsin were as effective as bromelain. Using a laser thrombosis model, the in vivo effects of orally and intraveneously applied bromelain on thrombus formation in rat mesenteric vessels were studied. Bromelain, orally applied at 60 mg/kg body weight, inhibited the thrombus formation in a time dependent manner, the maximum being after 2 hours in 11% of arterioles and 6% of venoles. Intravenous application at 30 mg/kg was slightly more active in reducing thrombus formation in arterioles (13%) and venoles (5%), suggesting that orally applied bromelain is biologically active. These results may help to explain some of the clinical effects observed after bromelain treatment in patients with thrombosis and related diseases.

Adult↗