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PubMed1996

Hybrids of chicken cystatin with human kininogen domain 2 sequences exhibit novel inhibition of calpain, improved inhibition of actinidin and impaired inhibition of papain, cathepsin L and cathepsin B.

Chicken cystatin and human kininogen domain 2 are members of the cystatin superfamily of protein-type cysteine proteinase inhibitors. They show structural and functional similarities, but only human kininogen domain 2 inhibits calpain. Using recombinant chicken cystatin as a scaffold for hybrid cassette analysis, the known reactive-site regions (N-terminus, first hairpin loop and second hairpin loop) were substituted by the corresponding sequences of human kininogen domain 2 in a single and combined manner. Seven hybrids were expressed, purified to homogeneity, characterized protein-chemically, and their inhibition of papain, actinidin, human cathepsin B, human cathepsin L and calpain (80-kDa subunit of rabbit skeletal muscle calpain II and porcine erthrocyte calpain 1) was determined. Strong but temporary inhibition of calpain by chicken cystatin hybrids carrying the N-terminus alone (variant sc1-KD2) or the N-terminus together with the first hairpin loop (variant sc1/2-KD2) was observed; hybrids of the second hairpin loop (sc3-KD2, sc1/3-KD2, sc2/3-KD2, sc1/2/3-KD2) were less strong calpain inhibitors. These data indicate that the inhibiton of calpain by human kininogen domain 2 requires the correct conformation and combination of several contact sites, and suggest that the N-terminus and the first hairpin loop play a major role in this ensemble. Remarkably, hybrid sc2-KD2 exhibited 5 or 150 times stronger inhibition of actinidin compared to native chicken cystatin or to proteolytically isolated human kininogen domain 2, respectively. This indicates an important role of the first hairpin loop of cystatins in the interaction with actinidin. Along with the impaired inhibition of cathepsin L, papain, actinidin, cathepsin B and calpain by the hybrids sc1/3-KD2, sc2/3-KD2 and sc1/2/3-KD2, these results support our hypothesis that all three predicted contact regions of kininogen domain 2 contribute to binding in the active-site clefts of papain-like enzymes in a finely balanced manner.

Amino Acid Sequence↗

PubMed1999

Purification of large quantities of human salivary cystatins S, SA and SN: their interactions with the model cysteine protease papain in a non-inhibitory mode.

OBJECTIVES: Our aim was to purify large quantities of human salivary cystatins S, SA and SN in order to determine whether these salivary cystatins have a stable interaction with cysteine proteases at a second binding site, other than the protease active site. This property may affect their availability to act as cysteine protease inhibitors within the oral environment. METHODS: Salivary cystatins S, SA and SN were purified from human submandibular sublingual saliva to homo- geneity by column chromatography. Formation of stable complexes between the model cysteine protease papain in the absence of reductant was assessed by SDS-PAGE and probing Western blots with antibody to human salivary cystatin SN. Proteolytic activity of the complex was determined in the gel after electrophoresis. RESULTS AND CONCLUSIONS: Only cystatin SN (14.3 kD) was found to form a stable complex with papain (22 kD) that could be separated by SDS-PAGE producing a Coomassie stained band at (37 kD). After western transfer this same band (37 kD) cross-reacted with antibody to SN. In the presence of E64, an active site inhibitor of cysteine proteases, the same complex was formed, suggesting that SN is able to bind to papain at a site other than the active site. Activity staining of the gel confirmed that this complex (-E64) retained proteolytic activity. Such complex formation between cystatin SN and cysteine proteases in a non-inhibitory mode may reduce its availability to act as an effective cysteine protease inhibitor in the oral environment.

Blotting, Western↗

PubMed1978

Papain solubilization of the Epstein-Barr virus-induced membrane antigen.

The Epstein-Barr virus (EBV)-induced membrane antigen (MA) was successfully solubilized from the membranes of viable EBV-infected Raji cells by treatment with papain (5 to 6 U per 1 X 10(7) to 2 X 10(7) cells). The loss of MA from viable cells was monitored by membrane immunofluorescence and antibody-dependent cellular cytotoxicity. Soluble MA was demonstrated in papain digests through inhibition of antibody-dependent cellular cytotoxicity and by inhibition of the binding of anti-MA antibodies to cells as detected by use of 125I-labeled staphylococcal protein A. Approximately 75% of the MA activity in the extracts was not sedimentable at 100,000 X g,, indicating that the majority of EBV MA activity that was released by this procedure was associated with small-molecular-weight material. Antiserum prepared from an owl monkey immunized with these papain extracts contained antibody to MA and neutralizing antibodies, but lacked detectable antibodies against viral capsid antigens and EBV-induced early antigens.

Animals↗

PubMed1976

Thermoanalytical and histological study of intra-articular papain-induced degradation and repair of rabbit cartilage. II. Mature animals.

The effect of papain treatment on the cartilage tissue of the knee joint of mature rabbits was studied by histological and thermoanalytical methods. Changes taking place in the composition of cartilage in the course of ageing were shown. As a result of papain treatment a significant irreversible destruction of the ground substance took place. Papain-induced loss of proteoglycans and the intensity of repair reactions and age-related differences are discussed.

Aging↗

PubMed1977

The chemical and kinetic consequences of the modification of papain by N-bromosuccinimide.

Nonactivated papain was treated with N-bromosuccinimide at pH 4.75. The N-bromosuccinimide-modified enzyme was characterized by (1) the change in absorbance at 280 nm, (2) amino acid analysis, (3) separate chemical determinations of tryptophan and tyrosine (4) difference spectroscopy, and (5) an N-terminal residue determination. It is concluded that N-bromosuccinimide in sevenfold molar excess oxidizes one tryptophan and two to three tyrosine residues per molecule of nonactivated papain, without causing peptide chain cleavage. Kinetic studies with several substrates and competitive peptide inhibitors were performed at pH6 using the N-bromosuccinimide-modified papain. In addition, the kinetics of the modified enzyme with the substrate alpha-N-benzoyl-L-arginine ethl ester were studied in the region of pH 3.5-9.0. All substrates (and inhibitors) test, with the exception of alpha-N-benzyoyl-L-arginine p-nitroanilide, displayed approximately a two fold decrease in both kcat and Km (or Ki), relative to the native enzyme. It is concluded that the key tryptophan residue which is probably Trp-177.

Amino Acids↗

PubMed1976

Papain inhibition by serum.

Sera from seven animal species (rat, cow, cat, dog, human, rabbit, and hamster) were tested and found to inhibit the papain-catalyzed hydrolysis of alpha-N-benzoyl-L-arginine-p-nitroaniline-HCl (L-BAPA). The relative concentration of inhibitor in each serum sample was expressed in terms of its papain inhibitory capacity (PIC) defined as the number of units of papain inhibited per ml of serum. Rat serum contained the highest concentration of inhibitor while hamster serum contained the lowest concentration. The inhibitor appears to be competitive with respect to L-BAPA and is heat labile and nondialyzable. Antipapain activity can be separated from antitrypsin activity by (NH4)2SO4 fractionation.

Ammonium Sulfate↗

PubMed1986

Lung mechanics in papain-treated rabbits.

To further investigate the effects of airway cartilage softening on static and dynamic lung mechanics, 11 rabbits were treated with 100 mg/kg iv papain, whereas 9 control animals received no pretreatment. Lung mechanics were studied 24 h after papain injection. There was no significant difference in lung volumes, lung pressure-volume curves, or chest wall compliance. Papain-treated rabbits showed increased lung resistance: 91 +/- 63 vs. 39 +/- 22 cmH2O X l-1 X s (mean +/- SD; P less than 0.05), decreased maximal expiratory flows at all lung volumes, and preserved density dependence of maximal expiratory flows. We conclude that increased airway wall compliance is probably the mechanism that limited maximal expiratory flow in this animal model. In addition the increased lung resistance suggests that airway cartilage plays a role in the regulation of airway caliber during quiet tidal breathing.

Airway Resistance↗

PubMed1988

Regulation of human IgE synthesis by soluble factors. Papain treatment of a FcE receptor-positive B-cell line (RPMI-1788) releases regulatory factors for IgE synthesis.

A novel mechanism for the release of helper and suppressor factors for human IgE synthesis is described. When FcE receptor-positive RPMI-1788 cells are treated with papain, a helper factor(s) for human IgE synthesis is released. At the same time a significant decrease in the number of cell surface FcE receptors is observed. The immunoglobulin synthesis-enhancing activity is IgE isotype-specific inasmuch as the same supernatant suppresses the synthesis of human IgA myeloma cells. When the FcE receptor-positive RPMI-1788 cells are treated with tunicamycin and then with papain, a suppressor factor(s) for human IgE synthesis is released. The mechanism by which these factors affect human myeloma IgE synthesis is unclear at present. Our results indicate that enhanced IgE synthesis is not due to increased numbers of secreting cells nor to an increased release of presynthesized IgE. In summary, papain treatment of FcE receptor-positive, but not FcE receptor-negative cells, generates a factor that regulates IgE synthesis. These results also provide evidence for the close relationship between the IgE regulatory factors and the low affinity receptors for IgE present on lymphocytes.

B-Lymphocytes↗

PubMed2001

Immobilization of papain on cold-plasma functionalized polyethylene and glass surfaces.

Polyethylene and glass surfaces were functionalized under dichlorosilane-RF-cold-plasma environments and were employed as substrates for further in situ derivatization reactions and immobilization of papain. Surface functionality changes of RF-plasma-exposed surfaces were monitored under 40 kHz continuous discharge environments. The nature and morphology of derivatized substrates and the substrates bearing the immobilized enzyme were analyzed using survey and high resolution ESCA, ATR-FTIR, and fluorescence of chemical derivatization techniques. Spacer molecules intercalated between the substrates and the enzyme significantly increased the enzyme activity (comparable with the that of the free enzyme). Computer-aided conformational modeling of the substrate-spacer systems corroborated with experimental data indicated that an optimal distance might exist between the enzyme and the substrate. The activity of free and immobilized papain was monitored using benzoyl arginine ethyl ester assay. The pH data were recorded every 0.3 s over 25 min. The Michaelis-Menten kinetic constants were evaluated for immobilized enzymes. It was shown, that the immobilized papain retains most of its activity after several washing/assay cycles.

Arginine↗

PubMed1976

Effect of human alpha-antitrypsin in papain-induced emphysema in the hamster.

The possibility that human alpha1-antitrypsin could effectively prevent development of emphysematous lesions produced in hamsters 7 days after exposure to aerosolized papain (3% for 3 hours) was investigated. Pretreatment with intratracheal human alpha1-antitrypsin prevented the appearance of these lesions in a dose-dependent manner. On the other hand, systemic administration of large doses of human alpha1-antitrypsin failed to prevent papain-induced pulmonary lesions, despite a significant increase in serum trypsin inhibitory capacity. These results suggest that intra-alveolar rather than serum concentrations of human alpha1-antitrypsin are critical for preventing the development of pulmonary emphysema in this animal model. It is interesting that although the mechanism by which human alpha1-antitrypsin prevented the papain-induced lesions is unknown, intratracheally administered human alpha1-antitrypsin similarly prevented the development of pulmonary emphysematous lesions induced by 0.1 mg of porcine pancreatic elastase given intratracheally.

Animals↗

PubMed1994

On the mechanism of inactivation of active papain by ascorbic acid in the presence of cupric ions.

An inactivation mechanism of active papain (EC 3.4.22.2) by the Cu(2+)-ascorbic acid (AsA) system was examined. Incubation of active papain, which contains an active sulfhydryl (SH) group, with the Cu(2+)-AsA system under aerobic conditions resulted in an irreversible loss of enzyme activity. The enzyme was not inactivated at a molar ratio of enzyme to Cu2+ of 1:< 1, whereas at a molar ratio of 1:1-2, the extent of inactivation showed the same dependence on the extent of oxidation of AsA. Saturation kinetics were observed with respect to the concentration of AsA. The degree of inactivation was dependent on the decrease in SH content of the enzyme. Catalase at a low concentration partially protected the enzyme from inactivation, but did not affect the oxidation of AsA. In addition, catalase at a high concentration completely protected both the enzyme from inactivation and AsA from oxidation. The present results suggest that an additional function of H2O2, besides producing hydroxyl radicals (.OH), is to promote the conversion of Cu+ into Cu2+, and that an active SH group of papain is site-specifically modified by the .OH, resulting in inactivation of the enzyme.

Amino Acids↗

PubMed1992

[Effect of SL-1010 (sodium hyaluronate with high molecular weight) on experimental osteoarthritis induced by intra-articularly applied papain in rabbits].

Sodium hyaluronate (HA) with a molecular weight of approximately 600,000-1,200,000 is reportedly effective against osteoarthritis (OA). However, since HA with higher molecular weight is expected to be more effective against OA, we investigated the effects of HA (SL-1010) newly produced by fermentation with a molecular weight of 1,800,000-2,100,000 on the experimental OA induced by intraarticular injection of papain, into the knee joint of the rabbit, in comparison with those of HA with a molecular weight of about 950,000 (HA-95). When 0.4, 0.8, and 1.6% papain (0.5ml) was injected into the knee joint of the animal twice with a 3-day interval, there were dose-dependent degenerative changes and a decrease in sulfated glycosaminoglycan (S-GAG) in the articular cartilage with slight synovial inflammatory changes 6 weeks after the final injection of papain. In this OA model, intraarticular application of SL-1010 slightly reduced the degeneration of articular cartilage, compared with the injections of HA-95 or saline (control). SL-1010 also caused a significant recovery in the S-GAG level which was decreased in the cartilage of the OA model, compared with the control. In addition, SL-1010 inhibited the release of 35S-GAG from the cartilage obtained from normal and OA model joints. These results suggest that SL-1010 is effective in inhibiting the degeneration of cartilage in the OA model, probably due to the recovery of the S-GAG level by reducing the release of S-GAG from the cartilage.

Animals↗

PubMed2004

Studies on the interaction of papain with human placental cystatin by UV, fluorescence and CD spectroscopy.

The interaction of activated papain with low molecular weight cystatin (Mr 12500) purified from human placenta has been studied. Analysis of inhibition of caesinolytic activity of papain by cystatin showed stoichiometry of 1:1. Kinetic studies gave an inhibition constant (K(i)) value of 5.5 x 10(-8) M and association rate constant (K(+1)) value of 3.4 x 10(4) (M(-1) s(-1)). All spectroscopic studies showed conformational changes in both papain and cystatin on formation of complex. The data suggest perturbation of environment of aromatic residues and change of their native structure and conformation thereby shedding light on the behaviour of cystatins, especially interaction of placental cystatin with thiol protease inhibitors.

Circular Dichroism↗

PubMedDate not supplied

Papain anaphylaxis: a case report.

We report a case which illustrates occupational sensitivity to Papain with life-threatening anaphylaxis. In addition to urticaria and angioedema and glottic symptoms, the patient demonstrated annoying intermittent problems mimicking rheumatoid, gastrointestinal, cardiac, and pulmonary disease. All symptoms and signs disappeared after he removed himself from direct occupational exposure to Papain, and he was able to continue working in a setting five stories removed from the powder. The ubiquitous presence of Papain in our daily lives, in the bathroom, dining room, hospital, or workplace establishes it as a significant contaminant. This case emphasizes the value of including papaya as a scratch testing antigen in clinical practice.

Adult↗

PubMed1996

Plasma cholesterol-suppressing effect of papain-hydrolyzed pork meat in rats fed hypercholesterolemic diet.

The effects of papain-hydrolyzed pork meat on plasma and liver cholesterol levels were studied in rats fed a cholesterol-enriched diet. In rats fed the low-molecular-weight fraction of papain-hydrolyzed pork meat, the plasma cholesterol concentration, more particularly the VLDL and LDL cholesterol concentrations, were significantly lower (p < 0.01) than in the rats fed untreated pork meat or soybean protein. Feeding with this fraction rather than with untreated pork meat also led to a significantly lower liver cholesterol concentration (p < 0.01) and increased fecal excretion of neutral and acidic steroids. The low-molecular-weight fraction contained peptides with molecular weights of 3,000 or less and had an amino acid composition similar to that of pork meat itself. This study suggests that peptides produced by papain-hydrolysis of pork meat have a hypocholesterolemic activity through their interference with the steroid absorption process.

Animals↗

PubMed1975

Increased sensitivity of the rosette-forming reaction of human T lymphocytes with sheep erythrocytes afforded by papain treatment of the sheep cells.

Human T lymphocytes are known to adhere to sheep erythrocytes. This affinity reaction was found to be consistently increased following treatment of the sheep cells with papain. Reactions with treated erythrocytes were more stable and involved higher percentages of lymphocytes. Using a double marker system of papain-treated sheep erythrocytes together with Fc rosettes as a presumptive B-cell marker, there was an overlap population of only 1% lymphocytes. Sheep erythrocytes treated with papain therefore appear to remain specifically adherent to human T lymphocytes.

Animals↗

PubMed2000

Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry.

The pK(a) of (Asp(158))-CO(2)H of papain (EC 3.4.22.2) was determined as 2.8 by using 4-chloro-7-nitrobenzofurazan (Nbf-Cl) as a reactivity probe targeted on the thiolate anion component of the Cys(25)/His(159) nucleophilic-acid/base motif of the catalytic site. The possibility of using Nbf-Cl for this purpose was established by modelling the papain-Nbf-Cl Meisenheimer intermediate by using QUANTA/CHARMM and performing molecular orbital calculations with MOPAC interfaced with Cerius 2. A pH-dependent stopped-flow kinetic study of the reaction of papain with Nbf-Cl established that the striking rate maximum at pH 3 results from reaction in a minor ionization state comprising (Cys(25))-S(-)/(His(159))-Im(+)H (in which Im represents imidazole) produced by protonic dissociation of (Cys(25))-SH/(His(159))-Im(+)H with pK(a) 3.3 and (Asp(158))-CO(2)H. Although the analogous intermediate in the reaction of caricain (EC 3.4.22.30) with Nbf-Cl has similar geometry, the pH-k profile (k being the second-order rate constant) lacks a rate maximum under acidic conditions. This precludes the experimental determination of the pK(a) value of (Asp(158))-CO(2)H of caricain, which was calculated to be 2.0 by solving the linearized Poisson-Boltzmann equation with the program UHBD ('University of Houston Brownian dynamics'). A value lower than 2.8 had been predicted by consideration of the hydrogen-bonded networks involving Asp(158) and its microenvironments in both enzymes. The difference between these pK(a) values (values not previously detected in reactions of either enzyme) accounts for the lack of the rate maximum in the caricain reaction and for the differences in the electronic absorption spectra of the two S-Nbf-enzymes under acidic conditions. The concept of control of cysteine proteinase activity by multiple electrostatic modulators, including (Asp(158))-CO(2)(-), which modifies traditional mechanistic views, is discussed.

Aspartic Acid↗

PubMed2000

beta-Lactam derivatives as enzyme inhibitors: N-substituted derivatives of (S)-4-oxoazetidine-2-carboxylate as inhibitors of elastase and papain.

N-Alkyl and N-acyl substituted 4-oxoazetidine-2-carboxylates are synthesized and evaluated as inhibitors of the proteases porcine pancreatic elastase (PPE) and papain. The compounds are obtained by alkylation or acylation at the nitrogen of benzyl (S)-4-oxoazetidine-2-carboxylate, which is synthesized by a modified literature procedure. The enzymatic assays prove some derivatives to be effective inhibitors of PPE and/or papain. The N-BOC protected amino acid derivatives 10 and 13 inhibit PPE reversibly with KI-values in the micromolar range. On the other hand, papain is inactivated irreversibly by benzyl (S)-2-(benzyloxycarbonyl)azetidin-1-acetate (6).

Animals↗