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PubMed2004

Two polymorphs of a covalent complex between papain and a diazomethylketone inhibitor.

The three-dimensional structure of two polymorphs of a ZLFG-CH2-papain covalent complex has been determined by X-ray crystallography. The structures indicate that: (i) the methylene carbon atom of the inhibitor is covalently bound to the Sgamma atom of Cys25 of papain; (ii) the hydrophobic S2 pocket formed by Pro68, Val133, Val157, and Asp158 is occupied by the inhibitor's phenylalanyl P2 side chain; (iii) extensive hydrogen bonding and hydrophobic interactions are responsible for the interaction of the inhibitor with the enzyme. Comparison with similar structures suggests that in covalent complexes preservation of main chain-main chain interactions between the enzyme and the inhibitor may have higher priority than the P-S interactions.

Binding Sites↗
PubMed1979

The influence of ionic strength and incubation time on the sensitivity of methods using papainized red cells.

The sensitivity of a combination of various papain methods with normal and low ionic strength (lis) of the serum-cell mixture for the detection of red cell antibodies was tested after varying periods of incubation of the mixture. The mixture was centrifuged before reading when the period of incubation was short. The maximum sensitivity without centrifugation was always reached after a period of 60 min. With a longer incubation the sensitivity decreased. A sensitivity that equalled the maximum sensitivity without centrifugation could be obtained after only 10 min of incubation, if it was followed by a centrifugation before reading. Lis consistently increased the sensitivity of the papain methods, especially after 45--60 min of incubation.

Antibodies↗
PubMed1978

Evidence for multiple reactive forms of papain.

The pH-dependence of the second-order rate constants of acylation and alkylation reactions of the -SH group of papain were determined by using neutral and charged reactants under identical conditions. From these pH rate profiles, in contrast to previous claims, different pKa values were obtained with different groups of reactants. In the case of charged reactants, like chloroacetate (pKa = 3.6) and arginine derivatives (pKa = 4.3), the pKa differences can be attributed to electrostatic effects. However, the fact that a pKa of 3.25 was found with methyl and ethyl bromoacetate, and a pKa of 4.0 was obtained with bromoacetamide and a number of neutral substrates, is inconsistent with the theories put forth hitherto for the meaning of such pKa values, because they all consider only one reactive enzyme form. The different pKa values obtained here with neutral reactants are explained in terms of various reactive papain forms. The perturbation of pKa by electrostatic effects was examined by reacting simple thiol compounds containing different charges, like 2-mercaptoacetate, 2-mercaptoethylamine, 2-mercaptoethanol and glutathione, with the neutral chloroacetamide and with the negatively charged chloroacetate. Differences in pKa can be interpreted in terms of intramolecular and intermolecular electrostatic interactions.

Acylation↗
PubMed1982

Dimerization of deoxyribonuclease I, lysozyme and papain. Effects of ionic strength on enzymic activity.

Transition of bovine ribonuclease A from its monomeric to a dimeric form changes the pattern of enzymic activity response to ionic strength [Sorrentino, S., Carsana, A., Furia, A., Doskocil, J., and Libonati, M. (1980) Biochim. Biophys. Acta. 609, 40-52]. To see whether this phenomenon could be common to other enzyme-substrate systems, the action of various dimeric and monomeric enzymes (ox pancreas deoxyribonuclease, hog spleen acid deoxyribonuclease, bovine seminal ribonuclease, egg-white lysozyme, and papain) on polyelectrolytic substrates has been studied under different conditions of ionic strength. Dimerization of ox pancreas deoxyribonuclease, lysozyme and papain was obtained by cross-linkage with dimethyl suberimidate. The main results of the investigation, similar to those obtained with ribonuclease A, are the following. 1. Enzyme monomers and dimers show markedly different patterns of activity response to ionic strength at given pH values: the reactions catalyzed by monomeric enzymes are highly modulated by salt, whereas those catalyzed by dimeric enzymes are not. In particular, at the reaction optimum the monomeric form of an enzyme is significantly more active than the dimeric one. 2. The optimum of the reaction catalyzed by a dimeric enzyme is shifted to higher ionic strengths in comparison with that of the reaction catalyzed by a monomeric enzyme. A model is proposed that could explain these results on the basis of the influence of ionic strength on the intramolecular dynamics of the enzyme molecule and its non-specific interactions with polyelectrolytic substrates.

Animals↗
PubMed1976

The component fragments obtained by acid dissociation of papain-solubilized H-2 molecules.

H-2Kk and H-2Dd molecules were specificially purified from a radioiodinated H-2a preparation obtained by papain digestion of spleen cell membranes of A/J strain mice. The molecules were isolated by binding to H-2 alloantisera of the corresponding private specificity followed by precipitation with rabbit anti-mouse IgG antiserum. The specifically precipitated radioiodinated H-2Kk and H-2Dd molecules were dissociated by acid treatment into large and small components of about 37,000 and 11,000 respectively. These were separated by gel filtration at acid pH or by gel isoelectric focusing in the presence of 6 M urea. Each component separated by gel filtration of the acid-dissociated H-2 molecules showed a high degree of size homogeneity as determined by sodium dodecyl sulphate-acrylamide gel electrophoresis. Upon gel isoelectric focusing, however, the small components showed two peaks of radioactivity closely located together at pH 7-8, both of which had a restricted pH range, while the large components gave one peak of a relatively wide pH range of pH 5-6. The H-2Kk and H-2Dd molecules gave essentially the same pattern in terms of the numbers and the positions of the radioactivity bands. Under the iodination conditions used the large components of H-2Kk molecules contained more radioactivity than the small components, while the reverse was true in case of H-2Dd molecules. Such a difference was also found with H-2Kk and H-2Dd molecules isolated by use of alloantisera of the respective public specificity. The assay of binding of the isolated components with H-2 alloantisera of defined specificity revealed that the large components retain most of the allospecificities of the parental H-2 molecules. No H-2 allospecificities were found on the small components. The small components showed extensive binding with rabbit antiserum against mouse beta2-microglobulin. The same antiserum did not show any binding with the large components. On the other hand, both of the components did bind with rabbit antiserum against papain-solubilized H-2 molecules.

Animals↗
PubMed1966

Papain-treated globulins in specific and cross-reacting immunofluorescent staining.

Jones, Wallis L. (Communicable Disease Center, Atlanta, Ga.), and J. W. Foster. Papain-treated globulins in specific and cross-reacting immunofluorescent staining. J. Bacteriol. 91:984-986. 1966.-Cross-reaction staining with preimmune and specific fluorescein conjugates to Corynebacterium diphtheriae was studied by use of papain-treated antisera. Enzyme-treated antisera were labeled and chromatographically separated into three fractions. Fractions were tested by both direct and inhibition fluorescent-antibody procedures. The cross-reaction staining was found in all three fractions, whereas specific staining was found in only two fractions.

Animals↗
PubMed1993

Identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus.

The murine coronavirus mouse hepatitis virus gene 1 is expressed as a polyprotein, which is cleaved into multiple proteins posttranslationally. One of the proteins is p28, which represents the amino-terminal portion of the polyprotein and is presumably generated by the activity of an autoproteinase domain of the polyprotein (S. C. Baker, C. K. Shieh, L. H. Soe, M.-F. Chang, D. M. Vannier, and M. M. C. Lai, J. Virol. 63:3693-3699, 1989). In this study, the boundaries and the critical amino acid residues of this putative proteinase domain were characterized by deletion analysis and site-directed mutagenesis. Proteinase activity was monitored by examining the generation of p28 during in vitro translation in rabbit reticulocyte lysates. Deletion analysis defined the proteinase domain to be within the sequences encoded from the 3.6- to 4.4-kb region from the 5' end of the genome. A 0.7-kb region between the substrate (p28) and proteinase domain could be deleted without affecting the proteolytic cleavage. However, a larger deletion (1.6 kb) resulted in the loss of proteinase activity, suggesting the importance of spacing sequences between proteinase and substrate. Computer-assisted analysis of the amino acid sequence of the proteinase domain identified potential catalytic cysteine and histidine residues in a stretch of sequence distantly related to papain-like cysteine proteinases. The role of these putative catalytic residues in the proteinase activity was studied by site-specific mutagenesis. Mutations of Cys-1137 or His-1288 led to a complete loss of proteinase activity, implicating these residues as essential for the catalytic activity. In contrast, most mutations of His-1317 or Cys-1172 had no or only minor effects on proteinase activity. This study establishes that mouse hepatitis virus gene 1 encodes a proteinase domain, in the region from 3.6 to 4.4 kb from the 5' end of the genome, which resembles members of the papain family of cysteine proteinases and that this proteinase domain is responsible for the cleavage of the N-terminal peptide.

Amino Acid Sequence↗
PubMed2005

The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity.

Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.

Amino Acid Sequence↗
PubMed2005

The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme.

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M(-1)s(-1), 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.

Amino Acid Sequence↗
PubMed1984

Hyaline articular cartilage dissected by papain: light and scanning electron microscopy and micromechanical studies.

Papain was used to digest the hyaline femoral condylar cartilages of 30 adult Wistar rats. Matrix proteoglycan degradation was assessed by the light microscopy of paraffin sections stained with toluidine blue. The extent of surface structural change was estimated by scanning electron microscopy, and the structural integrity of the hyaline cartilage tested by the controlled impact of a sharp pin. The results demonstrated an early loss of cartilage metachromasia, increasing with time of papain digestion, and an alteration in the shape of the cartilage splits produced mechanically. Accompanying the loss of matrix material was a conspicuous sequence of morphological changes in cartilage surface structure. The results confirm a close relationship between matrix integrity, mechanical properties, and fine structure.

Animals↗
PubMed1986

Effect of intravenous papain on tracheal pressure-volume curves in rabbits.

To investigate the effects of airway cartilage softening on tracheal mechanics, pressure-volume (PV) curves of excised tracheas were studied in 12 rabbits treated with 100 mg/kg iv papain, whereas 14 control animals received no pretreatment. The animals were killed 24 h after the injection and the excised specimens studied 24 h later. Treated tracheas exhibited decreased ability to withstand negative transmural pressures, reflected in increased collapse compliance: 6.2 +/- 2.1 vs. 2.0 +/- 0.5% peak volume (Vmax)/cmH2O means +/- SD, P less than 0.001, (Vmax = extrapolated maximal tracheal volume), increased kc (exponential constant that reflects the shape of collapse limb of the PV curve): 0.244 +/- 0.077 vs. 0.065 +/- 0.015 (P less than 0.001). The distension limb of the PV curve greater than 2.5 cmH2O transmural pressure (Ptm) was no different. Compliance between 0 and 2.5 cmH2O Ptm was increased in papain-treated rabbits: 4.97 +/- 1.73 vs. 2.30 +/- 0.31% Vmax/cmH2O (P less than 0.001). Tracheal volume, and therefore mean diameter, was decreased at 0 Ptm: 2.7 +/- 0.26 vs. 3.2 +/- 0.27 mm (P less than 0.001). We conclude that airway cartilage softening increases the compliance of the trachea at pressures less than 2.5 cmH2O Ptm.

Animals↗
PubMed1992

Assessment of papain-induced lung injury in isolated lungs by measurements of aerosol deposition and mixing.

Aerosol bolus inspirations were used to assess lung injury in 15 isolated dog lungs exposed to low (0-375 units) or high doses (600-1,200 units) of papain. Effective air space size (EAD) was determined from aerosol deposition during a 5-s breath hold. Convective mixing was assessed by the spreading of the expired bolus with respect to expired volume, quantified by a coefficient of dispersion (CD) equal to the square root of the difference in the variances of the expired and inspired boluses divided by the volumetric penetration of the bolus. After exposure, CD measured with deeply penetrating boluses increased by an average of 2.5% in the low-exposure group (P greater than 0.05) and 28.0% in the high-exposure group (P less than 0.0001). CD measured with shallowly penetrating boluses decreased by 4.3% (P less than 0.0001) in the low-exposure group and increased by an average of 18.3% in the high-exposure group (P less than 0.05). Papain exposure caused EAD to increase in some lungs and decrease in others. For deep bolus penetrations, EAD changed by an average of -0.8% in the low-exposure group (P greater than 0.05) and +21.1% in the high-exposure group (P greater than 0.05). Both EAD and CD appeared to be sensitive to lung injury. However, changes in EAD were less consistent than those in CD, possibly due to changes caused by lung injury in the regional distribution of inspired aerosol.

Aerosols↗
PubMed1982

Changes in pulmonary connective tissue after a single intratracheal instillation of papain in the rat.

The effect of a single intratracheal instillation of 2 mg of papain on metabolic changes of collagen and elastin in lungs was studied in rats labeled with [14C]proline in vivo. The total amount, concentration, specific and total radioactivity of both proteins were examined during the 28 days after papain administration. In the early period (1 to 3 days) a loss of elastin content (about 50% of control; p less than 0.01) was found, whereas collagen content increased (p less than 0.05). Simultaneously, the specific as well as the total radioactivity of both proteins was enhanced (p less than 0.01) which indicated an accelerated biosynthesis and metabolic turnover. This fact was reflected in the increased amounts of both collagen and elastin in the lungs 14 and 28 days after instillation of the enzyme. The changes in connective tissue proteins accompanied a large increase in lung weight (200% that of the control rats; p less than 0.01) immediately after the exposure. Toward the end of the 28-day period, the increase was smaller. The enzymatic damage probably induced the formation of nonfunctional elastin and fibrous scars as a consequence of the healing process.

Animals↗
PubMed1972

Effects of elastase, collagenase, and papain on structure and function of rat lungs in vitro.

Present concepts of the roles of collagen and elastin in lung elastic behavior and maintenance of lung structure have been largely inferred from anatomical observations or from studies of isolated fibers in vitro. Based on the intimate association of elastin and collagen it has been postulated that elastin contributes little to elastic behavior and that collagen is the major determinant of lung structure. Using clostridial collagenase, pancreatic elastase, and papain we have selectively degraded these fibers and studied the resulting changes in elastic behavior and structure of rat lungs in vitro.Pressure-volume curves were recorded during continuous slow air inflation and deflation (10.5 ml/min) before and after the intratracheal instillation of 0.5 ml of control or enzyme solution. Surface tension-lowering activity of lavaged material was studied. All lungs were fixed inflated at 25 cm H(2)O pressure and whole lung sections were stained for elastin, collagen, and reticulin. Collagenase produced a marked susceptibility to pleural rupture but did not alter elastic behavior or lung structure. Elastase and papain produced segments of lung with increased compliance; this change was not due to alteration in surface forces but was associated with decreased tissue elastic recoil. Histologically, altered tissue recoil correlated well with evidence of damaged elastin fibers. In contrast to previous concepts these results suggest that elastin is the major connective tissue determinant of lung structure and elastic behavior.

Animals↗
PubMed1994

Effect of radical scavengers on the inactivation of papain by ascorbic acid in the presence of cupric ions.

Incubation of papain (EC 3.4.22.2) with ascorbic acid (AsA) and Cu2+ in acetate buffer (pH 5.6) results in an irreversible loss of enzyme activity by site-specific generation of free radicals [H. Kanazawa, S. Fujimoto, A. Ohara, Biol. Pharm.Bull., 16, 11 (1993)]. In this study, the effect of some compounds, known free radical scavengers, on the relationship between the inactivation of papain by the Cu(2+)-AsA system and the oxidation of AsA was investigated. Catalase completely protected the enzyme from inactivation by the Cu(2+)-AsA system, although hydrogen peroxide (H2O2) by itself, known to be generated during the autoxidation of AsA, did not inactivate the enzyme. The oxidation of AsA was unaffected by catalase. Both thiourea and sodium thiocyanate completely protected the enzyme from inactivation, while AsA was partially oxidized only in the initial stage. In the presence of potassium iodide, both the inactivation of the enzyme and the oxidation of AsA were characterized by a rapid initial phase followed by a stable phase where no reaction took place and, subsequently, a slower phase. Histidine partially prevented the inactivation of the enzyme and the oxidation of AsA. The present results suggest that H2O2 serves as a source of secondary, highly reactive species, probably hydroxyl radicals, which are responsible for the inactivation, and that the protection from inactivation by some radical scavengers, such as thiourea, sodium thiocyanate, potassium iodide, and histidine, is based on the removal of metal ions (Cu2+ or Cu+) at the specific site of inactivation.

Ascorbic Acid↗
PubMed1979

Fluorimetric determination of DNA in papain digests of cartilage, using ethidium bromide.

A fluorimetric procedure for the determination of the DNA content of cartilage is described. The tissue is initially solubilised by digestion with papain, and ethidium bromide is used for the subsequent quantitation of DNA. The basis of the procedure is the enhancement of fluorescence which occurs when ethidium bromide complexes with native nucleic acids, fluorescence due to DNA being distinguished from that due to RNA through the use of ribonuclease. The method provides reproducible results, allowing determination of DNA in papain digests containing greater than 1.25 microgram DNA/ml, and is a rapid alternative to more laborious colorimetric or fluorimetric methods, which require the separation of DNA from other tissue components. The procedure is highly specific for DNA and is useful in metabolic studies in which various parameters of chondrocyte activity are being studied.

Animals↗
PubMed1992

Ferrocenopapain, an organometallic protein formed by site-specific inactivation of papain using chloroacetylferrocene.

Papain is rapidly, specifically and irreversibly inactivated by chloroacetylferrocene in a time-dependent, two-stage process which involves initial, physical complexation between the enzyme and the organometallic reagent followed by chemical reaction. The kinetics of the process show saturation kinetics with respect to inactivator concentration. The modification occurs with a 1:1 stoichiometry and the degree of loss of enzymatic activity is directly reflected in the loss of thiol groups in the active-site that are accessible to Ellman's reagent. The modification is faster at higher pH. The ferrocenium ion of the modified papain can be generated at low pH by anaerobic treatment with ferric nitrate and shows a 180 percent increased lifetime at pH 3.3 compared to the acetylferrocenium ion under identical conditions. The spectral properties of the ferrocenopapain indicate that the ferrocene is in an unusual environment at the protein active-site and the implications of this are discussed.

Cysteine↗
PubMed1982

Papain-induced mitosis of chondrocytes in adult joint cartilage: an experimental study in full-grown rabbits.

When diluted papain is injected intra-articularly into the knee joints of adult rabbits, slowing progressing, degenerative changes develop in the articular cartilage. After intravenous injection of crude papain no significant degenerative changes are found. With the aid of radioactive thymidine, it is possible to demonstrate that the chondrocytes recover their ability to divide before any degenerative changes can be found. At the same time the matrix loses its staining properties with Safranin-O and toluidine blue.

Animals↗