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At least 487 records · Page 27Linked to original sources
PubMed1998

Papain proteolysis releases a soluble NADPH dependent diaphorase activity from bovine neutrophil membranes.

An NADPH dependent cytochrome c reductase has been purified from resting bovine neutrophil membranes. A high degree of purification, approaching homogeneity, is indicated by the presence of a single 75 kDa protein band on silver stained SDS-PAGE (10%). The purified protein catalyzes as well an NADPH dependent reduction of iodonitrotetrazolium violet (INT). Limited papain digestion of the purified preparation produces a 65 kDa product which retains both enzymatic activities. In a similar fashion papain digestion of the plasma membrane bound protein generates a fully active soluble NADPH dependent INT and cytochrome c reductase preparation (65 kDa). Proteolytic cleavage would appear to occur at a protein-membrane anchor remote from the proteins catalytic site. The cytochrome c reductase acts independently of the O2-generating cytochrome b558, a leukocyte plasma membrane protein which also catalyzes an NADPH dependent INT reduction.

Animals↗
PubMed1998

Human cathepsin X: a novel cysteine protease of the papain family with a very short proregion and unique insertions.

A novel cDNA encoding a cysteine protease of the papain family named cathepsin X was obtained by PCR amplification from a human ovary cDNA library. The cathepsin X cDNA is ubiquitously expressed in human tissues and contains an open reading frame of 912 nucleotides encoding a predicted protein of 303 amino acids. All highly conserved regions in papain-like cysteine proteases including the catalytic residues are present in cathepsin X. The mature part of cathepsin X is 26-32% identical to human cathepsins B, C, H, K, L, O, S and W. The cathepsin X sequence contains several unique features: (i) a very short proregion; (ii) a three amino acid residue insertion in a highly conserved region between the glutamine of the putative oxyanion hole and the active site cysteine; and (iii) a second insertion of 15 amino acid residues that can be aligned with the occluding loop region in cathepsin B.

Amino Acid Sequence↗
PubMed1999

Cathepsin J, a novel murine cysteine protease of the papain family with a placenta-restricted expression.

A novel mouse cysteine protease of the papain family was identified by searching the dbEST database. A 1.28 kb full-length cDNA was obtained which contains an open reading frame of 999 nucleotides and encodes a predicted polypeptide of 333 amino acids. The deduced polypeptide exhibits features characteristic of cysteine proteases of the papain type including the highly conserved residues of the catalytic triad, and was hence named cathepsin J. Cathepsin J represents the murine homologue of a previously described rat cathepsin L-related protein. Mature cathepsin J shows 59.3% identity to mouse cathepsin L and contains the characteristic ER(F/W)NIN motif within the propeptide indicating that this protease belongs to the subgroup of cathepsin L-like cysteine proteases. Northern blot analysis of various tissues revealed a placenta-restricted expression. This expression pattern may suggest a role of cathepsin J in embryo implantation and/or placental function. Ctsj was mapped to mouse chromosome 13 in the vicinity of cathepsin L suggesting that cathepsin J may have arisen by gene duplication from cathepsin L or a common ancestral gene.

Amino Acid Sequence↗
PubMed1984

Chromatographic separation of papain evaluated by immunochemical methods.

The chromatographic separation of crude papain preparations on Sephadex G-50 (fine) enables pure papain to be obtained in a single step. Immunochemical techniques have been found to be very convenient for testing the purity of the individual chromatographic fractions. A general approach is presented that makes it possible to follow the course of the chromatographic purification of any immunogenic compound by simple qualitative immunochemical techniques that can be applied in any laboratory.

Chromatography, Gel↗
PubMed2000

Papain digestion of different mouse IgG subclasses as studied by electrospray mass spectrometry.

On-line liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) has been utilized to monitor the papain digestion of mouse monoclonal antibodies (mAb) of different subclasses (IgG1, IgG2a, IgG2b, and IgG3). The method is simple, rapid, sensitive, and allows for simultaneous determination of digestion products and the identification of microheterogenous fragments. The results provide important insight into the mechanism of papain digestion of mouse IgGs of different subclasses.

Animals↗
PubMed1998

The inactivation kinetics of papain by guanidine hydrochloride: a re-analysis.

The kinetic theory of the substrate reaction during modification of enzyme activity has been applied to study the inactivation kinetics of enzymes by denaturant. However, an important problem related to the determination of the inactivation rate constants has not been considered in a previous publication (Xiao, et al., Biochim. Biophys. Acta, 1164 (1993) 54-60). In most denaturation experiments, the high concentrations of denaturants may greatly affect the kinetic behavior of the system to preclude the use of the kinetic parameters determined in the absence of denaturant. In the present study, the kinetic equation of substrate reaction in presence of denaturant has been derived. A re-examination of the effect of high concentrations of guanidine hydrochloride on the inactivation of papain, taking into consideration the effect of high concentrations of guanidine hydrochloride on the Michaelis constant, showed that, for papain, the substrate gives no protection on its inactivation. It is the purpose of the present communication to stress the importance of observing the effect of the denaturant on the kinetic parameters for kinetic analysis of enzyme inactivation by denaturants generally.

Guanidine↗
PubMed1998

Evaluation of resins for on-bead screening: a study of papain and chymotrypsin specificity using PEGA-bound combinatorial peptide libraries.

TentaGel, ArgoGel and PEGA resins were evaluated for on-bead biological screening, using a fluorescently-labelled peptide attached to each and assayed for papain activity. Peptide attached to PEGA was cleaved in near quantitative yield at the expected sites, whilst an identical sequence on TentaGel and ArgoGel beads was hydrolysed in very low yields and nonspecifically on ArgoGel. The compatibility of PEGA with enzymes was further demonstrated by the determination of subsite specificities of papain and chymotrypsin using PEGA-bound peptide libraries, which proved to be similar to those observed in free solution.

Chymotrypsin↗
PubMed1985

Papain fragmentation of the (Na+,K+)-ATPase beta subunit reveals multiple membrane-bound domains.

Purified dog kidney (Na+,K+)-ATPase was reacted with tritiated sodium borohydride after treatment with neuraminidase and galactose oxidase. This procedure did not affect the ATPase activity of the enzyme, and all of the covalently bound radioactivity was found in the beta subunit (Mr 54 000). Papain digestion of the tritiated enzyme produced two labeled fragments of Mr 40 000 and 16 000. Further proteolysis generated an Mr 31 000 peptide from the larger fragment. Unlike the tryptic and chymotryptic sites of the alpha subunit, the sites of papain hydrolysis were insensitive to conformations of the (Na+,K+)-ATPase. Determination of the NH2-terminal sequences was used to arrange the fragments within the linear map of the beta chain. Finally, none of the labeled peptides was released from the membrane under nondenaturing conditions. These results are consistent with a model of the beta subunit containing a 40 000-dalton NH2-terminal piece and a 16 000-dalton COOH-terminal piece. Both fragments have extracellularly exposed carbohydrate and at least one membrane-bound domain.

Animals↗
PubMed1986

Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate.

The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Brønsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.

Anions↗
PubMed1989

Kinetics of binding of chicken cystatin to papain.

The kinetics of binding of chicken cystatin to papain were studied by stopped-flow fluorometry under pseudo-first-order conditions, i.e., with an excess of inhibitor. All reactions showed first-order behavior, and the observed pseudo-first-order rate constant increased linearly with the cystatin concentration up to the highest concentration that could be studied, 35 microM. The analyses thus provided no evidence for a limiting rate resulting from a conformational change stabilizing an initial encounter complex, in contrast with previous studies of reactions between serine proteinases and their protein inhibitors. The second-order association rate constant for complex formation was 9.9 X 10(6) M-1 s-1 at 25 degrees C, pH 7.4, I = 0.15, for both forms of cystatin, 1 and 2. This value approaches that expected for a diffusion-controlled rate. The temperature dependence of the association rate constant gave an enthalpy of activation at 25 degrees C of 31.5 kJ mol-1 and an entropy of activation at 25 degrees C of -7 J K-1 mol-1, compatible with no appreciable conformational change during the reaction. The association rate constant was independent of pH between pH 6 and 8 but decreased at lower and higher pH in a manner consistent with involvement of an unprotonated acid group with a pKa of 4-4.5 and a protonated basic group with a pKa of 9-9.5 in the interaction. The association rate constant was unaffected by ionic strengths between 0.15 and 1.0 but decreased somewhat at lower ionic strengths. Incubation of the complex between cystatin 2 and papain with an excess of cystatin 1 resulted in slow displacement of cystatin 2 from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Cystatins↗
PubMed1976

Resonance Raman evidence for substrate reorginization in the active site of papain.

Resonance Raman spectra were obtained for the acylenzyme 4-dimethylamino-3-nitro(alpha-benzamido)cinnamoyl-papain prepared using the chromophoric substrate methyl 4-dimethylamino-3-nitro(alpha-benzamido)cinnamate. These spectra contained vibrational spectral data of the acyl residue while covalently attached to the active site and could be used to follow directly acylation and deacylation kinetics. Spectra were obtained at pH values ranging from those where the acyl-enzyme is relatively stable (pH 3.0, tau 1/2 congruent to 800 s) to those where it is relatively unstable (pH 9.2, tau 1/2 congruent to 223 s). Throughout this range acyl-enzyme spectra differed completely from that of the free substrate or the product (4-dimethylamino-3-nitro(alpha-benzamido)cinnamic acid) indicating that a structural change occurred on combination with the active site. The spectra are consistent with rearrangement of the alpha-benzamido group in the bound substrate, -NH--C(==O)Ph becoming --N==C(--OX)Ph, where the bonding to oxygen is unknown. Superimposed on these large differences, small changes in acyl-enzyme spectra also occurred as pH was raised to decrease the half-life. All of the above spectral perturbations are consistent with a structural change in the acyl-enzyme which precedes the rate-determining step in deacylation. Thus, deacylation proceeds from an acyl residue structure differing from that of the substrate in solution. Upon acid denaturation the spectrum characteristic of the intermediate reverts to one closely resembling the substrate, demonstrating that a functioning active site is necessary to produce the observed differences. Spectra in D2O of native acyl-enzyme were identical with those in H2O, indicating that the observed differences in rate constant were not due to solvent-induced structural changes. Activated papain purified by crystallization or by affinity chromatography formed the acyl-enzyme. However, the kinetics of formation and deacylation differed between these materials, as did the spectral properties. Small differences in active-site structure are considered to be responsible for this effect, and it is suggested that such spectral perturbations may be useful in directly relating small differences in structure of the substrate in the active site with corresponding differences in kinetics.

Binding Sites↗
PubMed2002

Protein engineering of nitrile hydratase activity of papain: molecular dynamics study of a mutant and wild-type enzyme.

The mechanism of hydrolysis of the nitrile (N-acetyl-phenylalanyl-2-amino-propionitrile, I) catalyzed by Gln19Glu mutant of papain has been studied by nanosecond molecular dynamics (MD) simulations. MD simulations of the complex of mutant enzyme with I and of mutant enzyme covalently attached to both neutral (II) and protonated (III) thioimidate intermediates were performed. An MD simulation with the wild-type enzyme.I complex was undertaken as a reference. The ion pair between protonated His159 and thiolate of Cys25 is coplanar, and the hydrogen bonding interaction S(-)(25).HD1-ND1(159) is observed throughout MD simulation of the mutant enzyme.I complex. Such a sustained hydrogen bond is absent in nitrile-bound wild-type papain due to the flexibility of the imidazole ring of His159. The nature of the residue at position 19 plays a critical role in the hydrolysis of the covalent thioimidate intermediate. When position 19 represents Glu, the imidazolium ion of His159-ND1(+).Cys25-S(-) ion pair is distant, on average, from the nitrile nitrogen of substrate I. Near attack conformers (NACs) have been identified in which His159-ImH(+) is positioned to initiate a general acid-catalyzed addition of Cys-S(-) to nitrile. Though Glu19-CO(2)H is distant from nitrile nitrogen in the mutant.I structure, MD simulations of the mutant.II covalent adduct finds Glu19-CO(2)H hydrogen bonded to the thioimide nitrogen of II. This hydrogen bonded species is much less stable than the hydrogen bonded Glu19-CO(2)(-) with mutant-bound protonated thioimidate (III). This observation supports Glu19-CO(2)H general acid catalysis of the formation of mutant.III. This is the commitment step in the Gln19Glu mutant catalysis of nitrile hydrolysis.

Computer Simulation↗
PubMed1984

Actinidin hydrolysis of substituted-phenyl hippurates: a quantitative structure-activity relationship and graphics comparison with hydrolysis by papain.

The hydrolysis of 29 phenyl hippurates (XPhOCOCH2NHC(=O)C6H5) by the cysteine protease actinidin has been studied and a quantitative structure-activity relationship (QSAR) has been formulated: log 1/Km = 0.74 sigma + 0.50 pi'3 + 0.24MR4 + 2.90. In this expression Km is the Michaelis constant, sigma is the Hammett constant, pi'3 is the hydrophobic parameter for the more hydrophobic of the two meta substituents, and MR4 is the molar refractivity of para substituents. The QSAR for actinidin is compared with a similar one obtained for another cysteine plant protease papain. A color stereo computer graphics model constructed from the X-ray crystallographic coordinates of actinidin is compared with those of our previously reported models for papain.

Cysteine Endopeptidases↗
PubMed2004

Activity of immobilized papain dehydrated by n-propanol in low-water media.

A propanol-rinsed enzyme preparations (PREP) of papain showed an activity of 59 nmol min(-1) (mg powder)(-1) in tert-butanol at the optimal water activity of 0.2. The immobilized papain was stable in aqueous media for 3 d at 4 degrees C. Solid-state buffers (bases and their HCl salts) suspended in the organic medium decreased the initial rate in all cases tested. The operational stability during Z-Gly-Phe-NH2 synthesis was improved when solid cysteine was added, doubling the yield after 24 h.

1-Propanol↗
PubMed2002

Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme.

The leader proteinase (L(pro)) of foot-and-mouth-disease virus is an unusual papain-like cysteine proteinase. Synthesized without an N-terminal pro precursor region, it frees itself from the growing polypeptide chain by cleavage at its own C-terminus. It also possesses a unique electrostatic environment around the active site, essentially due to Asp(163), which orients the catalytic histidine residue, and Asp(164); the equivalent residues in papain are Asn(175) and Ser(176). The importance of these residues for L(pro) activity was examined by site-directed mutagenesis. Replacement of Asp(163) with asparagine reduced activity by five-fold towards a hexapeptide substrate and slightly delayed self-processing when expressed in rabbit reticulocyte lysates. However, no effect on the cleavage of the only known cellular substrate of L(pro), eukaryotic initiation factor 4GI (eIF4GI), was observed. In contrast, replacement of Asp(164) by either alanine, asparagine or lysine abrogated activity towards the hexapeptide. Furthermore, in all cases, the onset of both self-processing and eIF4GI cleavage were significantly delayed; the reaction rates were also diminished compared with those of the wild-type enzyme. The alanine-substituted enzyme was least affected, followed by those substituted with asparagine and lysine. The double mutant protein in which both aspartate residues were replaced by asparagine was most severely affected; it failed to complete either self-processing or eIF4GI cleavage within 3 h, compared with the 8 min required by the wild-type enzyme. Hence, we propose that the electrostatic charge of Asp(164), and to a lesser extent that of Asp(163), is extremely important for L(pro) to attain full activity upon synthesis.

Amino Acid Substitution↗
PubMed2001

Studies on the inactivation of soluble and immobilized papain by the ascorbic acid-Cu2+ system: a model to propose the effect of free radicals on membrane-bound enzymes in vivo.

Free radicals have been suggested to be widely implicated as the species responsible for harmful biological processes, such as aging, carcinogenesis and numerous other diseases. The mechanism of biological damage produced in such processes has been investigated in a wide variety of systems, including studies on proteins, enzymes, nucleic acids and carbohydrates. In the present study we selected an ascorbic acid-transition-metal ion (ASA-Cu(2+)) system in order to understand the mechanism of soluble and membrane-bound enzyme inactivation by generating free radicals. Papain, a thiol protease, was immobilized on an immobilized-metal-ion carrier and used as a model to examine the inactivation behaviour of membrane-bound enzymes. A comparison was made between the inactivation of soluble and immobilized papain by free radicals, and the potential of different radical scavengers to prevent the inactivation of enzyme was examined.

Ascorbic Acid↗
PubMed1971

Release of intestinal surface-membrane glycoproteins associated with enzyme activity by brief digestion with papain.

Rat intestinal surface-membrane glycoproteins were labelled by intraperitoneal injection of [1-(14)C]glucosamine 4h before the animals were killed. At this time, density-gradient centrifugation of disrupted brush borders indicated that glycoprotein radioactivity was distributed identically with sucrase, a plasma-membrane marker. Labelled brush borders were digested by papain for brief time-intervals known to release surface-enzyme particles without disruption of the unit membrane. Digestion for 5min released 90% of the surface sucrase, and almost one-half of the brush-border glycoprotein and label. On Sepharose 4B column chromatography most of the glycoprotein and label emerged as a single peak. This peak contained the most actively labelled glycoprotein in the brush border and was closely associated with maltase, sucrase, beta-naphthylamidase and alkaline phosphatase. The peak was partially resolved on polyacrylamide-gel electrophoresis into three bands. Each band contained a distinctive enzyme or enzyme pair, and was labelled by [1-(14)C]glucosamine. No periodic acid-Schiff-negative protein was observed in the peak material. Glycoproteins susceptible to brief digestion with papain are therefore closely linked to released surface-enzyme particles. Intestinal surface glycoproteins are heterogeneous with respect to molecular weight, electrophoretic mobility and function.

Alkaline Phosphatase↗