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Topological studies on the hydrolases bound to the intestinal brush border membrane. I. Solubilization by papain and Triton X-100.

Papain digestion of closed, right side out vesicles from pig, rat and rabbit jejunum brush border induces the release of the hydrolases bound to the membrane without grossly affecting the lipid bilayer limiting the vesicles. This observation definitely proves that intestinal hydrolases are surface components attached to the external side of the membrane. All proteins released by papain could be identified by electrophoresis and immunoelectrophoresis to already known intestinal hydrolases, with the exception of an unidentified substance strongly stained by the Schiff's reagent. The early observation that the aminopeptidase form released from pig bursh border by Triton X-100 is different from that released by papain was extended to other hydrolases from pig, rat and rabbit. In some cases, the Triton-released form could be converted by further proteolytic digestion into a new form similar to that liberated by papin. These facts may be related to the existence of hydrophobic anchors retaining the intestinal hydrolases to the membrane surface.

Animals↗

Influence of organic solvents on papain kinetics.

Papain activity was studied in water-organic solvent mixtures using the fluorogenic substrate Dabcyl-Lys-Phe-Gly-Gly-Ala-Ala-Edans. The increase of organic solvent (MeOH, EtOH, iPrOH, TFE, MeCN, (MeO)2Et and DMF) concentration in the mixture caused a substantial decrease the initial rate of papain-catalyzed hydrolysis. Moreover, the number of papain active sites decreased with the increase of DMF and MeOH concentration.

Amino Acid Sequence↗

A rosetting test with papain-treated erythrocytes (RBC) for the detection of a minor erythrocyte population (chimerism) with different antigenic expressivity.

A rosetting test with papain-treated erythrocytes was set up and evaluated for the detection of minor erythrocyte populations (chimerism), which can be distinguished by their antigenic differences. The test, for Rh system antigenic differences, has been able to detect erythrocyte populations with a 0.5: 1000 density and a sensitivity 3-4 times as high as that of the antiglobulin test. Critical elements for the optimization of the test are: the final ratio between papain-treated erythrocytes and erythrocytes examined (2:1); the specificity check of the antiserum used which, considering the increased sensitivity, must be performed by the rosetting test itself; the accurate and delicate resuspension of the final pellet. Some disadvantages are the impossibility to detect antigenicity that can be altered by papain and, above all, the scarce possibility of quantifying, if not roughly, the size of the minor population.

Antigens, Surface↗

1-Peptidyl-2-haloacetyl hydrazines as active site directed inhibitors of papain and cathepsin B.

Fifteen 1-peptidyl-2-haloacetyl hydrazines, which can be considered halometanes of azapeptides containing Phe in P2 and alpha-aza-Ala or alpha-aza-Gly in P1, were synthesized and tested as models of cysteine-proteases inhibitors. By use of kinetic methods, they proved to irreversibly inactivate papain and cathepsin B via a reversible enzyme-inhibitor intermediate. Second-order rate constants of inactivation in the range 26-23000 M-1s-1 were observed for papain and 2000-39600 M-1s-1 for cathepsin B. KI for the reversible EI adducts ranged from 230 to 0.16 microM for papain and from 11 to 0.37 microM for cathepsin B. Structure of possible reversible EI complex is proposed and used to discuss the effects of structural variation of the inhibitors on the kinetic parameters of inactivation. Title compounds proved to be selective for cysteine-proteases, since no inhibiting activity could be detected toward trypsin, chymotrypsin and porcine pancreatic elastase at 0.1 mM concentration, after 6 h incubation. Relatively low aspecific alkylating properties were also verified in tests using glutathione as the nucleophile.

Animals↗

Inhibition of tumor growth, invasion and metastasis in papain-immunized mice.

Papain-immunized mice possess serum antibodies which cross-react with cathepsin-B- and cathepsin-H-like endopeptidases isolated from B16 melanoma cells. The growth rate, invasion and metastasis of both the B16 melanoma and the Lewis lung carcinoma were inhibited in mice immunized with papain. These animals presented an increased mean survival time as compared to the tumor-bearing nonimmunized controls. Quantitative microscopy suggested that vasodilation and edema, associated with tumor invasion, are, at least partially, sustained by proteolytic enzymes, being strongly reduced when tumor cells were inoculated in papain-immunized mice.

Abdominal Muscles↗

Cell surface glycoproteins of rat lymphocytes. I. Correlation of mitogenic stimulation by periodate or neuraminidase and galactose oxidase with the presence of papain-sensitive glycoproteins.

Oxidation of viable rat lymph node lymphocytes with either periodate or a combination of neuraminidase and galactose oxidase (NGO), followed by reduction with tritiated sodium borohydride, labels similar sets of cell-surface molecules as assessed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Periodate and NGO induce blast transformation of lymph node lymphocytes (oxidative mitogenesis), and borohydride reduction inhibits the proliferative response. Thus, it is inferred that some or all of the glycoproteins that are labeled with tritiated borohydride may be involved in mediating the stimulation caused by the oxidizing agents. Treatment of lymph node lymphocytes with 5 units/ml papain abolishes the response to periodate or NGO but does not significantly affect the response to Con A. At the same time, papain treatment eliminates the labeled bands representing six high m.w. glycoproteins (175,000, 170,000, 160,000, 155,000, 100,000, and 70,000 daltons). No significant effect is seen on the labeling of the other components visualized in the slab gels. The results implicate the subset of six high m.w. papain-sensitive sialoglycoproteins in mediating oxidative mitogenesis of rat lymph node lymphocytes.

Aldehydes↗

[Correlation between the function and structure in papain-induced emphysema in dogs].

To examine the relationship between morphologic changes and the alteration of pulmonary function, papain solution was administered to 11 dogs by inhalation and to 5 dogs by a single intratracheal injection. Pulmonary function tests, including determination of lung volume subdivision, diffusion capacity (DLCO), pressure-volume relationship of the lung and arterial blood gas were performed before and after papain treatment. The dogs were then killed and the lungs processed for pathologic studies. The severity of emphysema was graded as percent of emphysema according to Dunill's method and we compared this to the antemortem pulmonary function data. Exponential constant K was determined by fitting lung pressure-volume data to the equation V = Vo (1-e-KP). After papain administration, DLCO decreased, K of the pressure-volume curve increased, and arterial blood oxygen pressure reduced significantly. The changes of residual volume, the ratio of residual volume to total lung capacity, DLCO per unit lung volume, and the values of the exponential functions of pressure-volume data were correlated with the severity of emphysema with statistical significance, but none of them showed excellent order correlation. Multiple regression equation using 2 to 4 of the parameters indicated that the severity of emphysema can be predicted more accurately. We conclude that recognition of the severity of emphysema requires a multivariate approach including the determination of lung volume, DLCO, and pressure volume relationship of the lung, because the relationship between the severity of emphysema and the values obtained with these measurements are nonlinear.

Animals↗

[Comparative analysis of the adjuvant action of fab-fragments of normal rabbit IgG obtained using pepsin and papain].

The capacity of Fab fragments of normal rabbit IgG to enhance the immune response to sheep erythrocytes in the homologous recipients was determined by the structure of the C-terminal part of the heavy chain Fd fragment. This followed from the fact that pepsin F(a')2 and Fab' fragments enhanced considerably the hemagglutinin production and proliferation of the antibody-forming cells in the spleen, whereas papain Fab fragments, used in the same dose as the pepsin fragments, possessed only negligible adjuvant activity. As shown the adjuvant activity of pepsin and papain fragments displayed an inverse correlation with the titres and papain homoreactants in the sera of the homologous recipients. The data obtained suggested that the target cells for Fab fragments were lymphocytes carrying cytophilic homoreactants as receptors for the fragments.

Adjuvants, Immunologic↗

Characteristics and application of immobilized papain in a continuous-flow reactor.

Papain was immobilized on an anion-exchange resin by physical adsorption followed by cross-linking with glutaraldehyde. The activity and rate of heat inactivation of the enzyme was determined at 37 to 95 degrees C. Activity increased with an increase in temperature up to 85 degrees C, then decreased slightly at 95 degrees C. Activation of the enzyme by cysteine in 2 mM EDTA increased proportionally with an increase in concentration from 0.004 to 0.12 M. Levels of dissolved oxygen in solutions of EDTA containing cysteine in a closed system and with nitrogen purging in the absence of cysteine in an open system were monitored at 51 degrees C. Both the presence of cysteine and purging with nitrogen were effective in depleting dissolved oxygen. The final oxygen level was essentially independent of cysteine concentrations of 0.01, 0.02, and 0.10 M, and was reached in 95, 85, and 55 min, respectively. However, final levels of oxygen were dependent upon the flow rate of nitrogen from 1 to 10 SLPM and were reached within 15 min. Immobilized papain in a continuous-flow stirred-tank (300 rpm) reactor with a 4-liter reaction volume accompanied by nitrogen purging of 4 SPLM was used to hydrolyze a 1.5% casein solution in the presence of 0.01 M cysteine in 2 mM EDTA at 51 degrees C. The reaction rate was 1 liter/h and activity was measured based on the content of trichloroacetic acid-soluble protein in the outflow. Papain activity was not observed to decrease during a 30-h reaction period.

Anion Exchange Resins↗

Hydrolysis of alkyl ester and amide substrates by papain.

The ratio of the rate constants of acylation of papain with some amino acid ester and amide substrates is unexpectedly low. The contribution to this low ratio by the N-acyl group and the amino acid side chain was studied by measuring the rate constants of substrates containing various acyl groups (benzoyl and benzyloxycarbonyl) and various side chains (glycine, alanine, norleucine, citrulline and arginine). The benzoyl esters were found to be less reactive than the corresponding benzyloxycarbonyl esters, whereas the benzoyl and corresponding benzyloxycarbonyl amides reacted with papain at similar rates. These findings can be explained by the dominance of hydrogen bond formation between the enzyme and amide substrates, which comprensates for the less favourable binding of the benzoyl group. It is also apparent from the similar acylation rate constants for norleucine, citrulline and arginine derivatives that the guanidyl group only slightly affects the reaction of arginine derivatives with papain.

Acylation↗

Identity of acyl group conformations in the active sites of papain and cathepsin B by resonance Raman spectroscopy.

Resonance Raman spectroscopic data provide conclusive evidence for the existence of an acyl-enzyme intermediate during the reaction of a thionoester substrate, N-methyloxycarbonylphenylalanylglycine methyl thionoester (CH3OC(=O)-Phe-NHCH2C(=S) OCH3), with cathepsin B from porcine spleen. The resonance Raman spectrum of CH3OC(=O)-Phe-NHCH2C(=S)S-cathepsin B, where the thiol S is from the active-site cysteine residue, is compared to that of the corresponding papain acyl-enzyme. Within the limits of experimental error (+/-2 cm-1 for peak positions), there are no detectable spectral differences. Since the resonance Raman spectrum is sensitive to the torsional angles in the glycinic bonds and the cysteine linkages, the conformations are identical in those parts of the acyl-enzymes where chemical transformation occurs. A conformational analysis of the model compound CH3OC(=O)-Phe-NHCH2C(=S)SC2H5 demonstrates that the dithioacyl group in both dithioacyl-enzymes is present as a single population of a form known as conformer B. Conformer B is characterized by a small torsional angle about the glycinic NHCH2-CS(thiol) bond such that the nitrogen and S (thiol) atoms are in close contact. This conformer is widespread among the dithioacyl intermediates of plant cysteine proteinases, and it is apparent that the same chemistry is retained in a mammalian cysteine proteinase. Steady-state kinetic parameters are also reported for CH3OC(=O)-Phe-NHCH2C(=S)OCH3 reacting with papain and cathepsin B. The similarity of the Kcat values, 0.53 and 1.15 s-1, for papain and cathepsin B, respectively, provides further evidence for a conserved deacylation process.

Animals↗

[Chemotherapeutic effectiveness of antibiotics in combination with papain in experimental septicemia].

The efficacy of papain, a proteolytic enzyme made in the USSR, was studied alone and in combination with antibiotics, such as benzylpenicillin, streptomycin, chloramphenicol, tetracycline, erythromycin and novobiocin in treatment of mice with septicemia caused by antibiotic-resistant staphylococci. It was shown that the enzyme increased the chemotherapeutic efficacy of the antibiotics on the average by 50 per cent. When used alone in a dose of 0.1 mg per mouse papain prevented death in 20 per cent of animals. The high chemotherapeutic efficacy of the antibiotic combinations with papain allowed recommending it for use in clinical practice for the treatment of infections caused by antibiotic-resistant staphylococci.

Animals↗

[Physicochemical properties and amino acid composition of papain fragments of immunoglobulin G isolated from blood of cows suffering from leukosis].

It is shown that the peculiar to the malignant growth protein isolated from blood of cattle suffering from leukosis is more stable to the effect of papain than the human protein characteristic of the malignant growth. Papain breaks the studied protein up into three components, the sedimentation constants of Fab- and Fc-fragments isolated from the papain hydrolysate are 3,9 and 3,6S and their molecular mass --55000 and 45000 Daltons, respectively. In the Fab-fragment the contents of lysine, glutaminic acid, leucine, serine are higher and those of arginine, alanine, valine, methionine, phenylalanine and isoleucine are lower as compared with the Fab-fragment isolated from immunoglobulin G of healthy animals. The content of amino acids changes more significantly in the Fab-fragment that in the unsplitted molecule of the protein under study. The polar-to-nonpolar amino acids ratio is 2.27 for the Fab-fragment and 2.48 for the unsplitted molecule. For the same structures of protein isolated from immunoglobulin G of health animals these values are 1.80 and 2.24, respectively. Evidently, peculiarities of the specific to the malignant growth protein isolated from blood of cattle suffering from leukosis are characteristic mainly of its Fab-fragment.

Amino Acids↗

Early ultrastructural changes in papain-induced experimental emphysema.

These studies show that very soon after exposure of canine lungs to crude papain mixed with a marker (India ink), the alveolar surface-active lining was both morphologically and functionally altered, and alveolar macrophages were destroyed in significant numbers. These changes occurred before other identifiable major alterations. The morphologic changes were characterized by replacement of the normal alveolar surface lining by an amorphous material adhering to the alveolar surfaces and penetrating the pores of Kohn, which were significantly enlarged. Type II epithelial cells and alveolar macrophages were increased in number, with a significant proportion of the latter observed to be disintegrating. Polymorphonuclear leukocytes were also noted in increased numbers, but this was also observed in control lungs (instilled only with saline plus India ink). Functionally the alveolar surface lining as studied in lung extracts showed an abnormal stability index after either in vivo or in vitro exposure to crude papain. These findings suggest that the early effects of this exogenous protease on the lung are interrelated, alveolar surface lining injury appearing to set the stage for accelerated macrophage lysis, with the probable release of phagocytosed exogenous protease as well as intracellular endogenous proteases. These events may represent early steps in the pathogenesis of experimental papain-induced pulmonary emphysema.

Animals↗

Synthesis of N alpha-protected aminoacyl 7-amino-4-methyl-coumarin amide by phosphorous oxychloride and preparation of specific fluorogenic substrates for papain.

We report an improved procedure for the synthesis of fully protected aminoacyl 7-amino-4-methylcoumarin amide (MCA) employing the phosphorous oxychloride anhydride method. Seven Boc-X-MCA [where X = Arg(NG Tos), Cys(S-Bzl), Thr(O-Bzl), Ser(O-Bzl), Phe, Leu and Gly] and Z-Tyr(O-Me) were synthesized using this procedure, with yields ranging from 50% to 75%. These aminoacyl-MCA derivatives were employed for the synthesis of epsilon-NH2-caproyl-Leu-X-MCA, a fluorescent peptide series, which were assayed as papain substrates. All of them were completely hydrolyzed by papain, indicating that all of the Boc-X-MCA derivatives obtained were practically free of racemization. Since epsilon-NH2-Caproyl-Leu-(S-Bzl)Cys-MCA is very susceptible to hydrolysis by papain, quite resistant to hydrolysis by chymotrypsin and not hydrolyzed by trypsin, it is recommended for assays of thiol-proteinases in which specificity is required.

Amino Acids↗

Structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors.

Cysteine proteinases are widely distributed among living organisms. According to the most recent classifications (Rawlings and Barrett, 1993, 1994), they can be subdivided on the basis of sequence homology into 14 or even 20 different families, the most important being the papain and the calpain families. The papain-like cysteine proteinases are the most abundant among the cysteine proteinases. The family consists of papain and related plant proteinases such as chymopapain, caricain, bromelain, actinidin, ficin, and aleurain, and the lysosomal cathepsins B, H, L, S, C and K. Most of these enzymes are relatively small proteins with Mr values in the range 20000-35000 (reviewed in Brocklehurst et al., 1987; Polgar, 1989; Rawlings and Barrett, 1994; Berti and Storer, 1995), with the exception of cathepsin C, which is an oligomeric enzyme with Mr approximately 200000 (Metrione et al., 1970; Dolenc et al., 1995). A number of cysteine proteinases are located within lysosomes. Four of them, cathepsins B, C, H and L, are ubiquitous in lysosomes of animals, whereas cathepsin S has a more restricted localisation (Barrett and Kirschke, 1981; Kirschke and Wiederanders, 1994). The enzymes, except cathepsin C, are endopeptidases (reviewed in Kirschke et al., 1995), although cathepsin B was found also to be a dipeptidyl carboxypeptidase (Aronson and Barrett, 1978) and cathepsin H also an aminopeptidase (Koga et al., 1992). Cathepsin C is a dipeptidyl aminopeptidase, but at higher pH it exhibits also dipeptidyl transferase activity (reviewed in Kirschke et al., 1995). Among the lysosomal cysteine proteinases, cathepsin L was found to be the most active in degradation of protein substrates, such as collagen, elastin and azocasein (Barrett and Kirschke, 1981; Maciewicz et al., 1987; Mason et al., 1989), arid cathepsin B the most abundant (Kirschke and Barrett, 1981). All the enzymes are optimally active at slightly acidic pH, although their pH optima for degradation of synthetic substrates vary from 5.5 for cathepsin L to 6.8 for cathepsin H (reviewed in Kirschke et al., 1995). Several other lysosomal cysteine proteinases, such as cathepsins N, T and K, are known, although their properties are less well characterised (reviewed in Kirschke et al., 1995). In particular cathepsin K has attracted recent interest (Bromme et al., 1996; Shi et al., 1995; Bossard et al., 1996; Drake et al., 1996) and was found to be expressed specifically in osteoclasts (Drake et al., 1996) with properties similar to cathepsin L (Bossard et al., 1996).

Animals↗

Mouse stefins A1 and A2 (Stfa1 and Stfa2) differentiate between papain-like endo- and exopeptidases.

Stefin A (Stfa) acts as a competitive inhibitor of intracellular papain-like cysteine proteases which play important roles in normal cellular functions such as general protein turnover, antigen processing and ovarian follicular growth and maturation. In the mouse there are at least three different variants of Stfa (Stfa1, Stfa2 and Stfa3). Recent genetic studies identified structural polymorphisms in Stfa1 and Stfa2 as candidates for Aod1b, a locus controlling susceptibility to day three thymectomy (D3Tx)-induced autoimmune ovarian disease (AOD). To evaluate the functional significance of these polymorphisms, recombinant allelic proteins were expressed in Escherichia coli, purified and characterized. The polymorphisms do not markedly alter the folding characteristics of the two proteins. Stfa1 and Stfa2 both act as fast and tight binding inhibitors of endopeptidases papain and cathepsins L and S, however their interaction with exopeptidases cathepsins B, C and H was several orders of magnitude weaker compared to human, porcine and bovine Stfa. Notwithstanding, the K(i) values for the interactions of Stfa1-b from AOD resistant C57BL/6J mice was 10-fold higher than that of the Stfa1-a allele from susceptible A/J mice for papain, cathepsins B, C and H but not L and S. In contrast, the inhibitory activities of Stfa2-a and Stfa2-b were found to be roughly equivalent for all targets peptidases.

Amino Acid Sequence↗

A model of interfacial inactivation for papain in aqueous organic biphasic systems.

A model was proposed to describe the effects of the main factors in aqueous-organic two-liquid-phase media on the stability of papain. The relationships between the half-life of papain activity and these factors including interfacial tension, stirring rate, phase volume ratio and temperature were investigated. The results showed that these factors had notable effects on papain stability except temperature. The correlation coefficient between the model and the experimental data were 0.829, which indicated the model is practicable.

Journal Article↗