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PubMed1990

[Peptide synthesis catalyzed by proteases. The effect of temperature on kinetics of acyl transfer catalyzed by papain].

The effect of temperature on the kinetics of papain-catalyzed peptide synthesis was studied. A characteristic feature of this process is that the peptide synthesis is accompanied both by the hydrolysis of the synthesized bond and by the further elongation of the peptide chain. These phenomena yield the maximum on the time dependence of the first synthetic product. A decrease in temperature is an effective way to increase the yield of this substance which reflects the increase in the nucleophil reactivity resulting from the temperature decrease. Moreover, the latter diminishes the contribution of secondary enzymatic reactions such as further hydrolysis of the reaction product and polypeptide chain elongation.

Catalysis↗
PubMed1990

Molecular cloning of a protein associated with soybean seed oil bodies that is similar to thiol proteases of the papain family.

A 34,000-Da protein (P34) is one of the four major soybean oil body proteins observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated organic solvent-extracted oil bodies from mature seeds. P34 is processed during seedling growth to a 32,000-Da polypeptide (P32) by the removal of an amino-terminal decapeptide (Herman, E.M., Melroy, D.L., and Buckhout, T.J. (1990) Plant Physiol, in press). A soybean lambda ZAP II cDNA library constructed from RNA isolated from midmaturation seeds was screened with monoclonal antibodies directed against two different epitopes of P34. The isolated cDNA clone encoding P34 contains 1,350 base pairs terminating in a poly(A)+ tail and an open reading frame 1,137 base pairs in length. The open reading frame includes a deduced amino acid sequence which matches 23 of 25 amino-terminal amino acids determined by automated Edman degradation of P34 and P32. The cDNA predicts a mature protein of 257 amino acids and of 28,641 Da. The open reading frame extends 5' from the known amino terminus of P34 encoding a possible precursor and signal sequence segments with a combined additional 122 amino acids. Prepro-P34 is deduced to be a polypeptide of 42,714 Da, indicating that the cDNA clone apparently encodes a polypeptide of 379 amino acids. A comparison of the nucleotide and deduced amino acid sequences in the GenBank Data Bank with the sequence of P34 has shown considerable sequence similarity to the thiol proteases of the papain family. Southern blot analysis of genomic DNA indicated that the P34 gene has a low copy number.

Amino Acid Sequence↗
PubMed1978

Dependence of the catalytic activity of papain on the ionization of two acidic groups.

The pH dependence of kcat/Km for the papain-catalyzed hydrolysis of ethyl hippurate, N-alpha-benzoyl-L-citrulline methyl ester, and the p-nitroanilide, amide, and ethyl ester derivatives of N-alpha-benzoyl-L-arginine was determined below pH 6.4. The value of kcat/Km was observed to be modulated by two acid ionizations rather than a single ionization as previously believed. For the five substrates studied, the average pK values for the two ionizations are 3.78 +/- 0.2 and 3.95 +/- 0.1 at T/2 0.3, 25 degrees C. The observation that similar pK values were obtained with different substrates was taken as evidence that the kinetically determined pK values are close in value to true macroscopic ionization constants for ionization of groups on the free enzyme.

Hydrogen-Ion Concentration↗
PubMed1988

Effects of phosphorylation, MgATP, and ionic strength on the rates of papain degradation of heavy and light chains of smooth muscle heavy meromyosin at the S1-S2 junction.

The effects of ionic strength, MgATP, and phosphorylation on the degradation rates of heavy meromyosin (HMM) by papain have been compared to their effects on the sedimentation coefficient (s20,w) to determine the relationship of the degradation rate to the equilibrium between the flexed and the extended forms (Suzuki, H., Stafford, W. F., Slayter, H. S., and Seidel, J. C. (1985) J. Biol. Chem. 260, 14810-14817). At 0.025 M NaCl, where HMM is predominantly in the flexed form, MgATP, Mg-adenylyl imidodiphosphate or MgADP reduce kH by 80-90%. MgATP exerts its optimal effect at this ionic strength, where at least 70% of HMM is flexed in the presence or absence of MgATP, suggesting that nucleotides reduce kH by decreasing the proteolytic susceptibility of the flexed form. At 0.5 M NaCl, where HMM is in the extended form, MgATP has no effect on kH. At low ionic strengths phosphorylation decreases kH but increases it in the presence of MgATP. Plots of kH against s20,w determined at various ionic strengths are linear, the data for phosphorylated and dephosphorylated HMM falling on the same line. Thus, raising the ionic strength or phosphorylating the 20-kDa light chain appears to alter kH by increasing the fraction of HMM in the extended form. The degradation rate of the 20-kDa light chain (kL) of dephosphorylated HMM responds to changes in ionic strength in essentially the same way as does kH, suggesting that the response of kL to changes in ionic strength can also be attributed to conversion of HMM to the extended form. However, kL for phosphorylated HMM measured in the presence of MgATP exhibits very little dependence on ionic strength.

Adenosine Triphosphate↗
PubMed1988

[Synthesis of peptides catalyzed by papain in organic solvents with minimal water content].

Synthesis of a model peptide, Z-Ala-Val-OBut, catalyzed by papain in acetonitrile containing 0-5% water was studied. Z-Ala-OCH2CONH2 was used as a carboxyl component. The optimal concentration of water was found to be 0.3-0.5%. Under these conditions the reaction rate is 50 to 100 times lower than in water but the yield of the peptide can be as high as 99.5%. Effect of reagent concentrations and temperature on the reaction was also studied.

Acetonitriles↗
PubMed1986

Benzoylamidoacetonitrile is bound as a thioimidate in the active site of papain.

13C NMR spectroscopy has been used to demonstrate that 13CN-labeled benzoylamidoacetonitrile forms a covalent adduct with the thiol group of cysteine 25 in the active site of papain. Spectral comparison with model compounds indicates that the adduct is a thioimidate. On the basis of a proposed mechanism for the formation of the thioimidate, it is concluded that the -CH2C(= NH)S--imino nitrogen does not sit in the active site in the same manner as the thiol ester carbonyl oxygen of the thiol acyl enzyme (or the oxyanion of the tetrahedral intermediate). Thus, in this sense the stabilization of the thioimidate does not reflect a similarity in structure between the bound thioimidate and the transition state.

Acetonitriles↗
PubMedDate not supplied

[Determination of papain activity].

A method is developed to determine the proteolytic activity of papain by the hydrolysis of protamine sulphate, a low-molecular weight protein rich in arginine. Sensitivity of the method is 20 times as high as sensitivity of the known methods for determination of the enzyme activity by the cleavage of hemoglobin and casein.

Arginine↗
PubMed1986

[Effect of papain and levamisole on the body's immunological indices and oxidation-reduction processes in experimental sensitization].

The effect of papain on sensitized animals was evident from activation of oxidation processes in the liver and adrenal glands, increased ability of the host to develop cellular immunity reactions, a moderate decrease in the level of humoral antibodies and inactivation of the monooxygenase system of the liver and adrenal glands. Levamisol had a favourable effect on the immunological reactions and monooxygenase system. However, it induced an increase in tissue hypoxia of the organs.

Adrenal Glands↗
PubMedDate not supplied

[Use of microfilaria treated with papain in the serodiagnosis of lymphatic filariasis caused by Wuchereria bancrofti (Var. pacifica)].

A standardised method for serodiagnosis of lymphatic filariasis W. bancrofti (var. pacifica) was performed. The authors used indirect fluorescent assay with papain treated microfilariae as source of antigen. Very high levels of antibodies were obtained in 96% microfilaremic patients. The geometric mean titers were 22.7 for healthy subjects and 605.5 for the filariasis patients. Cross reactivity was studied with two subcutaneous and one lymphatic filariasis. The geometric mean titers were 211.1 for Onchocerca volvulus, 105.6 for Loa-loa and 254 for Brugia malayi patients. No significant reactivity was observed with some other parasites.

Cross Reactions↗
PubMed1985

[Synthesis of leucine- and methionine-enkephalin using papain].

Thiol protease papain has been used for synthesis of leucine-and methionineenkephalin from methyl esters of N-protected amino acids. The synthesis was carried out in basic medium, minimizing the hazard of the secondary peptide hydrolysis. The reaction products remain in solution during the whole process. The yields at the final stage of the synthesis were 89% (leucineenkephalin) and 79% (methionineenkephalin).

Chemical Phenomena↗
PubMed1985

[Immobilized papain in the treatment of acute destructive lactation mastitis].

Experience with the surgical treatment of 123 patients has shown the positive effect of proteolytic enzyme Papain immobilized on fiber polymer materials. It shortened the period of purification of purulent wounds of the lactating mammary gland from devitalized tissues which facilitated putting early secondary sutures on the granulating wound without a dissection of wound margins and resulted in quicker healing (13,5 days).

Anti-Infective Agents, Local↗
PubMed1979

The use of resonance Raman spectroscopy to monitor catalytically important bonds during enzymic catalysis. Application to the hydrolysis of methyl thionohippurate by papain.

Resonance Raman spectra were obtained of the dithioacyl-enzyme intermediate produced during the papain-catalayzed hydrolysis of methyl thionohippurate. Intense resonance Raman features were observed in (formula; see text) and (formula; see text) stretching regions from the intermediate's (formula; see text) chromophore. These results demonstrate that by using a single atom replacement, i.e. sulfur for oxygen, the catalytically crucial bonds in the ester moiety can be monitored during enzymolysis via the resonance Raman spectrum. The method can be extended to other enzymes whose catalytic mechanisms involve the formation of a thiol-acyl intermediate.

Hippurates↗
PubMed1979

Purification and chemical characterization of papain-solubilized histocompatibility-2 antigens from mouse liver.

A large scale purification of histocompatibility-2 (H-2) antigens from mouse liver is described. The antigens were solubilized by a limited papain digestion of a crude preparation of liver membranes (strain A/J) and purified by ion exchange chromatography, gel filtration, affinity chromatography, and isoelectric focusing. The overall degree of purification of H-2Kk was 1,300-fold and that of H-2Dd was 1,500-fold; approximately 8 mg of purified H-2a antigens were obtained from 1 kg of liver. The purification was followed by a sensitive radioimmunoassay in which H-2a-containing fractions were used to inhibit the binding of 125I-labeled H-2a to appropriate antisera. H-2Dd and H-2Kk co-purified through all the steps but the concentration of H-2Kk was 2- to 3-fold higher than that of H-2Dd in the liver homogenate as well as in the purified H-2 preparation. beta 2-microglobulin was initially present in a 3- to 10-fold excess over H-2 in the liver homogenate, but the purified H-2 preparation contained approximately 2 mol of alloantigenic heavy chain/mol of beta 2-microglobulin. Isoelectric focusing and disc-gel electrophoresis showed a charge heterogeneity of H-2, with a mean isoelectric point of pH 4.9. Electrophoresis on sodium dodecyl sulfate gels showed one band. Denaturing conditions were required to remove beta 2-microglobulin and small amounts of impurities from H-2. The amino acid sequence of the first 27 residues of the isolated heavy chains was determined.

Amino Acid Sequence↗
PubMed1979

[Electrophoretic study of products of wall-eyed pollock and bovine rhodopsins fragmented by papain].

Fragmentation of wall-eyed pollock and bovine rhodopsins by papain in the photoreceptor membrane was studied by sodium dodecyl sulfate electrophoresis. A scheme of step-wise rhodopsin proteolysis is presented. The molecular weights and localization of the carbohydrate and chromophore components of the fragments formed were determined. The data obtained suggest that the photoreceptor membranes of both rhodopsins contain three sites accessible to water environment and are indicative of topographical similarity of the rhodopsins.

Animals↗
PubMed1983

The pH dependency of bovine spleen cathepsin B-catalyzed transfer of N alpha-benzyloxycarbonyl-L-lysine from p-nitrophenol to water and dipeptide nucleophiles. Comparisons with papain.

Cathepsin B has been shown to catalyze the transfer of the N alpha-benzyloxycarbonyl-L-lysyl residue from the corresponding p-nitrophenyl ester substrate to water and dipeptide nucleophiles. These reactions occurred through the formation of an acyl-enzyme intermediate. The pH dependency of the acylation and deacylation steps were determined from the increases in the maximum rate of appearance of p-nitrophenol on addition of glycylglycine or L-leucylglycine to the reaction. The second order acylation rate constant, kcat/Km was found to depend on the state of ionization of three groups in the enzyme having pKa values of 4.2, 5.5, and 8.6. Protonation of the group with pKa = 5.5 decreased but did not abolish enzymatic activity, resulting in the appearance of a second, active protonic form of the enzyme between pH 4.2 and pH 5.5. The first order rate constant for the hydrolysis of the acyl-enzyme intermediate was independent of pH between 4.0 and 7.5. In contrast, acyl group transfer from cathepsin B to glycylglycine and L-leucylglycine depended on a group with a pKa of about 4.5. These results are discussed in terms of possible structural and functional homologies between the active sites of cathepsin B and papain.

Animals↗
PubMed1978

The effect of a single injection of papain on growth of the limb bones of the mouse.

The chondrodystrophic mouse mutant stumpy (stm) shortens proximal limb bones more than distal ones and acts late in development (circa 14 days post partum). It was thought that a late disturbance in bone growth might cause more damage to epiphyses of proximal than distal bones. This was investigated using a single injection of papain on days 8, 10 and 12: shortening of proximal and distal bones was demonstrated, but was similar in all three experimental groups.

Achondroplasia↗
PubMed1977

Antigenic specificities detected on papainized human granulocytes by microgranulocytotoxicity.

A reliable and routine identification of human granulocyte antigens is possible with an improved microgranulocytotoxicity assay. Freshly isolated, papainized granulocytes, an extended incubation period, and alcohol to prevent the adherence of cells are important factors enhancing cellular reactivity. Absorption studies indicate an absence of B-lymphocyte alloantigens on granulocytes. Five tentative granulocyte specificities were defined with granulocytotoxic non-HLA alloantisera. Sib-pair studies suggest the independent chromosomal segregation of the granulocyte and HLA antigen systems.

B-Lymphocytes↗
PubMed1980

[Induction of partial immune tolerance in a closely-related species system by a single injection of papain-extracted, donor species specific, lymphocyte membrane antigens].

This paper reports of experimental xenografting of kidneys in closely related species (fox-dog) modifying primary graft rejection by single i.v. injection of donorspecies-specific lymphocyte antigen in soluble form. Papain treatment was used to separate histocompatibility antigens from lymphocytes of donors. This antigen, in vitro, is capable of reducing cytotoxicity of xeno-antisera by adsorption of their lymphocytotoxic antibodies. A single intraoperative i.v. injection of this antigen to the transplant recipients results in the subsequent failure of humoral antibody production, thus this antigen-mediated effect is thought to be a state of B-cell tolerance. Survival times are more than doubled in comparison to untreated controls and last in average 14.6 +/- 1.74 days (n = 5). Histological studies demonstrate rather well conserved renal parenchym with sporadic infiltrations of round cells.

Animals↗