Relationship between sensitivity and density of muscarinic receptors in single smooth muscle cells of guinea pig taenia caecum prepared under three conditions

The relationship between the sensitivity (the pD2 value) of carbachol and the density (the total concentration of receptors) of muscarinic receptors using single cells from the guinea pig taenia caecum prepared with a mixture of crude collagenase and trypsin inhibitor, purified collagenase alone, and a mixture of purified collagenase and papain was examined. The sensitivity of the single cells prepared with a mixture of purified collagenase and papain was about 10 times more effective than that of the single cells prepared under other conditions. The dissociation constant of [3H]quinuclidinyl benzilate (QNB) and Hill's coefficient did not change in the single cells prepared under the three conditions, though the maximum binding sites were significantly greater in the cells prepared with the mixture of purified collagenase and papain than in those prepared by other means. These results suggest that the increase in the sensitivity of carbachol obtained in the single cells prepared with this mixture is due to the increase in the density of muscarinic receptors and also suggest that the effects of this enzyme mixture may be due to an increase in the incorporation of newly synthesized receptors and (or) changes in receptor turnover.     

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.     

Mercaptan-induced fragmentation of a subunit-like proteolytic fragment of immunoglobulin M

Limited papain hydrolysis of immunoglobulin M (IgM) produces a subunit-like proteolytic fragment designated IgM(p) (Inman & Hazen, 1968). In the presence of mercaptans, IgM(p) partially dissociated into Fc(mu)-like and Fab(mu) fragments. Treatment of residual IgM (that remaining after a papain digestion) with 2mm-mercaptoethylamine resulted in fragmentation of the same type that occurs in a routine limited digestion of IgM with papain, although exogenous enzyme was not added to the mixture. When IgM was hydrolysed with (14)C-labelled papain, a small quantity of the enzyme was found to be associated with the residual IgM and IgM(p) fractions. IgM and IgM 7S subunit (IgM(s)) that had been exposed to papain in the absence of activating mercaptan and separated from the enzyme by gel filtration also fragmented when subsequently treated with 2mm-mercaptoethylamine. The fragments resembled those produced during a typical limited papain digestion of IgM. It was concluded that mercaptoethylamine induced fragmentation of IgM(p) by activating adsorbed papain.     

甲醇溶液对木瓜蛋白酶催化活性的影响

试验结果表明,木瓜蛋白酶在一定浓度甲醇溶液中水解酪蛋白的活性显著上升;动力学测定表明,该酶在甲醇溶液中Km下降;在甲醇溶液中木瓜蛋白酶催化酪蛋白水解反应的最适pH为8.5,最适温度为70℃;紫外差示光谱研究表明,在甲醇溶液作用下木瓜蛋白酶的二级结构发生了变化;荧光发射光谱研究表明,木瓜蛋白酶在甲醇溶液中发射峰位向低波长移动,荧光峰值明显增高。     

Non-enzymatic glycation of IgG: an in vivo study.

The IgG glycation level of 30 healthy subjects and 60 type 2 diabetic patients with different degrees of metabolic control was evaluated by matrix-assisted laser desorption ionization mass spectrometry, a technique allowing the determination of mass increase of the IgG molecule. When applied to the digested mixture obtained by the action of papain on the plasma protein fraction, the same method established the mass increase of Fab and Fc fragments of IgG; for the former, a higher mass increase was found, possibly explained by its high reactivity to glucose. Experimental results were confirmed by molecular modeling calculations. Results suggest that the immunodeficiency observed in diabetic patients may be due to the inhibition of molecular recognition between antibody and antigen as a result of a change in functionality of the modified Fab fragment of IgG.     

A necessary modification to the preparation of papain from any high-quality latex of Carica papaya and evidence for the structural integrity of the enzyme produced by traditional methods

A method of preparation of papain (EC 3.4.22.2) from relatively soluble types of latex of Carica papaya, including spray-dried latex produced by a controlled and relatively mild process, was devised. Spray-dried latex dissolves easily in water up to 350mg/ml at 22 degrees C, which corresponds to approx. 230mg of protein/ml. When the usual method of preparation of crystalline papain contaminated only by its oxidation products, developed by Kimmel & Smith [J. Biol. Chem. (1954) 207, 515-531], is applied to spray-dried latex, the result is a preparation of papain heavily contaminated by chymopapains A and B (EC 3.4.22.6), and to a lesser extent by papaya peptidase A. This applies also to other types of papaya-latex currently commercially available, which, though less soluble than spray-dried latex, are more soluble than the types of latex available when the method of Kimmel & Smith (1954) was developed. This contamination is avoided by adjusting the concentration of the initial latex extract to 65mg of protein/ml (or less) before salt fractionation. For spray-dried latex this corresponds to 100mg of latex/ml. Papain isolated from spray-dried latex was characterized by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as thiol-specific reactivity probes and alpha-N-benzoyl-l-arginine ethyl ester as substrate. Papain isolated from this source appears to have the same catalytic-centre characteristics as papain isolated previously from latex produced by harsher methods. The catalysis of the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester by the mixture of thiol proteinases extracted from spray-dried latex by application of the method of Kimmel & Smith (1954) appears to obey Michaelis-Menten kinetics. The presence of the other enzymes results in an increase in the value of K(m) and a decrease in the catalytic-centre activity (k(cat.)) relative to the values for the catalysis by papain.     

Papain kinetics in the presence of a water-miscible organic solvent

The effects of various concentrations of a water-miscible organic solvent [a 7:3 (v/v) mixture of N, N dimethylformamide and dimethylsulfoxide] on the kinetics of papain have been investigated. The parameters k(cat) and K(m) for the amidase and esterase activity of papain using N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and N-alpha-benzoyl-L-arginine ethyl ester (BAEE) as substrates were determined. For both types of activity, k(cat) initially increased (up to about 15% solvent), and then decreased with increasing concentrations of organic solvent. In contrast, K(m) increased sharply with the organic solvent concentration. Active site titration at 0 and 50% solvent indicated no change in the amount of active enzyme. Fluorometric measurements of the emission spectrum of papain did not indicate any major conformational changes with increasing concentrations of organic solvent.     

Cysteine proteinase activity regulation. A possible role of heparin and heparin-like glycosaminoglycans.

Papain is considered to be the archetype of cysteine proteinases. The interaction of heparin and other glycosaminoglycans with papain may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions that can regulate the function of this class of proteinases in vivo. The conformational changes in papain structure due to glycosaminoglycan interaction were studied by circular dichroism spectroscopy, and the changes in enzyme behavior were studied by kinetic analysis, monitored with fluorogenic substrate. The presence of heparin significantly increases the alpha-helix content of papain. Heparin binding to papain was demonstrated by affinity chromatography and shown to be mediated by electrostatic interactions. The incubation of papain with heparin promoted a powerful increase in the affinity of the enzyme for the substrate. In order to probe the glycosaminoglycan structure requirements for the papain interaction, the effects of two other glycosaminoglycans were tested. Like heparin, heparan sulfate, to a lesser degree, was able to decrease the papain substrate affinity, and it simultaneously induced alpha-helix structure in papain. On the other hand, dermatan sulfate was not able to decrease the substrate affinity and did not induce alpha-helix structure in papain. Heparin stabilizes the papain structure and thereby its activity at alkaline pH.     

Effects of Various Gases on the Survival of Dried Bacteria During Storage

Salmonella newport and Pseudomonas fluorescens were dried together in papain digest broth and sucrose-glutamate, and stored in several gases at various water activities (a(w)) between 0.00 and 0.40 at 25 C for various periods up to 81 weeks. Both S. newport and P. fluorescens, dried in papain digest broth and stored in air, died rapidly if the conditions were very dry (0.00 a(w)) or moist (0.40 a(w)). Storage in carbon dioxide and argon gave greater survival than storage in air but lower survival than did storage in nitrogen or in vacuo. When the organisms were dried in a sucrose-glutamate mixture the differences between the gases were very small, and variations in residual water were less important. Of the inert gases, argon gave the best survival when the organisms were dried in papain digest broth, especially at 0.00 a(w); the survival in neon and krypton was lower and in xenon and helium it was much lower.     

Mechanism of solvent induced thermal stabilization of papain

In the present study an attempt is made to elucidate the effects of various cosolvents, such as sorbitol, sucrose, xylose and glycerol, on papain. The stabilizing effects of these cosolvents on the structure and function of papain is determined by the activity measurements, fluorescence spectroscopy and differential scanning calorimetry (DSC). The enzyme activity measurements indicate several fold increase in the thermal stability of the enzyme in all the cosolvents used. The thermal denaturation studies of papain in presence of various concentrations of cosolvents indicated a shift in the apparent thermal denaturation temperature (app Tm) suggesting increased thermal stability of papain in presence of cosolvents. The app Tm shifted from a control value of 83+/-1 degrees C to a value of >90+/-1 degrees C in presence of 40% sorbitol. The DSC thermogram for native papain can be clearly deconvoluted into two transitions corresponding to left and right domain and in presence of cosolvents both transitions A and B shift to higher temperature. Maximum stabilization was seen in case of 30% sorbitol where the thermal transition temperatures increased compared to control. The results from partial specific volume measurements of papain in presence of cosolvents suggest that the preferential interaction parameter (xi3) was negative in all cosolvents and maximum hydration was observed in the case of glycerol where the preferential interaction parameter was 0.165g/g. These above results suggest that there is a considerable increase in the thermal stability of papain in presence of these cosolvents as a result of preferential hydration.     

Stability of protease in organic solvent: structural identification by solid-state NMR of lyophilized papain before and after 1-propanol treatment and the corresponding enzymatic activities

Lyophilized enzyme powder is often used in organic solvents. However, the enzymatic activity decreases during the reaction process. In the present study, the relation between structural stability and enzymatic activity in an organic solvent was investigated. 13C cross-polarization magic angle spinning NMR spectroscopy was used to determine the secondary structure of lyophilized papain in the solid-state. Deconvolution of the peaks of the backbone carbonyl carbons suggested that the proportion of beta-sheet conformation increased after lyophilization from a phosphate buffer solution. The esterification of N-benzyloxycarbonyl phenylalanylalanine amide was attempted using the lyophilized papain as a catalyst in anhydrous 1-propanol. The yield of ester was 46.1% after 48 h at 50 degrees C, but this reaction slowed remarkably after 48 h. When the lyophilized papain was suspended in anhydrous 1-propanol for 7 days without the substrate, the proportion of beta-sheet conformation was further increased and the suspended papain had no activity. These results suggest that the increase in beta-sheet conformation caused inactivation of papain. The increase in beta-sheet conformation caused by both lyophilization and suspension in propanol was found, which was related to a decrease in enzymatic activity.     

Subsite differences between the active centres of papaya peptidase A and papain as revealed by affinity chromatography. Purification of papaya peptidase A by ionic-strength-dependent affinity adsorption on an immobilized peptide inhibitor of papain.

An affinity column consisting of the specific peptide inhibitor of papain, Gly-Gly (O-benzyl)Tyr-Arg, attached to Sepharose was found to bind the active thiol proteinase papaya peptidase A specifically, but only at an ionic strength significantly higher than the one at which papain is bound. When a mixture of active papaya peptidase A and its irreversibly oxidized contaminant was applied to the column, the active enzyme was bound whereas the inactive material was not. The bound enzyme was released by deionized water and found to contain 1 mol of SH group/mol of protein. The different conditions required for the binding of the two enzymes to the immobilized peptide was shown to reflect different ionic-strength-dependences of the affinity of the two enzymes for the peptide in solution. Whereas the affinity of papain for the inhibitor appears to be insensitive to ionic strength over the range studied, that of papaya peptidase A is ionic-strength-dependent and always lower than that of papain. A rate assay is devised for papaya peptidase A with N-benzyloxycarbonylglycine p-nitrophenyl ester as the substrate at pH 5.5. After calibration against an active-site titration the assay yields the thiol-group concentration without interference from inactive contaminants. For the papaya peptidase A-catalysed hydrolysis of N-benzyloxycarbonylglycine p-nitrophenyl ester at pH 5.5 kcat. was found to be 16.7s-1, which is about 3 times the value found for the same reaction catalysed by papain.     

Activation of glutaraldehyde-crosslinked chymotrypsinogen-A. Enzymatic activity and circular dichroism studies

Phenylhydrazine does not inactivate papain or glyceraldehyde-3-phosphate dehydrogenase under anaerobic conditions. The inactivation of papain and glyceralde-hyde-3-phosphate dehydrogenase under aerobic conditions is ascribed to the oxidation of phenylhydrazine by O₂ which generates phenyldiimide and H₂O₂, both of which react with the essential sulfhydryl groups and inactivate the enzymes. Phenyldiimide generated from methyl phenylazoformate inactivates both of the sulfhydryl enzymes under anaerobic conditions. The inactivation of papain and GPD with aerobic, aqueous solutions of [¹⁴C]phenylhydrazine introduces a small amount of radioactivity into the enzymes which is discharged by dithiothreitol. The amount of radioactivity bound to papain is increased when cyanide is present in the inactivation mixture.When the β-[¹⁴C]thiocyanoalanine derivative of papain is treated with phenylhydrazine the radioactivity is discharged from the enzyme. Cyanide evidently reacts with the sulfenic acid derivative of papain to form a thiocyanate derivative. Phenylhydrazine presumably displaces cyanide from the thiocyanate derivative to form a sulfenyl hydrazide derivative to account for the increased incorporation of [¹⁴C]phenylhydrazine when papain is inactivated with aerobic solutions of [¹⁴C]-phenylhydrazine in the presence of cyanide. When the sulfhydryl group of papain is oxidized to a sulfenic acid with H₂O₂ and then treated with [¹⁴C]phenylhydrazine, ¹⁴C is not incorporated into the enzyme. These experiments suggest that the H₂O₂ in the aerobic solutions of phenylhydrazine oxidizes the sulfhydryl group at the active site of papain to a sulfenic acid. The [¹⁴C]phenyldiimide in these solutions reacts to some extent with the active sulfhydryl group to form a sulfenyl hydrazide derivative.     

Enzymatic hydrolysis of nitriles by an engineered nitrile hydratase (papain Gln19Glu) in aqueous-organic media

A mutant of the cysteine protease papain, displaying nitrile hydratase and amidase activities, was expressed in Pichia pastoris and used for the hydrolysis of peptide nitriles in aqueous-organic media. The rate of hydrolysis of these nitriles is lowered by increasing acetone concentration. This is caused by an increase of the Michaelis constant, and a variation of Vmax proportional to the amount of water in the mixture. The hydrolysis of the amide is less affected by the increase in co-solvent, which results in lower accumulation of this intermediate product. With the peptide nitrile tested, high nitrile concentrations could be used to promote the production of the amide and prevent its hydrolysis to the acid by diminishing the relative rate of amide hydrolysis. A number of non-peptidyl nitriles were also tested as potential substrates but activity was detected for only one compound with structural resemblance to peptide nitriles.     

Preparation of fully active ficin from Ficus glabrata by covalent chromatography and characterization of its active centre by using 2,2''-depyridyl disulphide as a reactivity probe.

1. Fully active ficin (EC 3.4.22.3) containing 1 mol of thiol with high reactivity towards 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) at pH4.5 per mol of protein was prepared from the dried latex of Ficus glabrata by covalent chromatography on a Sepharose-glutathione-2-pyridyl disulphide gel. 2. Ficin thus prepared is a mixture of ficins I-IV and ficin G, in which ficins II and III predominate. The various ficins exhibit similar reactivity characteristics towards 2-Py-S-S-2-Py. 3. Use of 2-Py-S-S-2-Py as a reactivity probe demonstrates (a) that in ficin, as in papain (EC 3.4.22.2), the active-centre thiol and imidazole groups interact to provide a nucleophilic state at pH values of approx. 6 additional to the uncomplicated thiolate ion that predominates at pH values over 9, and (b) a structural difference between ficin and papain that leads to a much higher rate of reaction of 2-Py-S-S-2-Py with ficin than with papain at pH values 3-4. This difference is suggested to include a lack in ficin of a carboxyl group conformationally equivalent to that of aspartic acid-158 in papain. 4. The high electrophilicity of the 2-Py-S-S-2PyH+ monocation allows directly the detection of the exposure of the buried thiol group of ficin at pH values below 4.     

Activation of lipoprotein lipase by blood platelets

When an homogenate of human blood platelets is added to preparations of partially purified adipose tissue lipoprotein lipase, the lipase activity is markedly enhanced. The degree of activation is related to the amount of platelet homogenate in the reaction mixture. The lipoprotein lipase activator in platelets is heat labile, nondialyzable, partially resistant to papain, and present mostly in the particulate fraction of platelet homogenates.     

Effects of exercise training on papain-induced pulmonary emphysema in Wistar rats.

The purpose of the present study was to evaluate the role of exercise training on the development of papain-induced emphysema in rats. Our hypothesis was that the increase in pulmonary tissue stretching associated with exercise could increase the severity of a protease-induced emphysema. Wistar rats were randomly assigned to four groups (n = 10 for each group) that received, respectively, intratracheal infusion of papain (6 mg in 1 ml of 0.9% NaCl) or vehicle and were submitted or not to a protocol of exercise on a treadmill. Rats exercised at 13.3 m/min, 6 days/wk, for 9 wk (increasing exercise time, from 10 to 35 min). We measured respiratory system elastance and resistance, the size and weight of the heart, and pulmonary mean linear intercept (Lm). After 9 wk of exercise training, there were no differences in respiratory system resistance and elastance values among the four experimental groups. Volume of the heart was significantly greater in rats submitted to exercise training (P = 0.007) compared with sedentary rats due to increases in volumes of both right and left cardiac chambers. Lm was significantly greater in rats that received papain compared with saline-infused rats (P = 0.025). Surprisingly, this was true, even though there was no significant decrease in elastance, possibly due to connective tissue remodeling. However, Lm was significantly greater in papain + exercise rats compared with rats that received papain and were not submitted to exercise. We conclude that exercise training can increase alveolar damage induced by papain infusion.     

Structural similarity of chymopapain forms as indicated by circular dichroism.

Four chymopapain forms were isolated by high-resolution liquid chromatography on a cation-exchange column. The three major forms possess nearly identical secondary and tertiary structures, as judged from their c.d. spectra; these components showed similar proteolytic activity and Mr values close to that of papain. The fourth isolated component seems to be a mixture of modified proteins.     

A simple method to separate cellulose-binding domains of fungal cellulases after digestion by a protease

Core-binding domains of fungal cellulases from Trichoderma reesei were purified using a new and simple technique. Cellulases were hydrolysed with papain and the binding domains were then separated from the digested mixture by ultrafiltration. The enzymatic digestion process was monitored using capillary electrophoresis. This methodology produced a yield of 85% of binding domains.     

Reaction of the purple membrane with a carbodiimide.

Purple membrane was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.5 and 8.0. At pH 4.5, the reaction yields cross-linked bacteriorhodopsin. The cross-linking is inhibited by pretreatment of the membrane with papain, or by the presence of carbohydrazide or glycine ethyl ester in the reaction mixture. The product of the pH 8.0 reaction is not cross-linked, but it displays altered properties. Two measures of photochemical activity (light-induced change in proton binding (delta h) and decay of photointermediate M) show changes indicative of slowed proton uptake. The delta h is increased by ethyl dimethylaminopropylcarbodiimide. This increase is unaffected by pretreatment of the membrane with papain, and it is not reversed by NH2OH. However, the reaction is inhibited by millimolar concentrations of CaCl2. The altered delta h is not apparent in detergent-solubilized membranes. Ethyl dimethylaminopropylcarbodiimide does not appear to cause a large alteration in the membrane surface charge, as measured by Ca2+ binding. We conclude that (1) at acid pH, ethyl dimethylaminopropylcarbodiimide can be used for cross-linking or for attachment of specific probes to the C-terminal region of bacteriorhodopsin, and hence to the cytoplasmic side of the purple membrane, and (2) at alkaline pH, ethyl dimethylaminopropylcarbodiimide reacts at a diffent type of site and appears to inhibit the proton pump.