Purification of cathepsin B by a new form of affinity chromatography.

Human cathepsin B was purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal linked to Sepharose 4B, with elution by 2,2'-dipyridyl disulphide at pH 4.0. The product obtained in high yield by the single step from crude starting material was 80-100% active cathepsin B. The possibility that this new form of affinity chromatography may be of general usefulness in the purification of cysteine proteinases is discussed.     

Evidence that the active centre of chymopapain A is different from the active centres of some other cysteine proteinases and that the Br?nsted coefficient (beta nuc.) for the reactions of thiolate anions with 2,2'-dipyridyl disulphide may be decreased by reagent protonation.

The active centres of chymopapains A and B (jointly designated EC 3.4.22.6) and papaya (Carica papaya L.) peptidase A were investigated by using 2,2'-dipyridyl disulphide and 5,5'-dithiobis-(2-nitrobenzoic acid) as thiol-specific reactivity probes. Whereas the first active-centre pKa values for chymopapain B and papaya peptidase A are less than 5, is as the case for papain (EC 3.4.22.2) and ficin (EC 3.4.22.3), that for chymopapain A is about 6.8. The reason why the reactions of thiols of pKa approx. 6.5 with 2.2'-dipyridyl disulphide are essentially pH-independent in the pH range around the thiol pKa is delineated. The value of the Brønsted coefficient (beta nuc.) for the reactions of thiolate ions with the 2,2'-dipyridyl disulphide monocation appears to be smaller than its value for the corresponding reactions with the neutral disulphide.     

Differences in the chemical and catalytic characteristics of two crystallographically ''identical'' enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment.

The characteristics of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2), two cysteine proteinases whose catalytic-site regions appear to superimpose to a degree that approaches atomic co-ordinate accuracy of both crystal structures, were evaluated by determining (a) the pH-dependence in acid media of the acylation process of the catalytic act (k+2/Ks) using N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-Nan) as substrate and (b) the sensitivity of the reactivity of the catalytic-site thiol group and its pH-dependence to structural change in small, thiol-specific, two-protonic-state reactivity probes (2,2'-dipyridyl disulphide and methyl 2-pyridyl disulphide) where enzyme-probe contacts should be restricted to areas close to the catalytic site. Distortion of the catalytic sites of the two enzymes at pH less than 4 was evaluated over time-scales appropriate for both stopped-flow reactivity probe kinetics (less than or equal to 1-2 s) and steady-state substrate catalysis kinetics (3-5 min) by using the 2,2'-dipyridyl disulphide monocation as a titrant for non-distorted catalytic sites. This permitted a lower pH limit to be defined for valid kinetic analysis of both types. The behaviour of the enzymes at pH less than 4 requires a kinetic model in which the apparently biomolecular reaction of enzyme with probe reagent is separated from the process leading to loss of conformational integrity by a potentially reversible step. The acylation of actinidin with L-Bz-Arg-Nan in acidic media occurs in two protonic states, one produced by raising the pH across pKa less than 4 which probably characterizes the formation of -S-/-ImH+ ion pair (pKa approx. 3) and the other, of higher reactivity, produced by raising the pH across pKa 5.5, which may characterize rearrangement of catalytic-site geometry. The pH-dependence of the acylation of papain by L-Bz-Arg-Nan is quite different and is not influenced by protonic dissociation with pKa values in the range 5-6. The earlier conclusion that the acylation of papain depends on two protonic dissociations each with pKa approx. 4 was confirmed. This argument is now more firmly based because titration with 2,2'-dipyridyl disulphide permits the loss of conformational integrity to be taken into account in the analysis of the kinetic data at very low pH. Methyl 2-pyridyl disulphide was synthesized by reaction of pyridine-2-thione with methyl methanethiolsulphonate and its pKa at I = 0.1 was determined by spectral analysis at 307 nm to be 2.8.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reactivities of neutral and cationic forms of 2,2''-dipyridyl disulphide towards thiolate anions. Detection of differences between the active centres of actinidin, papain and ficin by a three-protonic-state reactivity probe.

The second-order rate constants (k) for the reactions of 2,2'-dipyridyl disulphide (pKa2,45) with 2-mercaptoethanol (pKa9.6) and with benzimidazol-2-ylmethanethiol (pKa values 5.6 and 8.3) were determined at 25 degrees C at I 0.1 by stopped-flow spectral analysis over a wide range of pH. These were used to calculate the pH-independent second-order rate constants (k) for the reactions of neutral 2,2'-dipyridyl disulphide and of its monocation with the 2-mercaptoethanol thiolate anion (associated pKa9.6) and with the benzimidazol-2-ylmethanethiol zwitterion (associated pKa5.6). For both thiolate ions, the rate-enhancement factor (kmonocation/kneutral disulphide) is about 1.5x10(3). The dependence on pH in acidic media of k for the reaction of 2,2'-dipyridyl disulphide with actinidin, the thiol proteinase from Actinidia chinensis, was shown to differ from the forms of pH-dependence observed for the analogous reactions with papain (EC 3.4.22.2) and ficin (3.4.22.3). The reactivity of the 2,2'-dipyridyl disulphide dication and its apparent sensitivity to the presence and location of a positive charge in the attacking thiol are discussed.     

Chymopapain A. Purification and investigation by covalent chromatography and characterization by two-protonic-state reactivity-probe kinetics, steady-state kinetics and resonance Raman spectroscopy of some dithioacyl derivatives.

Chymopapain A was isolated from the dried latex of papaya (Carica papaya) by ion-exchange chromatography followed by covalent chromatography by thiol-disulphide interchange. The latter procedure was used to produce fully active enzyme containing one essential thiol group per molecule of protein, to establish that the chymopapain A molecule contains, in addition, one non-essential thiol group per molecule and to recalculate the literature value of epsilon 280 for the enzyme as 36 000 M-1 X cm -1. The Michaelis parameters for the hydrolysis of L-benzoylarginine p-nitroanilide and of benzyloxy-carbonyl-lysine nitrophenyl ester at 25 degrees C, and I 0.1 at several pH values catalysed by chymopapain A, papaya proteinase omega, papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were determined. Towards these substrates chymopapain A has kcat./km values similar to those of actinidin and of papaya proteinase omega and significantly lower than those of papain or ficin. The environment of the catalytic site of chymopapain A is markedly different from those of other cysteine proteinases studied to date, as evidenced by the pH-dependence of the second-order rate constant (k) for the reaction of the catalytic-site thiol group with 2,2'-dipyridyl disulphide. The striking bell-shaped component that is a characteristic feature of the reactions of S-/ImH+ (thiolate/imidazolium) ion-pair components of many cysteine-proteinase catalytic sites with the 2,2'-dipyridyl disulphide univalent cation is not present in the pH-k profile for the chymopapain A reaction. The result is consistent with the presence of an additional positive charge in, or near, the catalytic site that repels the cationic form of the probe reagent. Resonance Raman spectra were collected at pH values 2.5, 6.0 and 8.0 for each of the following dithioacyl derivatives of chymopapain A: N-benzoylglycine-, N-(Beta-phenylpropionl)glycine- and N-methoxycarbonylphenylalanylglycine-. The main conclusion of the spectral study is that in each case the acyl group binds as a single population known as conformer B in which the glycinic N atom is in close contact with the thiol S atom of the catalytic-site cysteine residue, as is the case also for papain and other cysteine proteinases studied. Thus the abnormal catalytic-site environment of chymopapain A detected by the reactivity-probe studies, which may have consequences for the acylation step of the catalytic act, does not perturb the conformation of the bound acyl group at the acyl-enzyme-intermediate stage of catalysis.     

Characterization of papaya peptidase A as a cysteine proteinase of Carica papaya L. with active-centre properties that differ from those of papain by using 2,2''-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. Use of two-protonic-state electrophiles in the identification of catalytic-site thiol groups.

1. The proteinase papaya peptidase A, one of the major components of the latex of Carica papaya L., was shown to contain 1 thiol group per molecule; this thiol group is essential for catalytic activity and is part of the catalytic site. 2. The usefulness of two-protonic-state reactivity probes coupled with modification/activity-loss data in assigning a thiol group as an integral part of the catalytic site as against merely 'essential' for activity is discussed. 3. The active centre of papaya peptidase A was investigated by using 2,2'-dipyridyl disulphide and 4-chloro-7-nitrobenzofurazan as reactivity probes. The presence in the enzyme in weakly acidic media of an interactive system containing a nucleophile S atom (pKI3.9,pKII7.9) was demonstrated. 5. Papaya peptidase A resembles ficin (EC 3.4.22.3) and actinidin (the cysteine proteinase from Actinidin chinenis) in that it does not appear to possess a carboxy group able to influence the reactivity of the thiol group by change of ionization state at pH values of about 4, a situation that contrasts markedly with that which obtains in papain. 6. Implications of the results for possible variations in cysteine proteinase mechanism are discussed.     

Amino acid sequences of the human kidney cathepsins H and L

The complete amino acid sequences of human kidney cathepsin H (EC 3.4.22.16) and human kidney cathepsin L (EC 3.4.22.15) were determined. Cathepsin H contains 230 residues and has an Mr of 25116. The sequence was obtained by sequencing the light, heavy and mini chain and the peptides produced by cyanogen bromide cleavage of the single-chain form of the enzyme. The glycosylated mini chain is a proteolytic fragment of the propeptide of cathepsin H. Human cathepsin L has 217 amino acid residues and an Mr of 23720. Its amino acid sequence was deduced from N-terminal sequences of the heavy and light chains and from the sequences of cyanogen bromide fragments of the heavy chain. The fragments were aligned by comparison with known sequences of cathepsins H and L from other species. Cathepsins H and L exhibit a high degree of sequence homology to cathepsin B (EC 3.4.22.1) and other cysteine proteinases of the papain superfamily.     

Breast cancer cells express cathepsins B and L but not cathepsins K or H

Lysosomal cysteine proteinases (cathepsins) are considered to play a role in bone degradation mediated by metastatic breast cancers. To evaluate which cathepsin contributes to the osteolysis, we quantitatively determined the expression levels of four cathepsins in two breast cancer cell lines, MCF-7 and MDA-MB-231, by competitive RT-PCR. Cathepsin K, which is the most abundant cathepsin in osteoclasts, was not detected in either cell lines. We also failed to detect cathepsin H mRNA. By contrast, we found significant expression of cathepsins B and L in both cell lines. By Northern blot analysis cathepsin B mRNA was detected in a single form in these cells, whereas osteoclasts contained multiple forms of the mRNA. Cathepsin B protein was also detected by Western blotting as a single immunoreactive band corresponding to its mature enzyme. These findings suggest that osteolysis associated with metastatic breast cancers takes place in a different way from osteoclast-mediated bone resorption.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cysteine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Human cathepsin H.

Cathepsin H was purified from human liver by a method involving autolysis and acetone fractionation, and chromatography on DEAE-cellulose, Ultrogel AcA 54, hydroxyapatite and concanavalin A-Sepharose. The procedure allowed for the simultaneous isolation of cathepsin B and cathepsin D. Cathepsin H was shown to consist of a single polypeptide chain of 28 000 mol.wt., and affinity for concanavalin A-Sepharose indicated that it was a glycoprotein. The enzyme existed in multiple isoelectric forms, the two major forms having pI values of 6.0 and 6.4; it hydrolysed azocasein (pH optimum 5.5), benzoylarginine 2-naphthylamide (Ba-Arg-NNap), leucyl 2-naphthylamide (Arg-NNap), (pH optimum 6.8). Arg-NNap and Arg-NMec, unlike Bz-Arg-NNap-, were not hydrolysed by human cathepsin B. Cathepsin H was similar to cathepsin B in being irreversibly inactivated by exposure to alkaline pH. Sensitivity to chemical inhibitors by 1 microM-leupeptin, which gave essentially complete inhibition of the other lysosomal cysteine proteinases, cathepsins B and L.     

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.     

Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells

Summary Cathepsins B and H are representative cysteine proteinases localized to lysosomes of a variety of mammalian cells. Previous studies indicated the presence of these enzymes also in secretory granules of endocrine cells. Therefore, the human endocrine pancreas and human insulinomas were investigated by light microscopical immunohistochemistry on serial semithin plastic sections immunostained sequentially for cathepsins B or H and pancreatic hormones. Out of the four established endocrine cell types, insulin (B-) and glucagon (A-) cells showed immunoreactivities for these cathepsins. Cathepsin B immunoreactivities showed a dot-like appearance in A- and B-cells and in insulinoma cells. Immunoreactivities for cathepsin H additionally were found in cell parts containing secretory granules of B-cells and insulinoma cells. By single and double immunoelectron microscopy the dot-like immunoreactivities for cathepsin B were identified as immunoreactive lysosomes of A- and B-cells and insulinoma cells. In addition, some of the secretory granules of A- and B-cells showed cathepsin B immunoreactivities. Cathepsin H immunoreactivities showed an other pattern: they were found regularly in the secretory granules of A- and B-cells and insulinoma cells, and in lysosomes of A-cells. These findings suggest that cathepsins B and H in lysosomes of A- and/or B-cells are involved in the degradation of lysosomal constituents. In secretory granules of these cells, these cystine proteinases may participate in the processing of the corresponding hormones from their precursor proteins.     

Chemical evidence for the pH-dependent control of ion-pair geometry in cathepsin B. Benzofuroxan as a reactivity probe sensitive to differences in the mutual disposition of the thiolate and imidazolium components of cysteine proteinase catalytic sites.

Benzofuroxan reacts with the catalytic-site thiol group of cathepsin B (EC 3.4.22.1) to produce stoichiometric amount of the chromophoric reduction product, o-benzoquinone dioxime. In a study of the pH-dependence of the kinetics of this reaction, most data were collected for the bovine spleen enzyme, but the more limited data collected for the rat liver enzyme were closely similar both in the magnitude of the values of the second-order rate constants (k) and in the shape of the pH-k profile. In acidic and weakly alkaline media, the reaction is faster than the reactions of benzofuroxan with some other cysteine proteinases. For example, in the pH region around 5-6, the reaction of cathepsin B is about 10 times faster than that of papain, 15 times faster than that of stem bromelain and 6 times faster than that of ficin. The pH-dependence of k for the reaction of cathepsin B with benzofuroxan was determined in the pH range 2.7-8.3. In marked contrast with the analogous reactions of papain, ficin and stem bromelain [reported by Shipton & Brocklehurst (1977) Biochem. J. 167, 799-810], the pH-k profile for the cathepsin B reaction contains a sigmoidal component with pKa 5.2 in which k increases with decrease in pH. This modulation of the reactivity of the catalytic-site -S-/-ImH+ ion-pair state of cathepsin B (produced by protonic dissociation from -SH/-ImH+ with pKa approx. 3) towards a small, rigid, electrophilic reagent, in a reaction that appears to involve both components of the ion-pair for efficient reaction, suggests that the state of ionization of a group associated with a molecular pKa of approx. 5 may control ion-pair geometry. This might account for the remarkable finding [reported by Willenbrock & Brocklehurst (1984) Biochem. J. 222, 805-814] that, although the ion-pair appears to be generated in cathepsin B as the pH is increased across pKa 3.4, catalytic competence is not generated until the pH is increased across pKa 5-6.     

Distribution of cathepsins B and H in rat tissues and peripheral blood cells

The concentrations of cathepsins B and H in various tissues and peripheral blood cells of rats were determined by means of sensitive immunoassays. The minimum detectable amounts of cathepsins B and H were 30 pg and 20 pg/assay, respectively, and the presence of endogenous thiol proteinase inhibitors did not interfere with the immunoassays. Cathepsin B was found at high levels in the kidney, vagina, spleen, and adrenal gland, and cathepsin H at high levels in the kidney, vagina, liver, lung, and spleen. Low levels of cathepsins B and H were present in the heart, skeletal muscle, and testis. The ratios of cathepsins B and H in various organs were different: the brain and adrenal gland contained much higher levels of cathepsin B than of cathepsin H, whereas the lung and liver contained higher levels of cathepsin H than of cathepsin B. In several organs such as the kidney, spleen, liver, and lungs, the level of cathepsins B plus H was much higher than that of thiol proteinase inhibitors (TPI-alpha + TPI-beta), whereas in tissues containing large amounts of TPI-alpha, such as the skin, esophagus and stomach, the level of inhibitors was higher than that of cathepsins B plus H. Of the peripheral blood cells tested, macrophages had the highest contents of cathepsins B and H, and so their level of cathepsins B plus H was much higher than that of TPI-alpha plus TPI-beta, whereas lymphocytes and neutrophils contained comparable amounts of proteinases and inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of thiols in degradation of proteins by cathepsins.

The effects of thiols on the breakdown of 125I-labelled insulin, albumin and formaldehyde-treated albumin by highly purified rat liver cathepsins B, D, H and L at pH 4.0 and 5.5 were studied. At both pH values degradation was strongly activated by the thiols cysteamine, cysteine, dithiothreitol, glutathione and 2-mercaptoethanol, and its rate increased with increasing thiol concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect the rate of degradation by cathepsin D or L, and determination of free thiol groups after incubation of the proteins in the presence of glutathione but without cathepsin showed that their disulphide bonds were stable under the incubation conditions. Sephadex G-75 chromatography of the acid-soluble products of insulin digestion by cathepsin D or L suggested that thiols can reduce disulphide bonds in proteins after limited proteolysis. The resultant opening-up of the protein structure would lead to further proteolysis, so that the two processes (proteolysis and reduction) may act synergistically. By using the osmotic protection method it was shown that, at a physiological pH, cysteamine, and its oxidized form cystamine, can cross the lysosome membrane and thus may well be the physiological hydrogen donor for the reduction of disulphides in lysosomes. The results are discussed in relation to the lysosomal storage disease cystinosis.     

A kinetic method for the study of solvent environments of thiol groups in proteins involving the use of a pair of isomeric reactivity probes and a differential solvent effect. Investigation of the active centre of ficin by using 2,2''- and 4,4''- dipyridyl disulphides as reactivity probes.

1. Whereas the second-order rate constants for the reaction of the thiolate ion of 2-mercaptoethanol with 4,4'-dipyridyl disulphide (k4PDS) and with 5,5'-dithiobis-2-nitrobenzoate dianion increase with decreasing dielectric constant of the solvent, or remain unchanged, the rate constant for the analogous reaction with 2,2'-dipyridyl disulphide (k2PDS) decreases. This anomalous solvent effect and other unusual physicochemical properties of 2,2'-dipyridyl disulphide are discussed. 2. The differential effect of solvent on the reactions of thiolate ion with the 2,2'- and 4,4'-dipyridyl disulphides is shown to provide a method of characterizing solvent environments of thiol groups in proteins by a reactivity-probe method that should not suffer from the usual drawback associated with the existence of steric or binding effects of unknown magnitude. Application of the method to ficin (EC 3.4.22.3) suggests that its active-centre thiol group resides in a relatively hydrophobic environment. 3. The pH-k profile for the reaction of ficin with 4,4'-dipyridyl disulphide is reported.     

Identification of cathepsin B,D and H in the larval midgut of colorado potato beetle,Leptinotarsa decemlineata say (Coleoptera: Chrysomelidae)

The larval midgut of the Colorado beetle, Leptinotarsa decemlineata contains cathepsin B, D and H activity detected by use of haemoglobin, synthetic substrates specific for each enzyme, pH at which the substrate was maximally hydrolysed and effects of potential activators and inhibitors on proteolytic activity. Cysteine proteases cathepsin B, and H were activated by thiol compounds and inhibited by iodoacetamide, TLCK and epoxysuccinyl-leucyl-amido(guanidino)butane (E-64) a cysteine specific proteinase inhibitor. Cathepsin B was distinguished from H by hydrolysis of benzoyloxycarbonyl-Ala-Arg-Arg-methoxynaphthylamide, a cathepsin B specific substrate and inhibition of substrate hydrolysis by leupeptin. Cathepsin H activity, detected using the specific substrate arginine-naphthylamide, was insensitive to leupeptin. Cathepsin D had maximal activity at pH 4.5 and was inhibited by pepstatin, an aspartic proteinase inhibitor.     

A marked gradation in active-centre properties in the cysteine proteinases revealed by neutral and anionic reactivity probes. Reactivity characteristics of the thiol groups of actinidin, ficin, papain and papaya peptidase A towards 4,4'-dipyridyl disulphide and 5,5'-dithiobis-(2-nitrobenzoate) dianion.

1. The kinetics of the reactions of the catalytic-site thiol groups of actinidin (the cysteine proteinase from Actinidia chinensis), ficin (EC 3.4.22.3), papain (EC 3.4.22.2) and papaya peptidase A (the other monothiol cysteine proteinase component of Carica papaya) with 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py) and with 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs22-) were studied in the pH range approx. 6-10. These studies provided the pH-independent second-order rate constants (k) for the reactions of the two probe reagents with the catalytic-site thiolate anions each in the environment of a neutral histidine side chain where an active-centre carboxy group would be ionized. 2. The ratio R equal to kNbs22-/k4-Py-S-S-4-Py provides an index of the catalytic-site solvation properties of the four cysteine proteinases and varies markedly from one enzyme to another, being 0.80 for papaya peptidase A (0.86 for the model thiol, 2-mercaptoethanol), 29 for actinidin, 0.18 for ficin and 0.015 for papain. These differences appear to derive mainly from the response of the enzyme to the negative charge on Nbs22-. 3. Possible implications of these results for (a) mechanisms of cysteine proteinase catalysis and (b) the possibility of using series of functionally related enzymes in the study of mechanism are discussed.     

Studies on cathepsin B in human articular cartilage.

The thiol proteinase cathepsin B (EC 3.4.22.1), previously called cathepsin B1, was assayed in human articular cartilage by its hydrolysis of the synthetic substrate alpha-N-benzoyl-DL-arginine 2-naphthylamide. The enzyme was activated by cysteine and EDTA and completely inhibited by iodoacetamide and HgCl2. It was also partially inhibited by whole human serum. Human osteoarthrotic cartilage had increased activity when compared with normal cartilage. Cathepsin B activity of normal cartilage was age-related, being high in juveniles and declining to low values in adult and elderly individuals. Cathepsin D and cathepsin B both exhibited a zonal variation through the cartilage depth; the surface cells appeared to contain more activity than those close to the subchondral bone.     

Incorporation of [18O]water in the formation of p-hydroxybenzyl alcohol by the p-cresol methylhydroxylase from Pseudomonas putida.

A simple spectrophotometric method for the detection of chymopapains A and B, contaminants in some preparations of papain (EC 3.422.2), was devised. If the amount of rapidly reacting thiol in the preparation, as determined by increase in A343 consequent on reaction with the two-protonic-state thiol titrant, 2,2' -dipyridl disulphide, is greater at PH8-9 than at pH4, contamination by the chymopapains is in dicated.