pH dependence of salt activation of human leukocyte elastase

The effects of pH on salt stimulation of the rates of hydrolysis of three substrates by human leukocyte elastase were studied. The enzyme was most active at pH 10.5, 8.0-8.5, and 9.5 for the hydrolyses of fluorescein isothiocyanate-labeled S-carboxymethylated bovine serum albumin (FITC-CM-BSA), succinyl-L-Ala-L-Pro-L-Ala-7-methylcoumaryl-4-amide (Suc-APA-MCA), and succinyl-L-Ala3-p-nitroanilide (Suc-Ala3-pNA), respectively, in the absence of NaCl. The enzyme was activated by 0.5 M NaCl similarly at all pHs tested for the hydrolysis of Suc-Ala3-pNA, but more at neutral and alkaline pH values, respectively, for the hydrolyses of FITC-CM-BSA and Suc-APA-MCA. Thus, in the presence of 0.5 M NaCl, the enzyme was most active at pH 8.0 and 10.0 with FITC-CM-BSA and Suc-APA-MCA, respectively. In contrast, the proteolytic activity of porcine pancreatic elastase was somewhat inhibited by 0.5 M NaCl.     

Cathepsin L plays an important role in the lysosomal degradation of L-lactate dehydrogenase

A cystatin alpha-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin alpha as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.     

Purification and characterization of proteinase In, a trypsin-like proteinase, in Escherichia coli.

We previously found a trypsin-like proteinase which momentarily appears immediately before DNA synthesis in the cell cycle of Escherichia coli synchronized by phosphate starvation and which is closely related to the initiation of DNA replication (Kato, M., Irisawa, T., Morimoto, Y. and Muramatu, M., unpublished results). The proteinase was named proteinase In. It was purified approximately 2880-fold with a recovery of 15%. The isolated enzyme appeared homogeneous by gel filtration and electrophoresis. Its molecular mass was estimated by analytical gel filtration and SDS/PAGE as approximately 66 kDa. The isoelectric point of proteinase In is 4.9 and its optimal pH is approximately 9. Although protein In hydrolyzes fluorogenic substrate for trypsin, its hydrolytic activity seems markedly affected by amino-acid sequence lying towards the N-terminal from the P1 (lysine, arginine) residue. The proteinase does not hydrolyze N2-benzoyl-D,L-arginine-4-nitronanilide and fluorogenic substrates for chymotrypsin and elastase. The proteinase activity is inhibited by leupeptin, antipain and 4-nitrophenyl 4-guanidinobenzoate, but the effects of tosyl-L-lysine chloromethane, diisopropylfluorophosphate, benzamidine and pentamidine isethionate on the proteinase activity are weak or not inhibitory. Its activity is strongly affected in the presence of NaCl and KCl, and at a concentration of 1.5 M, these increase the activity 14-fold and 13-fold, respectively, above that without salt. Proteinase In was strongly inhibited by various esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, and their inhibitory effects were roughly correlated with those on growth of E. coli. Proteinase activity was found in the cytoplasmic fraction.     

Intracellular distribution of fish muscle alkaline proteinase

1. Intracellular distribution of a muscle alkaline proteinase was investigated on four kinds of fish. 2. The total activity of the muscle proteinase of carp, Cyprinus carpio, was larger in both myofibrillar (Mf) and microsomal (Mic) fractions than the activity in mitochondrial, lysosomal, and supernatant fractions. The activity found in Mf fraction seemed to due to the Mic enzyme which was not separated from Mf fraction. 3. The relative specific activity was mostly found in Mic fraction in the species tested. 4. The results indicate that the distribution pattern of fish muscle alkaline proteinase is different from those of cathepsin D and acid phosphatase. 5. The Mic fraction hydrolyzed Mf and sarcoplasmic proteins. The rates were 40 and 55%, respectively, of the rate when casein was used as a substrate.     

Ca2+-dependent proteolysis of the Ah receptor

We previously reported (J. Biol. Chem. (1986) 261, 6352-6465) that the photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, upon incubation with the liver cytosol fraction from C57BL/6 mice, labeled in a 1:1 ratio two peptides that had apparent molecular masses of 95 and 70 kDa and similar proteolytic fragmentation patterns. In the cytosolic fraction of Hepa 1 cells, a cloned murine hepatoma cell line, the product of photoaffinity labeling is almost exclusively a 95-kDa peptide which is rapidly hydrolyzed by a Ca2+-dependent proteinase to a 70-kDa peptide as well as other fragments. Thus, the ligand binding unit of the Ah receptor in C57BL/6 mouse liver and Hepa 1 cell is a 95-kDa peptide, and the 70-kDa fragment is a proteolytic artifact. The Ca2+-dependent proteinase which hydrolyzes the 95-kDa peptide has the properties of calpain II: (i) an absolute requirement for Ca2+, with maximal activity at 0.5 to 1.0 mM Ca2+; (ii) a pH optimum of 7.5 to 8.0; (iii) inhibition by EDTA, iodoacetamide, leupeptin and L-trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by soybean trypsin inhibitor, aprotinin, or phenylmethanesufonyl fluoride. Upon chromatographic separation of the liver cytosol of C57BL/6 mice on DEAE-Sephacel, Ca2+-dependent proteinase activity (using casein or the labeled 95-kDa peptide as substrates) elutes with 0.25 M NaCl, and a specific proteinase inhibitor elutes with 0.15 M NaCl. Ca2+-dependent proteinase activity that hydrolyzes the 95-kDa peptide is found in the liver cytosols of several mammalian species.     

Proteolytic enzymes in sea urchin eggs: characterization, localization and activity before and after fertilization

The pH versus proteinase activity curve (casein or hemoglobin plus urea substrate) for homogenates of unfertilized Lytechinus eggs reveals two regions of maximum activity: one between pH 3.5 and 4.3, and another of far greater magnitude from pH 8.0 to 11.0. The two classes of proteinases can be separated on a sucrose density gradient. Both the acid and alkaline proteinases in homogenates prepared in isotonic monovalent salt solutions are remarkably stable at pH 7.4 and 0°C. Using synthetic peptide substrates, an enzyme with the specific esterase activity of chymotrypsin was demonstrated; this enzyme accounts for the major part of the proteinase activity at alkaline pH. In addition, an enzyme with specific esterase activity of trypsin was shown to be present, but of low activity. The proteinase activity at acid pH is largely due to an enzyme resembling cathepsin D. The data also suggest the presence of cathepsin B and cathepsin IV (or catheptic carboxypeptidase). When eggs are homogenized in isotonic NaCl plus KCl at pH 7.4, 0.02 M tris buffer at 0°C, all of the alkaline proteinase, and 85–90% of the acid proteinase activity is sedimented at 10,000 g. The presence of any proteinase activity in the supernatant phase represents an artifact of the preparative procedures used. The granules which possess the proteinase activity are contained entirely in the yolk fractions; and the acid proteinase is contained in a population of granules which sediment more readily than those which contain the alkaline proteinase. The acid proteinase resembles the lysosomal acid hydrolases in that it is readily released from the particulates; in contrast, the alkaline proteinase is bound relatively firmly. In contradistinction to reports in the literature, no changes in proteinase activity nor intracellular distribution could be detected following fertilization.     

The isolation and properties of a non-pepsin proteinase from human gastric mucosa.

1. A non-pepsin proteinase, proteinase 2, was successfully isolated free from pepsinogen (by repetitive chromatography on DEAE- and CM-celluloses) from the gastric mucosa of a patient with a duodenal ulcer and the uninvaded mucosa of a patient with a gastric adenocarcinoma. 2. Proteinases 1a and 1b, found in gastric adenocarcinoma, were not found in the gastic mucosa of these patients. 3. Proteinase 2 was shown to have an asymmetrical broad pH-activity curve with a maximum over the pH range 3.0-3.7. 4. Proteolytic activity of proteinase 2 was inhibited by pepstatin; the concentration of pepstatin giving 50% inhibition is of the order of 3nm. 5. Inhibition of proteolytic activity by carbenoxolone and related triterpenoids indicated that at pH 4.0 proteinase 2 possesses structural characteristics relating it to the pepsins and at pH 7.4 to the pepsinogens. 6. The sites of cleavage of the B-chain of oxidized insulin for proteinase 2 at pH 1.7 and pH 3.5 were shown to be similar to those previously established for human pepsin 3 and for the cathepsin E of rabbit bone marrow. 7. The non-pepsin proteinase 2 (cathepsin) of human gastric mucosa has properties more similar to cathepsin E than to the cathepsins D.     

Hydrolysis of succinyl-trialanine p-nitroanilide by two enzymes associated with human high-density lipoproteins

Succinyl-trialanine p-nitroanilide (Suc-Ala3-pNA), a synthetic substrate for the determination of elastase activity, was hydrolyzed in sequence by two enzymes that were found to be associated with human high-density lipoproteins. The enzymes involved in the sequence of reaction were separated by ion-exchange chromatography from apo-lipoprotein A-I and A-II, major apoproteins of high-density lipoproteins. One, designated as fraction MK, cleaves Suc-Ala3-pNA to succinyl-dialanine and alanine p-nitroanilide (Ala-pNA), and the other, designated as fraction U, cleaves Ala-pNA to alanine and p-nitroaniline. Fraction MK was inhibited by dithiothreitol, EDTA, and 1,10-phenanthroline, whereas fraction U was inhibited by 1,10-phenanthroline and bestatin. In addition to these findings, fraction MK also hydrolyzed 2,4-dinitrophenyl-prolyl-glutaminyl-glycyl-isoleucyl-alanyl-glycyl-glutaminyl- arginine (DNP-octapeptide), a specific substrate for the determination of vertebrate collagenase. Neither native elastin nor native collagen was hydrolyzed by a mixture of the two enzymes. Fraction U was very similar to aminopeptidase M with respect to its enzyme characteristics studied.     

Cysteine proteinase content of rat muscle lysosomes. Evidence for an unusual proteinase activity

Lysosomal cysteine proteinases were fractionated from partially purified rat muscle lysosomes. By gel filtration on Sephadex G75, cathepsin D was separated from two thiol-requiring proteolytic fractions of Mr 25 000 and 55 000, respectively. By chromatofocusing, the first fraction (Mr = 25 000) was resolved into three isoenzymic forms of cathepsin H, eluted at pH 5.8, 6.0 and 7.2, respectively, and two isoenzymic forms of cathepsin B, eluted at pH 5.5 and 5.25. Cathepsin H isoenzymes hydrolyzed Arg-NNap and BANA, were totally inhibited by 1 mM p-CMB and only to 60% by 5.10(-5) M leupeptin. The two forms of cathepsin B which degraded Z-Phe-Arg-NMec, Z-Arg-Arg-NNap and BANA were very sensitive to p-CMB and leupeptin. In addition to cathepsins B and H, a typical cathepsin-L- like activity was found in this fraction but only as a very minor component. The high Mr fraction (Mr = 55 000) contained a cysteine proteinase hydrolyzing, at pH 6.0, Z-Phe-Arg-NMec, and to a lesser extent Z-Arg-Arg-NNap and BANA. Unlike cathepsins B and H, it was very sensitive to p-CMB and HgCl2 and was fully activated only in the presence of 10 mM DTT, and inhibited to 93% by 2.10(-8) M leupeptin. By chromatofocusing, it was resolved into several isoenzymatic forms, eluted between pH 5.8 and 4.0.     

Cathepsin M: a lysosomal proteinase with aldolase-inactivating activity

A proteinase, designated cathepsin M, that catalyzes the limited modification and inactivation of fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) and fructose 1,6-bisphosphatase (EC 3.1.3.11) has been partially purified from rabbit liver. On the basis of its molecular size (M_r = 30,000), activation by sulfhydryl compounds and inhibition by leupeptin it has been characterized as a B-type cathepsin, but other properties distinguish it from cathepsins B, L, and H. Approximately 50% of the total cathepsin M activity is associated with membranes prepared from disrupted lysosomes; this fraction of the activity is also expressed by intact lysosomes. In the membrane-bound form the enzyme is active at neutral pH, but the soluble enzyme and the activity eluted from the membranes are maximally active at pH 5.0. Fasting increases the activity of cathepsin M; the increase is almost entirely in the membrane-bound fraction.     

Sites of cleavage of rabbit muscle aldolase by purified cathepsin M from rabbit liver

Rabbit liver cathepsin M, a sulfhydryl proteinase similar in catalytic properties to cathepsin B, causes a decrease in the activity of rabbit muscle aldolase assayed with fructose 1,6-bisphosphate but not with fructose 1-phosphate. Proteolytic modification of aldolase by cathepsin M is limited to the removal of small peptides from the COOH-terminus, including the COOH-terminal hexapeptide NH2-Ile-Ser-Asn-His-Ala-TyrOH. Correlation of loss of aldolase activity with COOH-terminal modification indicates that only three of the four subunits of muscle aldolase contribute to the catalytic activity of the tetrameric enzyme.     

Hydrolytic enzymes of Psoroptes cuniculi (Delafond)

Phosphatases, C4 and C8 esterases, leucine and valine aminopeptidases, N-acetyl-beta-glucosaminidase, beta-glucosidase, beta-galactosidase and beta-glucuronidase were detected in extracts of the parasitic mite Psoroptes cuniculi. Lipase, trypsin-like and chymotrypsin-like activities were not present. Haemoglobin was hydrolysed by a detergent-soluble fraction of the mite extracts with a maximum hydrolysis between pH 3 and 5. Acid proteinase activity was greater against haemoglobin than bovine serum albumin. Inhibitors of cysteine, serine and metallo-proteinases failed to inhibit the hydrolysis of H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH while pepstatin A inhibited its hydrolysis in a dose-dependent manner (IC50 8.02 x 10(-11) M (+/- 0.30 x 10(-11). Thermal inactivation of the proteolytic activity followed an exponential decay pattern. Typical K(m) and Vmax values were 7.2 x 10(-5) (+/- 0.7 x 10(-5) M-1 and 1.13 x 10(-3) (+/- 0.05 x 10(-3) OD unit-1 min-1 respectively. Acid proteinase activity eluted from a size exclusion column in a single, major peak representing a molecular weight range of 21-24.5 kDa. The major endoproteinase of P. cuniculi therefore appears to be a cathepsin D-like aspartic proteinase.     

Entamoeba histolytica: purification of cathepsin B

A cytotoxic cysteine proteinase with a molecular weight of 16,000 was isolated from axenically grown trophozoites of Entamoeba histolytica. The enzyme was purified from frozen-thawed strain HM-1 by ion-exchange chromatography on DEAE-cellulose, organomercurial agarose affinity chromatography, and size-exclusion chromatography. The purified enzyme had proteinase activity that could be demonstrated on azocasein (pH 5), hemoglobin (pH 5), or carbobenzoxy-L-arginyl--L-arginyl-7-amino-4-trifluoromethylcoumarin++ + (Z-arg-arg-AFC), a substrate specific for cathepsin B. Enzyme activity was stable to high pH, but not to 40 C for 1 hr or 56 C for 0.5 hr. As typical of cysteine proteinases, inhibition of activity on Z-arg-arg-AFC by p-chloromercuribenzoate or mercury was reversed by free sulfhydryl groups. Both the proteinase and cytotoxic activities of the purified amoebal cathepsin B were inhibited by leupeptin and serum and activated by free sulfhydryl groups, supporting the hypothesis that both activities are characteristics of amoebal cathepsin B. Virulent strains of E. histolytica (HM-1 and Rahman) had significantly more cathepsin B activity per milligram protein than less virulent strains (HK-9, Laredo, and Huff). The correlation between higher levels of cathepsin B activity in strains with greater virulence could indicate a role for amoebal cathepsin B in the pathogenesis of amoebiasis.     

The occurence of a neutral protease and its inhibitor in rat peritoneal macrophages.

The lysate of the glycogen-induced macrophages in rat peritoneal exudate was fractionated by centrifugation and extraction into a water extract, 1 M KCl extract and residue fractions. Approximately 50% of the neutral protease activity toward casein in the lysate was recovered in the KCl extract fraction, which was practically devoid of acid protease, cathepsin D. The pH optimum of the neutral protease toward casein and urea-denatured hemoglobin was pH 8.5. The activity was inhibited strongly by DFP or chymostatin and only partially by HgCl2 or PCMB. Addition of a salt to the reaction medium caused enhancement of the activity with an optimum concentration of 0.25 M: KCl, KBr, KI, NaCl, NaBr, NaI, and MgCl2 were all almost equally effective. When the enzyme preparation was filtered through a column of Sephadex G-75 gel in the presence of 1 M KCl, a larger molecular weight fraction at the void volume was obtained in addition to a smaller molecular weight fraction showing a caseinolytic activity insensitive to KCl concentration. The former was found to have a specific inhibitory effect on the latter activity.     

Monoclonal antibodies specific for neutrophil proteinase 4. Production and use for isolation of the enzyme

Four stable hybridoma cell lines producing monoclonal antibodies specific for neutrophil proteinase 4 (NP4) were established and one monoclonal antibody was chosen to produce an immunoaffinity-resin for the purification of NP4. In a precipitation assay system these antibodies bound NP4 in a dose-dependent manner, but did so neither with neutrophil elastase nor with cathepsin G. NP4 was purified and electrophoresis of the affinity-purified enzyme in sodium dodecyl sulfate polyacrylamide gels resulted in a single Mr = 30,000 polypeptide. The purified enzyme digested fibrin but not elastin and it cleaved Boc-Ala-ONp readily (Km = 0.47mM) at neutral pH, but had no effect on Suc-[Ala]3 Nan and N-Suc-[Ala]2-Pro-Phe-pNA. The proteolytic activity was inhibited by DFP, alpha 1 PI and alpha 2 M with a Ki of 10(-9)M for the NP4-alpha 1 PI complex. The NH2-terminal sequence and the amino-acid composition of NP4 were distinct from those of elastase and cathepsin G. Neutrophils contain large amounts of NP4 as judged by the comparable amounts of elastase- and NP4-alpha 1 PI complexes present in inflammatory exudates.     

Study of thiol proteases of normal human skin fibroblasts

The protease activity of cultured normal human skin fibroblasts was studied using the synthetic fluorigenic peptides, the modified protein 4-methylumbelliferyl-casein, the thiol inhibitors and the affinity for concanavalin A-Sepharose. The majority of the activity to N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methyl-coumarin and N-a-benzyloxycarbonyl-L-arginyl-arginyl-7-amido-4-methylcoumarin had a pH optimum of 6.0, and was thiol-dependent and inhibited by leupeptin and antipain. The activity toward N-benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methylcoumarin represents both cathepsin B and cathepsin L, whereas the activity towards 4-methylumbelliferyl-casein represent only cathepsin L. Cathepsin H could not be detected when assayed with L-arginine-7-amido-4-methylcoumarin substrate. Cathepsin D was present in comparatively small amounts when assayed with 4-methylumbelliferyl-casein. Activity towards 4-methylumbelliferyl-casein had pH optima at 3 and 6 and was stimulated by dithiothreitol. A proportion of the activity at pH 6.0 was not dependent on thiols and not inhibited by leupeptin, and had the general characteristics of a carboxyl proteinase. Over 70 per cent of the activity was in the lysosomal fraction and showed structure-linked latency. All the detectable protein emerged from the immobilized concanavalin A column and the fractions eluted by alpha-methyl-D-mannoside were significantly hydrolysed the synthetic peptides. Only that fraction which bound to concanavalin A was active towards 4-methylumbelliferyl-casein. Cathepsin B had no affinity for concanavalin A-Sepharose due to the absence of glycoprotein content, unlike cathepsin L which showed a strong affinity for concanavalin A-Sepharose.     

Identification of leukocyte cationic proteins that interact with ceruloplasmin

Proteins from leukocytes were investigated for their ability to interact with ceruloplasmin (Cp), a copper-containing glycoprotein of human plasma. Extract from leukocytes was subjected to affinity chromatography on Cp-Sepharose, after which proteins were eluted from the resin with 0.5 M NaCl in Tris-HCl, pH 7.4. SDS-PAGE of the eluate revealed protein bands with molecular weights 78, 57, 40, 30, 16, and 12 kD. Among these, Western blotting detected myeloperoxidase (57, 40, and 12 kD) and lactoferrin (78 kD). Also, the 30-kD component had a sequence (1)I-(2)I/V-(3)G-(4)G-(5)R/H at the N-terminus that is likely to indicate the presence of neutrophilic elastase, cathepsin G, proteinase 3, and azurocidin (CAP 37) - all from the family of serprocidins. Mass spectrometry of tryptic fragments indicated the presence of the 16-kD eosinophilic cationic protein (seven peptides), 27-kD cathepsin G (eleven peptides), 27-kD azurocidin (eight peptides), 29-kD neutrophilic elastase (seven peptides), and 27-kD proteinase 3 (six peptides). Myeloperoxidase was represented by 57-, 40-, and 12-kD fragments (thirteen, ten, and four peptides, respectively). Thus, interaction with Cp of five cationic proteins, i.e. of eosinophilic cationic protein, cathepsin G, neutrophilic elastase, proteinase 3, and azurocidin is reported for the first time.     

Characterization of cathepsin B proteinase (AcCP-2) in eggs and larvae stages of hookworm Ancylostoma caninum

Cathepsin B proteinase constitutes a large multigenes family in parasitic and non-parasitic nematodes. The localization of cathepsin B proteinases (AcCP-1 and AcCP-2) in adult worm of Ancylostoma caninum has been characterized (Harrop et al., 1995), but the localization and function in eggs and larval stages remained undiscovered. Here we described the expressing of cathepsin B proteinase (AcCP-2) in Escherichia coli, and immuno-localization of cathepsin B proteinase in eggs and larvae stages of A. caninum. A cDNA fragment encoding a cathepsin B proteinase (AcCP-2) was cloned from A. caninum and expressed in E. coli. Gelatin digestion showed that recombinant cathepsin B proteinase (AcCP-2) has protease activity. The protein level of cathepsin B proteinase in larval and adult worm was detected by western blot. The immuno-localization of cathepsin B proteinase in eggs and larval stages was characterized. The expression of cathepsin B proteinase was more abundant in eggs and larvae stages of A. caninum. It implied that cathepsin B proteinase might play roles in the early development of A. caninum.     

Human cathepsin G. Catalytic and immunological properties.

1. The specificity of cathepsin G, a neutral proteinase from human spleen, was examined by use of low-molecular-weight substrates. The enzyme was found to hydrolyse several synthetic substrates also hydrolysed by chymotrypsin, but with different kinetic constants. 2. Maximal activity against benzoyl-DL-phenylalanine 2-naphthol ester and azo-casein was in the range pH 7.5-8.0. 3. The sensitivity of cathepsin G to the action of potential inhibitors was determined, and compared with those of bovine chymotrypsin and subtilisin. Cathepsin G showed the characteristics of a serine proteinase, but was less affected by the chloromethyl ketone of tosylphenylalanine than was chymotrypsin. 4. A rabbit anti-(human cathepsin G) serum was raised, and precipitin lines formed in agarose gel were stained for activity of the enzyme. 5. Cathepsin G was shown to be immunologically identical with the chymotrypsin-like enzyme of the azurophil granules of the neutrophil granulocytes.