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Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.

The lysosomal cysteine proteinases cathepsin L and cathepsin B were examined for their effect on the neutrophil elastase inhibitory activity of human alpha 1-proteinase inhibitor (alpha 1PI). Human cathepsin L catalytically inactivated human alpha 1PI by cleavage of the bonds Glu354-Ala355 and Met358-Ser359 (the serine proteinase inhibitory site). Cathepsin B did not inactivate alpha 1PI, even when equimolar amounts of enzyme were employed. Cathepsin L is the first human proteinase shown to catalytically inactivate alpha 1PI. These findings, in conjunction with other reports, suggest that alpha 1PI contains a proteolytically sensitive region encompassing residues 350-358. Taken together with the discovery of the elastinolytic activity of cathepsin L (Mason, R. W., Johnson, D. A., Barrett, A. J., and Chapman, H. A. (1986) Biochem. J. 233, 925-927), the present findings emphasize the possible importance of cathepsin L in the pathological proteolysis of elastin and diminish the role that can be attributed to cathepsin B in such processes.

Rhodamine B is used as a marker dye in herbicide sprays. There is evidence that spray operators and others may absorb rhodamine B through the skin. This study was undertaken to investigate the in vivo mutagenicity of rhodamine B, to compare the in vitro mutagenicity of two commercial preparations of the dye with that of known mutagens including rhodamine 6G and to elucidate the pharmacokinetics of rhodamine B in the rabbit. Following the i.v. treatment of adult female New Zealand White rabbits with rhodamine B (1 mg/kg body wt), the plasma concentration of rhodamine B decreased rapidly and was accompanied by the appearance of at least four fluorescent polar metabolites. These metabolites, as well as a very small amount of rhodamine B, were also present in the urine which, when tested in the Ames assay was not significantly mutagenic against Salmonella typhimurium strains TA98 and TA100 either with or without metabolic activation. Urine from a human subject who had been contaminated with marker dye was also non-mutagenic. Both commercial preparations of rhodamine B were found to be weakly mutagenic, using the same assay system. It is concluded that while appropriate hygiene measures should be exercised by users of products containing this dye, the results do not support the hypothesis that rhodamine B is a genotoxic hazard in the mammalian organism.

A series of peptidyldiazomethanes was synthesized and tested as inactivators of the cysteine proteinases calpain II, cathepsin L and cathepsin B. Inactivators that react rapidly and that show a degree of selectivity between the enzymes were identified. Z-Tyr(I)-Ala-CHN2 (where Z represents benzyloxycarbonyl) reacts rapidly with cathepsin L and more slowly with cathepsin B, but does not inhibit calpain II. Z-Leu-Leu-Tyr-CHN2 reacts rapidly with cathepsin L and calpain II but very slowly with cathepsin B. Boc-Val-Lys(epsilon-Z)Leu-Tyr-CHN2 (where Boc represents t-butyloxycarbonyl) reacts more rapidly with calpain II than with cathepsin L or cathepsin B. The discriminating inhibitory effects of these compounds make them potentially useful for investigation of enzyme functions in vivo. The data presented also provide insights into the subsite specificity of calpain.

The major excreted protein (MEP) purified from Kirsten-virus-transformed 3T3 fibroblasts and mature human cathepsin L were compared in respect to a number of catalytic criteria and found to be similar. The Mr of MEP is 39,000, whereas that of mature human cathepsin L is 30,000. Sequence data suggested that MEP could be a pro-form of mouse cathepsin L. Both enzymes acted on the synthetic substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide with similar catalytic constants and acted optimally at pH 5.5. Both were rapidly inactivated by the active-site-directed inhibitors benzyloxycarbonyl-Phe-Phe-diazomethane and L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane, and furthermore, 3H-labelled L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-acetamid o)butane, which binds covalently to the heavy chain of mature cathepsin L, also bound to MEP. MEP autolyses rapidly at pH 3.0 to give lower-Mr (35,000 and 30,000) forms, but all forms react with the radiolabelled inhibitor. No autolysis occurred above pH 5.0. MEP hydrolysed azocasein at pH 5.0, demonstrating that it is capable of hydrolysing protein substrates without autolytic activation. Unlike mature forms of cathepsin L, MEP is stable, but not active, at neutral pH. The present work shows that cathepsin L can be secreted as a higher-Mr precursor that is stable in extracellular fluids but only active where local pH values fall below 6.0. These results suggest that the extra N-terminal peptide on MEP is not an activation peptide, but is a regulatory peptide affecting the pH-stability and activity of mouse cathepsin L.

Human liver cathepsin L consists of a heavy chain and a light chain with Mr values of 25,000 and 5000 respectively. The chains have been purified and their N-terminal amino acid sequences have been determined. The 40 amino acids determined from the heavy chain and 42 amino acids sequenced in the light chain are homologous with the N-terminal and C-terminal regions respectively of the superfamily of cysteine proteinases. Therefore it is likely that the two chains of cathepsin L are derived by proteolysis of a single polypeptide precursor. Of the amino acids sequenced, 81% are identical with the homologous portions of a protein sequence for a major cysteine proteinase predicted from a cDNA clone from a mouse macrophage cell line. This is the closest relative amongst the known sequences in the superfamily and strongly indicates that the protein encoded by this mRNA is cathepsin L. The mouse protein is also probably the major excreted protein of a transformed cell line [Gal & Gottesman (1986) Biochem. Biophys. Res. Commun. 139, 156-162]. The heavy chain is identical in only 71% of its residues with the sequence of ox cathepsin S, providing further evidence that this latter enzyme is probably not a species variant of cathepsin L. The relationship with a second unidentified cathepsin cDNA clone from a bovine library is much weaker (41% identity), and so this clone remains unidentified.

Cathepsin L variants purified from sheep and ox liver are shown to have similar catalytic properties to those from rat, rabbit and man with regard to activity against the substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide and inhibition by benzyloxycarbonyl-Phe-Phe-diazomethane, thus identifying cathepsin L in these species for the first time. All five variants of cathepsin L are shown to be immunologically related by their interaction with antibodies raised to the human enzyme. Sheep liver was found to yield more enzyme than any other species, suggesting that this tissue is a good source of cathepsin L. Cathepsin S, a closely related enzyme, could not be detected in livers of any of these species.

Cathepsin L was purified to apparent homogeneity from human liver obtained post mortem. It was necessary to treat the homogenate at pH 4.2 and 37 degrees C to release active enzyme. The purification procedure involved ion-exchange chromatography on carboxymethyl-Sephadex and the Mono S column of a Pharmacia fast-protein-liquid-chromatography system. The enzyme was found to consist of two polypeptide chains of Mr 25 000 and 5000. The larger chain was shown to contain the active-site cysteine residue. Human cathepsin L proved to be similar to the rat and rabbit enzymes in regard to kinetic constants for the substrate benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide and rates of inactivation by the active-site-directed reagents benzyloxycarbonylphenylalanylphenylalanyldiazomethane and benzyloxycarbonylphenylalanylalanyldiazomethane. Thus clear characteristics of cathepsin L are now emerging, and these should simplify the identification of the enzyme in other tissues and species.

Cathepsin L was purified from rabbit liver by a method involving whole-tissue homogenization, pH precipitation, ammonium sulphate fractionation and chromatography on CM-Sephadex C-50, phenyl-Sepharose and Sephadex G-75. Pure enzyme was obtained without the necessity of laborious subcellular fractionation techniques. The Mr of the enzyme was determined to be 29 000 by gel filtration, and affinity for concanavalin A-Sepharose indicated that it was a glycoprotein. A novel technique for detection of enzyme activity in agarose isoelectrofocusing gels showed that the enzyme existed in multiple isoenzymic forms with pI values ranging from 5.0 to 5.9. The enzyme catalysed the hydrolysis of azocasein, collagen and Z-Phe-Arg-NMec (where Z and NMec indicate benzyloxycarbonyl and N-methylcoumarin derivative respectively) optimally at pH 5.2, 3.3 and 6.0 respectively. In addition, cathepsin L was found to degrade benzoyl-Phe-Val-Arg-NMec and 3-carboxypropionyl-Ala-Phe-Lys-NMec. However, cathepsin B also cleaved all of these substrates. One major difference between these two enzymes was in their Michaelis constants for Z-Phe-Arg-NMec; cathepsin B had Km 75 microM whereas that of cathepsin L was 0.7 microM. Cathepsin L was inhibited by all of the usual chemical inhibitors of thiol proteinases as well as the more specific inhibitors Z-Phe-Phe-CHN2, Z-Phe-Ala-CHN2, compound E-64 and compound Ep-475. Active-site titration with compound E-64 showed that the purified sample contained 80% active protein, which had kcat. 20s-1 for the substrate Z-Phe-Arg-NMec. Antibodies were raised to active cathepsin L, and these did not cross-react with cathepsin B, thus demonstrating that these two enzymes are immunologically distinct.

The structural basis for the biological specificity of human cystatin C has been investigated. Cystatin C and other inhibitors belonging to family 2 of the cystatin superfamily interact reversibly with target peptidases, seemingly by independent affinity contributions from a wedge-shaped binding region built from two loop-forming inhibitor segments and a binding region corresponding to the N-terminal segment of the inhibitor. Human cystatin C variants with Gly substitutions for residues Arg-8, Leu-9, and/or Val-10 of the N-terminal binding region, and/or the evolutionarily conserved Trp-106 in the wedge-shaped binding region, were produced by site-directed mutagenesis and Escherichia coli expression. A total of 10 variants were isolated, structurally verified, and compared to wild-type cystatin C with respect to inhibition of the mammalian cysteine peptidases, cathepsins B, H, L, and S. Varying contributions from the N-terminal binding region and the wedge-shaped binding region to cystatin C affinity for the four target peptidases were observed. Interactions from the side chains of residues in the N-terminal binding region and Trp-106 are jointly responsible for the major part of cystatin C affinity for cathepsin L and are also of considerable importance for cathepsin B and H affinity. In contrast, for cathepsin S inhibition these interactions are of lesser significance, as reflected by a Ki value of 10(-8) M for the cystatin C variant devoid of Arg-8, Leu-9, Val-10, and Trp-106 side chains. The side chain of Val-10 is responsible for most of the affinity contribution from the N-terminal binding region, for all four enzymes. The contribution of the Arg-8 side chain is minor, but significant for cystatin C interaction with cathepsin B. The Leu-9 side chain confers selectivity to the inhibition of the target peptidases; it contributes to cathepsin B and L affinity by factors of 200 and 50, respectively, to cathepsin S binding by a factor of 5 only, and results in a 10-fold decreased affinity between cystatin C and cathepsin H.

The ability of benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 to label cysteine proteinases in a variety of human tissues was investigated. The inhibitor bound only to cathepsin B in tissues homogenized at pH 5.0. When liver was autolysed at pH 4.0 for up to 4 h, the inhibitor also bound to a protein of Mr 25,000. This was identified immunologically and chromatographically as cathepsin L. Both cathepsins B and L were found primarily in kidney, liver and spleen. In spleen, an additional protein of Mr 25,000 was also labelled. This protein could not be precipitated by antibodies to any of cathepsins B, H and L. This protein has tentatively been identified as human cathepsin S by its tissue distribution, chromatographic properties and molecular size. This work clearly shows that peptidyldiazomethanes are specific probes for cysteine proteinases, and that benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 binds to three such enzymes in human tissues.

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.

Cathepsin L was partially purified from lysates of freshly isolated macrophages lavaged from lungs of apparently healthy adults and found to be chromatographically and catalytically identical with liver cathepsin L. Western-blotting analysis showed that lung macrophages contain significant levels of a precursor of cathepsin L (43 kDa) in addition to mature enzyme (25 kDa). After culturing for a further 24 h, the precursor disappeared and a new band, corresponding to 34 kDa, appeared, suggesting that the precursor had been processed to an intermediate form of cathepsin L. Biosynthetic labelling of macrophages in vitro with [35S]methionine followed by immunoprecipitation with the cathepsin L antibody confirmed that the cells synthesize cathepsin L as a 43 kDa precursor that is then processed to the mature form (25 kDa) via a 34 kDa intermediate. The precursor, but not the processed forms, was released into the culture medium. During culture in vitro the 34 kDa intermediate accumulated, and little enzyme was processed to the 24 kDa form, consistent with the immunoblot data. Human lung macrophages contain a 1.5 kb transcript of cathepsin L mRNA, whereas none is detectable in human monocytes. These results establish that differentiation of human macrophages within the lung is accompanied by synthesis and expression of an elastinolytic enzyme, cathepsin L. The altered processing of cathepsin L observed during cultivation in vitro suggests caution in the assessment of the elastinolytic potential of human macrophages based on assay in vitro.

The major active forms of cathepsins B and L were identified in Kirsten-virus-transformed mouse fibroblasts by the use of a specific radiolabelled inhibitor, benzyloxycarbonyl-Tyr(-125I)-Ala-CHN2. No other proteins were labelled, demonstrating the specificity of this inhibitor for cysteine proteinases. Cathepsins B and L were distinguished by the use of specific antibodies. One active form of cathepsin B, Mr 33,000-35,000, and two active forms of cathepsin L, Mr 30,000 and 23,000, were identified. The intracellular precursors of these proteins had higher Mr values of 39,000 and 36,000 for cathepsins B and L respectively, as shown by pulse-chase experiments with [35S]methionine-labelled proteins. These did not react with the inhibitor under our culture conditions. The precursor of cathepsin L was secreted whereas the precursor of cathepsin B was not, demonstrating that secretions of the two enzymes are regulated differently. In contrast with results found previously for the purified protein [Mason, Gal & Gottesman (1987) Biochem. J. 248, 449-454], the secreted precursor form of cathepsin L did not react with the inhibitor either, indicating that it is not active and therefore, as such, cannot be directly involved in tumour invasion. The secreted protein did react with the inhibitor when incubated at pH 3.0, showing that the protein can be activated, although this did not occur under our culture conditions.

The pituitaries from steers fed for 125 days on rations varying in level of protein and carotene were assayed for thyrotropic hormone content. No effect of ration on thyrotropic hormone content of the pituitary glands was found. One lot of steers to which 10 mg. diethylstilbestrol daily per steer was included in the ration showed thyrotropic hormone content of the pituitary glands not altered by the hormone-like material.