The effects of proteolytic enzymes on the mechanical properties of adult human articular cartilage.

The effects of the lysosomal proteinase cathepsin D on the mechanical properties of adult human articular cartilage were examined in detail in 7 joints within the age range 21 to 72 years. The results of a preliminary study on the effects of the lysosomal proteinase cathepsin B1 and clostridial collagenase on the mechanical properties of cartilage are also presented. Cartilage which had been incubated with either cathepsin D or cathepsin B1 showed increased deformation in uniaxial compression perpendicular to the articular surface. The enzyme-treated cartilage also showed decreased tensile stiffness at low values of stress. This effect was more pronounced in specimens from the deeper zone of cartilage than in specimens from the superficial zone. It was also more pronounced in specimens which were aligned perpendicular to the predominant alignment of the collagen fibres in the superficial zone than in specimens which were parallel to the collagen fibres. At higher stresses the tensile stiffness of the treated cartilage was not significantly different from that of the untreated tissue. The tensile fracture stress of the cartilage was also not significantly reduced by the action of cathepsin D. In contrast to the effects observed with the cathepsins, the preliminary results obtained by incubating cartilage for 24 h with clostridial collagenase showed that both the tensile stiffness and the fracture stress were considerably lower than the corresponding values for the untreated tissue. Biochemical analysis of the incubation media, and the specimens, revealed that a large proportion of the proteoglycans was released from the cartilage by each of the three enzymes. The proportion of the total collagen which was released from the cartilage was different for each enzyme: cathepsin D released between 0 and 1.5 per cent, cathepsin B1 released between 2.3 and 4.3 per cent and collagenase released between 5.3 and 27.8 per cent of the collagen after 24 h.     

The effects of proteolytic enzymes on the mechanical properties of adult human articular cartilage

The effects of the lysosomal proteinase cathepsin D on the mechanical properties of adult human articulage were examined in detail in 7 joints within the age rangee 21 to 72 years. The results of preliminary study on the effects of the lysosomal proteinase cathepsin B1 and clostridial collagenase on the mechanical properties of cartilage are also presented.Cartilage which had been incubated with either cathepsin D or cathepsin B1 showed increased deformation in unixial compression perpendicular to the articular surface.The enzyme-treated cartilage also showed decreased tensile stiffness at low values of stress. This effect was more pronounced in specimens from the deeper zone of cartilage than in specimens from the superficial zone. It was also more pronounced in specimens which were aligned perpendicular to the predominant alignment of the collagen fibres in the superficial zone than in specimens which were parallel to the collagen fibres.At higher stresses the tensile stiffness of the treated cartilage was not significantly different from that of the untreated tissue. The tensile fracture stress of the cartilage was not significantly reduced by the action of cathepsin D.In contrast to the effects observed with the cathepsins, the preliminary results obtained by incubating cartilage for 24 h with clostridial collagenase showed that both the tensile stiffness and the fracture stress were considerably lower than the corresponding values for the untreated tissue.Biochemical analysis of the incubation media, and the specimens, reveled that a large proportion of the proteoglycans was released from the cartilage by each of the freeze enzymes. The proportion of the total collagen which was released from the cartilage was different for each enzyme: cathepsin D released between 0 and 1.5 per cent, cathepsin B1 released between 2.3 and 4.3 per cent and collagenase relesed between 5.3 and 27.8 per cent of the collagen after 24 h.     

Cathepsin D from Atlantic cod (Gadus morhua L.) liver. Isolation and comparative studies

The isolated cathepsin D-like enzyme from Atlantic cod (Gadus morhua L.) liver was shown to be a monomer with a molecular mass of approximately 40 kDa. It was inhibited by Pepstatin A and had an optimum for degradation of haemoglobin at pH 3.0. The purified enzyme had lower temperature stability than bovine cathepsin D. Antibodies raised against the purified enzyme and against two C-terminal peptides of cod cathepsin D recognized a 40 kDa protein in immunoblotting of the samples from the purification process. Both antisera showed cross reactivity with a similar sized protein in liver from cod, saithe (Pollachius virens L.), Atlantic herring (Clupea harengus L.) and Atlantic salmon (Salmo salar L.). A protein of same size was detected in wolffish (Anarhichas lupus L.) liver with the antibody directed against the purified enzyme. This antibody also recognized the native enzyme and detected the presence of cathepsin D in muscle of cod, saithe, herring and salmon. These antibodies may be useful in understanding the mechanisms of post mortem muscle degradation in fish by comparing immunohistochemical localization and enzyme activity, in particular in cod with different rate of muscle degradation. They may also be used for comparing muscle degradation in different fish species.     

Effect of cartilage proteoglycans on human collagenase activities.

No significant inhibition of purified rheumatoid synovial collagenase was found when this enzyme was assayed in the presence of porcine or human cartilage proteoglycans. Reaction mixtures containing up to twice the amount of proteoglycan compared to that of collagen, w/w, had little effect on collagen degradation as judged by the reconstituted [4C]collagen fibril assay and polyacrylamide gel electrophoresis. Proteoglycans were not degraded by the synovial collagenase preparation. Although the human collagenases derived from rheumatoid synoviam, gastric mucosa, skin and granulocytes showed some reduction in activity when exposed to aggregated proteoglycans at high concentrations, disaggregated proteoglycans had no inhibitory effect. It is concluded that cartilage proteoglycans do not directly inhibit human collagenases in vitro, but in vivo they may provide some physical barriers which might limit the accessibility of the enzyme to its collagen substrate.     

Effect of cartilage proteoglycans on human collagenase activities

No significant inhibition of purified rheumatoid synovial collagenase was found when this enzyme was assayed in the presence of porcine or human cartilage proteoglycans. Reaction mixtures containing up to twice the amount of proteoglycan compared to that of collagen, w/w,, had little effect on collagen degradation as judged by the reconstituted [¹⁴C] collagen fibril assay and polyacrylamide gel electrophoresis. Proteoglycans were not degraded by the synovial collagenase preparation. Although the human collagenases derived from rheumatoid synovium, gastric mucosa, skin and granulocytes showed some reduction in activity when exposed to aggregated proteoglycans at high concentrations, disaggregated proteoglycans had no inhibitory effect. It is concluded that cartilage proteoglycans do not directly inhibit human collagenases in vitro, but in vivo they may provide some physical barriers which might limit the accessibility of the enzyme to its collagen substrate.     

Alteration of Cartilage Lysosomal Enzyme Activities during Development

Cartilage cathepsin D, cathepsin B and acid phosphatase activities decreased with maturation of Sprague-Dawley rats. Although this phenomenon may largely be due to an age-dependent decrease in cell concentration at young ages (1–8 weeks), in older (8–25 weeks) rats there appeared to be a decrease in enzyme activity per cell. The dimunition in cartilage cathepsin D activity coincided with an apparent decrease in its concentration. In addition, the inverse correlation between rat age and cartilage lysosomal enzyme activities was, at least in part, tissue specific as the pattern of liver lysosomal enzyme activities was quite different from that noted with cartilage. Interestingly, hypophysectomy greatly diminished age-related modulations in lysosomal enzyme activities suggesting that one or more pituitary hormones may be involved in the mechanism of this age-dependent phenomenon. In addition, cartilage growth rate appeared to be correlated with the level of cartilage lysosomal enzyme activities, indicating that these enzymes may be related to the biochemical mechanism of cartilage growth and development.     

The effects of selective matrix degradation on the short-term compressive properties of adult human articular cartilage.

The effects of proteoglycan and collagen digestion on the transient response of human articular cartilage when tested in unconfined compression were determined. Small cylindrical specimens of cartilage, isolated from the femoral head of the hip joint and from the femoral condyles of the knee joint, were subjected to a suddenly applied compressive load using a test apparatus designed to yield a transient oscillatory response. From this response values of the elastic stiffness and the viscous damping coefficient were determined. Cathepsin D and cathepsin B1 were used to digest the proteoglycan in some specimens, while in other specimens leukocyte elastase was used to attack the non-helical terminal regions of the Type II tropocollagen molecules and possibly the Type IX collagen molecule and thereby disturb the integrity of the collagen mesh. The results showed that proteoglycan digestion alone reduced the viscous damping coefficient but it did not significantly alter the elastic stiffness as determined from the oscillatory response. In contrast, the action of elastase reduced both the damping coefficient and the elastic stiffness of the cartilage. The results demonstrated the role of proteoglycans in regulating fluid transport in cartilage and hence controlling the time-dependent viscous properties. The elastic stiffness was shown to be dependent on the integrity of the collagen fibre network and not on the proteoglycans.     

Cathepsin E from rat neutrophils: its properties and possible relations to cathepsin D-like and cathepsin E-like acid proteinases

An extract of rat neutrophils was found to contain a high hemoglobin-hydrolyzing activity at pH 3.2, about 70% of which does not cross-react with anti-rat liver cathepsin D antibody. A neutrophil non-cathepsin D acid proteinase was successfully isolated from cathepsin D and characterized in comparison with the properties of rat liver cathepsin D. The neutrophil enzyme differed from cathepsin D in chromatographic and electrophoretic behaviors as well as immunological cross-reactivity, and its molecular weight was estimated to be 98,000 by gel filtration on Toyopearl HW 55. These findings strongly suggest that the neutrophil enzyme could be classified as cathepsin E. The enzyme, now designated rat cathepsin E, had an optimal pH at 3.0-3.2, preferred hemoglobin to albumin as substrate, and was markedly resistant to urea denaturation. Rat cathepsins D and E cleaved the insulin B-chain at six and eight sites, respectively; five sites were common for both enzymes. Possible relations among cathepsin E and cathepsin D-like or E-like acid proteinases reported so far were discussed.     

Replacement of proteoglycans in embryonic chick cartilage in organ culture after treatment with testicular hyaluronidase

Explants of cartilage from tibiae of 11-12 days chick embryos were grown in organ culture. To one group hyaluronidase was added to the medium during the first 2 days of culture; the treated tissue was then cultured in medium without enzyme for a further 4 days. Control explants grown in hyaluronidase-free medium for 6 days grew rapidly in size and the total hexosamine content more than doubled during this time. After exposure to hyaluronidase, much of the hexosamine was lost from treated cartilage and appeared in the culture medium, but it was mostly replaced in the tissue during the subsequent recovery period. Analysis of cartilage and medium showed that net synthesis of hexosamine increased greatly in treated cartilage. The proteoglycans were extracted by two procedures from control and treated cartilage after 2, 4 and 6 days in culture. The hydrodynamic sizes of the purified proteoglycans were compared by gel chromatography and the composition of the gel-chromatographic fractions was determined. The proteoglycans from controls did not change during culture, but after exposure to hyaluronidase the proteoglycans from treated cartilage were of much smaller size and lower chondroitin sulphate content. During recovery, even though new proteoglycans were formed, they were nevertheless of smaller size and lower chondroitin sulphate content than control proteoglycans. They gradually became more like control proteoglycans during recovery from treatment, but even after 4 days they were not yet the same. After 2 days of treatment with the enzyme, the chondroitin sulphate in the cartilage was of shorter chain length than in controls but during recovery after 4 and 6 days in culture, the chain lengths in control and treated cartilage were similar. It is concluded that the proteoglycans formed in embryo cartilage in response to their depletion by enzyme treatment contained fewer chondroitin sulphate chains attached to the protein moiety of proteoglycans. This may have resulted from a failure under stress to glycosylate the protein moiety to the usual extent; alternatively the synthesis of normal proteoglycans of low chondroitin sulphate content may have increased, thus changing the proteoglycan population.     

Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species.

Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.     

Isolation, and catalytic and immunochemical properties of cathepsin D-like acid proteinase from rat erythrocytes

An erythrocyte membrane-associated cathepsin D-like acid proteinase, termed "EMAP," was purified to homogeneity from freshly collected rat blood in a yield of 60-65%. The molecular weight of the enzyme was determined to be 80,000-82,000 by Sephadex G-100 gel filtration. The enzyme was inhibited strongly by pepstatin and partially by HgCl2, Pb(NO3)2, and iodoacetic acid. The preferred substrate for the enzyme was hemoglobin. The enzyme also hydrolyzed serum albumin and casein, but to lesser extents, with an optimum pH of 3.5-4.0. However, it could not hydrolyze leucyl-2-naphthylamide, benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide or other synthetic substrates at pH values ranging from 3.5 to 9.5. The enzyme was very similar to human EMAP in a number of enzymatic properties, whereas it differed from rat cathepsin D in several respects, such as pH stability, molecular weight, isoelectric point, and chromatographic properties. Immunologically, the enzyme cross-reacted with the rabbit antibody prepared against human EMAP. The patterns of immunoelectrophoresis, immunoblotting, and immunoprecipitation of the enzyme were remarkably similar, if not identical, to those of human EMAP. In contrast, rat EMAP showed no reaction with the rabbit antibody raised to rat spleen cathepsin D. These results indicate that EMAP is a unique cathepsin D-like acid proteinase different from ordinary cathepsin D.     

The dermatan sulfate proteoglycans of bovine sclera and their relationship to those of articular cartilage. An immunological and biochemical study

Dermatan sulfate proteoglycans were isolated from adult bovine sclera and adult bovine articular cartilage. Their immunological relationships were studied by enzyme-linked immunosorbent assays using polyclonal antibodies raised against the large and small dermatan sulfate proteoglycans from sclera and a polyclonal and monoclonal antibody directed against the small dermatan sulfate proteoglycans from cartilage. The small dermatan sulfate proteoglycans from sclera and cartilage displayed immunological cross-reactivity while there was no convincing evidence of shared epitope(s) with the larger dermatan sulfate proteoglycans, nor did these larger proteoglycans share any common epitopes with each other. A hyaluronic acid binding region was detected immunologically on the larger scleral dermatan sulfate proteoglycan but was absent from the larger dermatan sulfate proteoglycan of cartilage and both the small dermatan sulfate proteoglycans. These antibodies were used in immunofluorescence microscopy to localize the scleral proteoglycans and molecules containing these epitopes in the eye. The large scleral dermatan sulfate proteoglycan was restricted to sclera while molecules related to the small scleral and cartilage proteoglycans were found in the sclera, anterior uveal tract, iris, and cornea. Amino acid sequencing of the amino-terminal regions of the core proteins of the small dermatan sulfate proteoglycans from sclera and articular cartilage showed that all the first 14 amino acids analyzed were identical and the same as reported earlier for the small bovine skin and tendon dermatan sulfate proteoglycans. These studies demonstrate that the larger dermatan sulfate proteoglycans of sclera and cartilage are chemically unrelated to each other and to the smaller dermatan sulfate proteoglycans isolated from these tissues. The latter have closely related core proteins and probably represent a molecule with a widespread distribution in which the degree of epimerization of glucuronic acid and iduronic acid varies between tissues.     

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.     

Comparisons of antibody reactivity and enzyme sensitivity between small proteoglycans from bovine tendon, bone, and cartilage

Preparations of small proteoglycans from bovine tendon, bone, and cartilage have been compared for sensitivity to various enzymes and reactivity with different polyclonal antibodies. Chondroitinase ABC digestion of all proteoglycans generated a core protein preparation that migrated similarly in sodium dodecyl sulfate-polyacrylamide electrophoresis as a doublet band with Mr approximately equal to 45,000. The small proteoglycans of cartilage were divided into two populations based upon electrophoretic migration of the intact molecules (Rosenberg, L. C., Choi, H. U., Tank, L-H., Johnson, T. L., Pal, S., Webber, C., Reiner, A., and Poole, A. R. (1985) J. Biol. Chem. 260, 6304-6313). The core preparations of tendon, bone, and the faster-migrating (PG II) proteoglycans of cartilage all interacted in Western blot/enzyme-linked immunosorbent assay analysis with polyclonal antibody raised against either the tendon or bone proteoglycans. The slower-migrating (PG I) proteoglycans of cartilage did not react with these antibodies. Digestion of the tendon small proteoglycan with Staphylococcus aureus V8 protease released glycosaminoglycan chains from the molecule and generated a 40-kDa protein fragment that was resistant to further rapid degradation by the enzyme. This large digestion fragment was also prominent following V8 protease digestion of the faster-migrating (PG II) population of small cartilage proteoglycans, but not the small proteoglycan of bone. The N-terminal amino acid sequence of the tendon (PG II) proteoglycan was determined. These observations provide additional evidence for heterogeneity among the chemically similar small proteoglycans from different tissues.     

Human erythrocyte membrane acid proteinase (EMAP): sidedness and relation to cathepsin D

An acid proteinase purified from human erythrocyte membranes (Yamamoto, K. & Marchesi, V.T. (1984) Biochem. Biophys. Acta 790, 208-218), now termed "EMAP," was further characterized with respect to its localization and relation to cathepsin D. The membrane-associated form of EMAP was shown to be latent by demonstrating that no activity was detectable in both resealed (right-side-out) ghosts and inside-out vesicles in the absence of detergents. The enzyme associated with the inside-out vesicles was unstable when exposured to acidic pH between 4.0 and 4.5, whereas the enzyme associated with the resealed ghosts was stable in the wide pH range of 3.7 to 9.0. Tryptic digestion produced the loss of activity for the enzyme associated with the inside-out vesicles but not the resealed ghosts. The antibody to rat spleen cathepsin D, which cross-reacted weakly but detectably with EMAP, selectively bound to the inside-out vesicles. These results indicate the location of EMAP on th inner surface of the membranes. Comparison of a number of enzymatic properties of EMAP with rat cathepsin D showed significant differences between these two enzymes. EMAP was less stable in the pH range of 3.5 to 6.0 than cathepsin D. The enzymes were distinguished from each other by differences in their elution profiles on DEAE-Sephacel and chromatofocusing columns and by differences in the extent of inhibition by a few specific inhibitors. Both enzymes revealed significant differences in the amino acid composition and specific activity towards bovine hemoglobin. The immunological relationship between these two enzymes is discussed.     

Evidence that extracellular cathepsin D is not responsible for the resorption of cartilage matrix in culture

Cathepsin D, the major lysosomal aspartic proteinase, is responsible for the autolysis of cartilage at slightly acidic pH, and it has been suspected of making a significant contribution to the breakdown of the living tissue, such as is stimulated by retinol. Our finding, however, has been that neither inhibitory antibodies against cathepsin D, nor chemical inhibition with pepstatin, significantly decreases the rate of degradation of proteoglycan in the organ culture system. Most of the other proteinase inhibitors tested were similarly ineffective, although EDTA and 1,10-phenanthroline inhibited the resorption by a cytotoxic effect. We conclude that although cartilage matrix degradation has clear characteristics of a proteolytic process, the identity of the enzyme(s) responsible remains obscure.     

The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan heterogeneity and the pathway of proteolytic degradation.

1. CaCl2-extracted proteoglycan from bovine nasal cartilage was degraded by four tissue proteinases till no further decrease in hydroynamic size was obtained. The proteoglycan and its final degradation products were then fractionated by Sepharose 2B chromatography. 2. The average size of the degradation products was least for cathepsin B and lysosomal elastase, and greatest for cathepsin D and cathepsin G. The latter two proteinases also produced degradation products that showed the widest range of sizes. 3. The structure of the degradation products ranged from peptides containing a single glycosaminoglycan chain to those containing twelve or more chains. Of the four proteinases, only cathepsin B produced peptides that contained a single chondroitin sulphate chain. 4. The proteoglycan was very heterogeneous with respect to size and chemical composition. Its behaviour on electrophoresis suggested that at least two genetically distinct core proteins might exist. 5. Irrespective of their structural variations, all proteoglycan molecules were able to interact with hyaluronic acid. In contrast, none of the degradation products were capable of this type of interaction. 6. A pathway for the proteolytic degradation of proteoglycans is postulated in which the sites of initial cleavage may be common to the majority of proteinases, whereas the production of the final clusters is dependent on the specificity of the proteinase. Only those proteinases of broadest specificity can produce single-chain chondroitin sulphate-peptides.     

Elucidation of cathepsin B-like activity associated with extracts of human myelin basic protein

Myelin basic protein (MBP) extracted from human delipidated white matter was found to be degraded at pH 3.0 by endogenous proteolytic activities of extracts. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Based on pH, activation by EDTA and DTE, and inhibition by p-CMPS, E-64 and, in particular, by leupeptin, the protease involved was tentatively identified as cathepsin B or a cathepsin B-like enzyme. As pepstatin failed to inhibit acid proteolysis of MBP cathepsin D was ruled out.     

Interaction of dinitrophenyl-pepstatins with human cathepsin D and with anti-dinitrophenyl antibody. Development of potential reagents for the localization in vivo of active proteinases at sites of tissue injury.

Extracellular cathepsin D has been observed by various cytochemical methods at sites of tissue injury. However, the role of this enzyme in connective tissue matrix degradation is uncertain because there are no histochemical methods for determining whether or not the cathepsin D is active at such sites in living tissues. We considered that the combined use of a labelled tight-binding inhibitor with immunoprecipitation of the enzymes might overcome this problem. We have explored the application of derivatives of the inhibitor pepstatin, as only active cathepsin D binds pepstatin tightly. A series of N-pepstatinyl-N'-dinitrophenyl-alpha, omega-diaminoalkanes were synthesized with alkyl-chain lengths of two, four and six carbon atoms. These compounds were tight-binding inhibitors of human cathepsin D. In fluorescence-quenching titrations the dinitrophenyl groups were also fully available to bind high-affinity anti-dinitrophenyl antibody. It was shown by immunodiffusion in gels and by gel permeation chromatography that N-pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane was a bifunction inhibitor able to bind cathepsin D and anti-dinitrophenyl antibody at the same time.     

Unique cathepsin D-type proteases in rat thoracic duct lymphocytes and in rat lymphoid tissues.

Two unique cathepsin D-type proteases apparently present only in rat thoracic duct lymphocytes and in rat lymphoid tissues are described. One, termed H enzyme, has an apparent molecular weight of similar to95,000; the other, termed L enzyme, has an apparent molecular weight of similar to45,000, in common with that of most cathepsins D from other tissues and species. Both enzymes differ from cathepsin D, however, by a considerably greater sensitivity to inhibition by pepstatin and by a smaller degree of inhibition by an antiserum which inhibits rat liver cathepsin D. H enzyme is converted to L enzyme by treatment with beta-mercaptoethanol; the relationship between the two enzymes remains unknown. H and L enzyme have been detected in rat lymphoid tissues and in mouse spleen, but they are not present in other rat tissues (liver, kidney, adrenals), rabbit tissues, calf thymus, bovine spleen, or human tonsils. As measured on acid-denatured bovine hemoglobin as substrate, both enzymes have pH activity curves identical with that of rat liver cathepsin D, with optimal activity at pH 3.6. Activity on human serum albumin is much less and also shows an optimum at pH 3.6; hence, neither enzyme has the properties of cathepsin E. Thiol-reactive inhibitiors have no effect on the activity of H and L enzyme; thus they do not belong to the B group of cathepsins. Additional information, discussed in this paper, leads us to conclude that partially purified H and L enzymes are cathepsin D-type proteases.