Effects of ATP, vanadate, and molybdate on cathepsin D-catalyzed proteolysis

The effects of ATP, vanadate, and molybdate on cathepsin D-catalyzed hydrolysis of proteins and peptides were examined. Hydrolysis of bovine serum albumin, hemoglobin, parathyroid hormone, and a synthetic octapeptide was activated by ATP. Degradation of the protein substrates all had similar ATP concentration dependence, but the magnitude of the activation varied. Kinetic constants for ATP activation were obtained with a synthetic substrate. ATP increased kcat from 0.4 to 2 s-1 but did not change KM. Kact for ATP was 800 microM. Studies with pepstatin-Sepharose confirm that ATP does not alter the substrate binding site on cathepsin D. Pepsin, a homologous aspartate protease, was not activated by ATP. It was also found that vanadate and molybdate inhibit cathepsin D-catalyzed proteolysis. However, this inhibition was dramatically dependent on substrate concentration and was eliminated at high substrate. Hydrolysis of the synthetic peptide was not inhibited at concentrations of molybdate below 50 microM, and above this concentration the peptide precipitated. Protein substrates were also found to precipitate in the presence of molybdate. The ATP dependence of the enzyme was not altered by molybdate or vanadate. These results suggest that inhibition by vanadate and molybdate is related to interactions with the substrate rather than with cathepsin D. It is concluded that ATP activation of cathepsin D may play a physiological role in regulation of proteolysis in lysosomes, but that vanadate and molybdate inhibition of lysosomal proteolysis does not establish ATP dependence.     

ATP activation of protein degradation by extracts of crude and purified lysosomal preparations

Activation of proteolysis by ATP was studied in lysates of crude and purified lysosomal preparations from liver and kidney at acid pH. In the crude system, from kidney, it was found that ATP activates proteolysis over a concentration range of 0.1-2 mM. Up to 4-fold activation was observed. GTP and CTP also activated proteolysis, but to a lesser extent. Proteolysis was inhibited by vanadate and molybdate. Fractionation of the kidney lysosomes on Percoll gradients produced two fractions containing lysosomal marker enzymes. Most of the acid phosphatase and the acid pyrophosphatase were found in the lighter band, while most of the beta-galactosidase and cathepsin activity was found in a more dense band. Proteolysis by lysates of both fractions was activated by ATP and inhibited by vanadate and molybdate. In the dense band proteolysis was also nearly totally blocked by pepstatin, and was enhanced by an inhibitor of pyrophosphatases, sodium fluoride. ATP also activates proteolysis in crude lysosomes from liver, but upon fractionation of this tissue it was found that all the lysosomal enzyme markers are present in the dense fraction obtained from the Percoll gradient. Again, proteolysis by lysates of the purified fractions was activated by ATP and inhibited by vanadate and molybdate. These data indicate that ATP can activate proteolysis at acid pH in a lysosomal milieu containing enzymes which also catalyze its breakdown. In the kidney there may be two lysosomal compartments which separate the enzymes catalyzing ATP breakdown from the proteolytic enzymes, but this is not essential for ATP activation as shown by the data from the liver and the crude lysosomal fractions.     

Vanadate inhibits the ATP-dependent degradation of proteins in reticulocytes without affecting ubiquitin conjugation

Reticulocytes contain a nonlysosomal, ATP-dependent system for degrading abnormal proteins and normal proteins during cell maturation. Vanadate, which inhibits several ATPases including the ATP-dependent proteases in Escherichia coli and liver mitochondria, also markedly reduced the ATP-dependent degradation of proteins in reticulocyte extracts. At low concentrations (K1 = 50 microM), vanadate inhibited the ATP-dependent hydrolysis of [3H]methylcasein and denatured 125I-labeled bovine serum albumin, but it did not reduce the low amount of proteolysis seen in the absence of ATP. This inhibition by vanadate was rapid in onset, reversed by dialysis, and was not mimicked by molybdate. Vanadate inhibits proteolysis at an ATP-stimulated step which is independent of the ATP requirement for ubiquitin conjugation to protein substrates. When the amino groups on casein and bovine serum albumin were covalently modified so as to prevent their conjugation to ubiquitin, the derivatized proteins were still degraded by an ATP-stimulated process that was inhibited by vanadate. In addition, vanadate did not reduce the ATP-dependent conjugation of 125I-ubiquitin to endogenous reticulocyte proteins, although it markedly inhibited their degradation. In intact reticulocytes vanadate also inhibited the degradation of endogenous proteins and of abnormal proteins containing amino acid analogs. This effect was rapid and reversible; however, vanadate also reduced protein synthesis and eventually lowered ATP levels in the intact cells. Vanadate (10 mM) has also been reported to decrease intralysosomal proteolysis in hepatocytes. However, in liver extracts this effect on lysosomal proteases required high concentrations of vanadate (K1 = 500 microM) and was also observed with molybdate, unlike the inhibition of ATP-dependent proteolysis in reticulocytes.     

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.     

Effect of vanadate, molybdate, and azide on membrane-associated ATPase and soluble phosphatase activities of corn roots

The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 × MO17) roots were investigated. Azide (0.1-10 millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 millimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase.

Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by K⁺. K⁺-stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K⁺ transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate.

     

Salt concentration-dependency of vitellogenin processing by cathepsin D in Xenopus laevis

The mechanism by which cathepsin D produces only limited proteolysis of vitellogenins (VTG) was studied in Xenopus oocytes. We first examined mature oocytes for the existence of cathepsin D; immunoblot and biochemical analyses revealed the existence of a 43kDa enzyme protein and its proteolytic activity in oocytes during and after the vitellogenesis. By determining the proteolytic activity of the fractions after subcellular fractionation of oocytes, we confirmed that cathepsin D is preserved in the yolk plasma of mature yolk platelets. The reaction of VTG with cathepsin D was examined in vitro at pH 5.6 as a function of NaCl concentrations. Lipovitellins generated from the VTG were preserved for several days at 37°C in the presence of the enzyme if the NaCl concentration was 0.15 mol/L or lower. The amount of lipovitellins decreased with increased molarity of the salt and at 0.5 mol/L NaCl they were rapidly degraded. The precipitates, growing in the reaction tube with 0.15 mol/L NaCl, included all constituents of yolk proteins and were ultrastructurally shown to have crystal structures perforated by empty cavities. No precipitates appeared at 0.5 mol/L NaCl. The results indicate that the limitation on proteolysis of the VTG by cathepsin D is due to the insolubility of yolk proteins at physiological salt concentrations, which explains why yolk can be stored stably in the presence of acid hydrolases over a long period.     

Purification and properties of cathepsin D from porcine spleen.

Cathepsin D was purified from porcine spleen to near homogeneity as determined by gel electrophoresis. The isolation scheme involved an acid precipitation of tissue extract, DEAE-cellulose and Sephadex G-200 chromatography, and isoelectric focusing. The end product represented about a 1000-fold purification and about a 10% recovery. The purified enzyme was the major isoenzyme, which represented 60% of cathepsin D present in porcine spleen. Two minor isoenzymes of cathepsin D were present in small amounts. The purified enzyme resembled porcine pepsin in molecular weight (35,000), amino acid composition, and inactivation by specific pepsin inactivators. The pH activity curve of the purified enzyme showed two optima near pH 3 and 4. The relative activities at these optimal pH values were affected by salt concentration. Experimental evidence indicated that the two-optima phenomenon is a property of a single enzyme species.     

Cathepsin D. Characteristics of immunoinhibition and the confirmation of a role in cartilage breakdown

1. Antisera were raised against lysosomal cathepsin D of man, chicken and rabbit. 2. The antisera were found to be specific and potent inhibitors of cathepsin D activity. 3. The immunological nature of the inhibition was established. 4. The inhibitory effect was studied by varying pH, antiserum/enzyme ratio, time of incubation, concentration of components and order of mixing, and by using purified antibody and univalent antibody fragments. 5. The specificities of the antisera were examined with respect to other enzymes, isoenzymes of cathepsin D and cathepsin D from different organs. 6. The antisera prevented the action of cathepsin D on isolated proteoglycans and on cartilage. 7. The antisera produced up to 90% inhibition of the autolysis of cartilage from chicks and rabbits, indicating that cathepsin D is the enzyme mainly responsible for the breakdown of proteoglycans in this system.     

Inactivation of fructose-1,6-bisphosphate aldolase by cathepsin L stimulation by ATP

Cathepsin L was capable of destroying rabbit muscle aldolase (d-fructose-1,6-bisphosphate d-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) activity towards the substrate fructase 1,6-bisphosphate. The rate of loss of activity towards this substrate was stimulated (approx. 2-fold) by physiological concentrations of ATP and to a lesser degree by GTP, CTP, UTP, ADP and cyclic AMP, while PP_i and P_i decreased the rate of inactivation. Other proteinases (cathepsin B, cathepsin D, trypsin and chymotrypsin) also decreased aldolase activity toward fructose 1,6-bisphosphate more rapidly in the presence of ATP and more slowly in the presence of P_i. Cathepsin L, at higher concentrations, was capable of inactivating aldolase activity towards fructose 1-phosphate and extensively degrading the enzyme; these reactions were not affected by ATP and P_i. The thermostability of aldolase was also unaffected by these ligands. ATP and P_i had no effect on the rates of hydrolysis of other proteins (hemoglobin, bovine serum albumin, casein and azocasein) by cathepsin L. These data indicate that the effects of ATP and P_i was due to interactions of these ligands with aldolase that make the enzyme more vulnerable to limited but not extensive proteolysis; these ligands do not directly affect cathepsin L activity.     

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.     

Acid-activated insulin-like growth factor binding protein protease activity of Cathepsin D in normal and malignant prostatic epithelial cells and seminal plasma

In this study, we demonstrate insulin-like growth factor binding protein (IGFBP) acid proteolysis in conditioned media (CM) from normal and malignant primary cultures of prostatic epithelial cells, prostatic cell lines, and in seminal plasma. We further demonstrate the absence of such activity in CM from prostatic stromal cells. Radio-labeled IGFBPs (1–6) were incubated with various acidified CM and seminal plasma. None of these media showed IGFBP proteolytic activity at neutral pH, but all CM from prostatic epithelial cells (PC-E) demonstrated strong IGFBP proteolysis at acidic pH. No acid-activated proteolysis was observed in the CM from stromal cell cultures. In order to ascertain the role of cathepsin D, anti-cathepsin antibodies were used to immunodeplete the media of the selected enzymes prior to incubation with IGFBPs. Depletion of cathepsin D greatly reduced the proteolytic activity of the PC-E CM. Additionally, purified cathepsin D yielded a digestion pattern identical to that produced by prostatic cell CM and seminal plasma, following acidic incubation with IGFBP-3. Remarkably, the proteolytic pattern generated by seminal plasma, when incubated with IGFBP-3 at neutral pH, corresponded to that produced by prostate-specific antigen (PSA), demonstrating the interpolation of both neutral and acid proteases from prostate cells into seminal plasma. In conclusion, prostatic epithelial cells secrete acid-specific IGFBP protease(s) related to cathepsin D. Although no significant statistical difference was observed in the degree of acid-specific proteolysis in the media from normal versus malignant primary epithelial cell cultures, physiologicalcharacteristics of the malignant state might facilitate increased cathepsin D activity. We suspect this proteolysis may play a role in prostatic cell proliferationand invasive tumor growth. J. Cell. Physiol. 171:196–204, 1997. © 1997 Wiley-Liss, Inc.     

Adenosine triphosphate hydrolysis by purified rubisco activase

Activation of ribulose bisphosphate carboxylase/oxygenase (rubisco) in vivo is mediated by a specific protein, rubisco activase. In vitro, activation of rubisco by rubisco activase is dependent on ATP and is inhibited by ADP. Purified rubisco activase hydrolyzed ATP with a specific activity of 1.5 mumol min-1 mg-1 protein, releasing approximately stoichiometric amounts of ADP and Pi. Hydrolysis was highly specific for ATP-Mg and had a broad pH optimum, with maximum activity at pH 8.0-8.5. ATPase activity was inhibited by ADP but not by molybdate, vanadate, azide, nitrate, or fluoride. Addition of rubisco in either the inactive or activated form had no significant effect on ATPase activity. Incubation of rubisco activase in the absence of ATP resulted in loss of both ATPase and rubisco activation activities. Both activities were also heat labile, with 50% loss in activity after 5 min at 38 degrees C and complete inhibition following treatment at 43 degrees C. Both activities showed a sigmoidal response to ATP concentration, with half-maximal activity at 0.053 mM ATP. Rubisco activation activity was dependent on the concentrations of both ATP and ADP. The results suggest that ATPase activity is an intrinsic property of rubisco activase.     

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.     

The vanadate complex of the calcium-transport ATPase of the sarcoplasmic reticulum, its formation and dissociation

Vanadate binding to different sarcoplasmic reticulum membrane preparations was determined by measuring bound vanadate colorimetrically and by phosphorylating the vanadate-free enzyme fraction with [gamma-32P] ATP. Colorimetry allowed the study of the dependence of equilibrium vanadate binding on ionized magnesium and the displacing effect of ionized calcium at vanadate concentrations greater than 0.1 mM only. At saturating magnesium concentration the enzyme binds 6-8 nmol vanadate/mg protein and half-maximum saturation is reached at 40 microM. Vanadate is displaced from the enzyme when its high-affinity calcium-binding sites are saturated and conversely calcium is solely displaced from its high-affinity binding sites by vanadate. The phosphorylation procedure allowed the measurement of equilibrium binding as well as the kinetics of vanadate binding and release at vanadate concentrations below 0.1 mM. Half-times of 30s and 3s were observed for vanadate release induced by 0.1 mM and 1 mM calcium respectively. Millimolar concentrations of ATP are required for vanadate displacement. Under equilibrium conditions the enzyme displays an affinity for vanadate of 1.6 X 10(6) M-1. The dependence on the concentration of vanadate of the rate of vanadate binding yielded an affinity of only 1 X 10(4) M-1. Closed vesicles bind vanadate much more slowly than calcium-permeable preparations. The initial rate of calcium-induced vanadate dissociation is accelerated considerably when the vesicles are made calcium permeable. The rate of vanadate dissociation from calcium-permeable vesicles reaches half-maximum values at 1-2 mM calcium indicating that the internal low-affinity calcium-binding sites must first be occupied in order to release bound vanadate. The results suggest that vanadate binding leads to a transition of the external high to internal low-affinity calcium-binding sites.     

Acidic pH as a physiological regulator of human cathepsin L activity.

Human cysteine protease cathepsin L was inactivated at acid pH by a first-order process. The inactivation rate decreased with increasing concentrations of a small synthetic substrate, suggesting that substrates stabilize the active conformation. The substrate-independent inactivation rate constant increased with organic solvent content of the buffer, consistent with internal hydrophobic interactions, disrupted by the organic solvent, also stabilizing the enzyme. Circular dichroism showed that the inactivation is accompanied by large structural changes, a decrease in alpha-helix content being especially pronounced. The high activation energy of the reaction at pH 3.0 (200 kJ.mol-1) supported such a major conformational change occurring. The acid inactivation of cathepsin L was irreversible, consistent with the propeptide being needed for proper folding of the enzyme. Aspartic protease cathepsin D was shown to cleave denatured, but not active cathepsin L, suggesting a potential mechanism for in-vivo regulation and turnover of cathepsin L inside lysosomes.     

The plasma membrane H(+)-ATPase from yeast. Effects of pH, vanadate and erythrosine B on ATP hydrolysis and ATP binding.

The H(+)-ATPase from the plasma membrane of Saccharomyces cerevisiae was isolated and purified. The rate of ATP hydrolysis and ATP binding was measured as a function of pH and the effect of the vanadate and erythrosine B inhibitors was investigated. The pH dependence of the rate of ATP hydrolysis forms a bell-shaped curve with a maximum at pH 6 and half-maximal rates at pH 5.0 and 7.4. Only the pH dependence between pH 6 and pH 7.6 is reversible. Above pH 7.6 and below pH 5.5, denaturation of the isolated enzyme is observed. The rate of ATP binding shows the same pH dependency as that of ATP hydrolysis. Both pH dependencies can be described by the dissociation of a monovalent acidic group with a pK of 7.4. It is concluded that the enzyme must be protonated before ATP binding. Vanadate does not inhibit ATP binding, ADP release or Pi release at concentrations where complete inhibition of ATP hydrolysis is observed. It is concluded that vanadate inhibits a step of the reaction cycle which occurs after Pi release. In contrast, erythrosine B inhibits ATP binding and thus affects the first step of the reaction cycle.     

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.     

Proteolysis of isolated mitochondria by myocardial lysosomal enzymes.

1. Solubilized mitochondria and lysosomal fractions were obtained from guinea-pig heart by differential centrifugation and selective membrane disruption. 2. Mitochondria incubated at 37 degrees C in the presence of lysosomal enzymes underwent proteolysis. The rate of protein degradation was inversely dependent on pH. 3. The use of proteinase inhibitors showed that at low pH the major enzyme involved in mitochondrial digestion was cathepsin D. 4. At neutral pH carboxyl proteinases were still active, but thiol proteinases accounted for most of the protein breakdown. 5. The role of lysosomal enzymes as mediators of mitochondrial damage in ischaemic myocardium is discussed.     

Vanadate inhibition of ATP and p-nitrophenyl phosphate hydrolysis in the fragmented sarcoplasmic reticulum.

The vanadate inhibition of the Ca(2+)-ATPase activity was analysed both in intact sarcoplasmic reticulum vesicles and in the presence of low concentrations of Tween 20, using ATP and p-nitrophenyl phosphate as substrates. The saturation of the internal low-affinity calcium-binding sites protects the enzyme against vanadate inhibition, because: (1) p-nitrophenyl phosphate hydrolysis is not inhibited by vanadate in intact vesicles, but inhibition developed after solubilization with detergents; (2) the vanadate inhibition of the p-nitrophenyl phosphate hydrolysis in solubilized preparations is prevented by free Ca2+ concentrations higher than 10(-3) M and vanadate competes with calcium (10(-5)-10(-3) M); and (3) the vanadate inhibition of ATP hydrolysis is decreased with an increase in vesicular Ca2+ concentration. The presence of magnesium ions is indispensable for the vanadate effect. The vanadate inhibition is non-competitive with respect to Mg-p-nitrophenyl phosphate and uncompetitive with respect to Mg-ATP. However, in the presence of dimethyl sulfoxide, which facilitates phosphorylation of the enzyme, the inhibition is converted to a competitive one with respect to a substrate. The results suggest, that in the process of enzyme operation vanadate interacts with the unliganded E form of Ca(2+)-ATPase, occupying probably an intermediate position between the E2 and E1 forms, with the formation of an E2 Van complex, that imposes the inhibition on the Ca(2+)-ATPase activity.     

Proteolysis of canine apolipoprotein by acid proteases in canine liver lysosomes.

Canine liver lysosomes were purified by sucrose discontinuous density gradient centrifugation and then ruptured by sonication to obtain the soluble fraction. This soluble lysosomal fraction, which contained a 25-fold increase in acid phosphatase activity per mg of total protein when compared with the original homogenate, was incubated with a subfraction (1.110 less than d less than 1.210 g/cm3, HDL3) of canine high density lipoproteins (HDL) at pH 3.8. HDL3 proteolysis by lysosomal proteases, measured as the release of peptides and amino acids by the ninhydrin reaction, followed hyperbolic curves with straight lines (r = 0.99) obtained on Lineweaver-Burk plots. Km calculated from the Lineweaver-Burk plot was 635 mug of HDL3 protein per 0.5 ml of incubation mixture. Optimum HDL3 proteolysis was observed from pH 3.8 to 4.5. Incubation with the other subcellular organelle fractions did not result in HDL3 proteolysis. To evaluate the effects of enzyme inhibitors, iodoacetate, p-chloromercuribenzoate (both specific for the endopeptidase, cathepsin B (EC 3.4.22.1)) and pepstatin (specific for the endopeptidase, cathepsin D (EC 3.4.23.5) were tested. Iodoacetate and p-chloromercuribenzoate inhibited HDL3 proteolysis 100% and bovine serum albumin proteolysis 65%. Pepstatin inhibited HDL3 proteolysis 45% and bovine serum albumin proteolysis 70%. The in vitro data presented support the hypothesis that hepatic lysosomes play an important role in HDL3 catabolism in the dog. Furthermore, results obtained from enzyme inhibition studies suggest that a specific lysosomal endopeptidase, cathepsin B, may play the key role in HDL3 proteolysis.