Bimane-labelled pepstatin, a fluorescent probe for the subcellular location of cathepsin D.

1. Pepstatinyl-cystamine was synthesized. The disulphide bond was cleaved and the pepstatin-bound thiol was made to react with monobromobimane. The fluorescent N-pepstatinyl-S-bimanyl-2-aminoethanethiol was purified. 2. Human cathepsin D showed tight binding of the bimane-labelled pepstatin at pH 3.5. The titration curves were used to determine the apparent dissociation constant, KD; values of approx. 1 x 10(-10) M were obtained. 3. Gel-chromatographic experiments showed that, like that of pepstatin, the binding of N-pepstatinyl-S-bimanyl-1-aminoethanethiol to cathepsin D was strongly pH-dependent. Binding was seen at pH 5.0, but could not be demonstrated at pH 7.4. 4. Cultured human synovial cells were fixed and incubated with the fluorescent inhibitor at pH 5.0 or pH 7.4. When examined by fluorescence microscopy the cells stained at pH 5.0 showed a punctate perinuclear distribution of bimane fluorescence. By contrast, the cells stained at pH 7.4 showed no fluorescence. 5. The distribution of cathepsin D, determined by indirect immunofluorescence at pH 7.4, closely resembled that of the fluorescent inhibitor seen at pH 5.0. 6. We conclude that N-pepstatinyl-S-bimanyl-2-aminoethanethiol is a fluorescent probe selective for the active conformation of cathepsin D.     

Dinitrophenyl-pepstatins as active-site-directed localization reagents for cathepsin D

1. N-Pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane, a potential active-site-directed localization reagent for cathepsin D, was found to bind non-specifically to immuno-precipitates containing cathepsin D. 2. Three new water-soluble localization reagents were synthesized, by using NN'-bis-(3-aminopropyl)piperazine, 3-oxa-1,5-diamino-pentane or 3,6-dioxa-1,8-diamino-octane, as spacer arms between the pepstatin and dinitrophenyl moieties. 3. The hydrophilic dinitrophenyl-pepstatins were all tight-binding inhibitors of cathepsin D at pH 3.5, but showed little or no binding to immuno-precipitates containing the inactive enzyme at pH 7.4. 4. Gel-chromatographic experiments showed that, at pH 5.0, all the dinitrophenyl-pepstatins were bifunctional reagents able to bind cathepsin D and anti-dinitrophenyl antibody at the same time. Enzyme-inhibitor-antibody complexes were not formed at pH 7.4, thus confirming that the reagents were active-site-directed. 5. Cultured human synovial cells were fixed and incubated with the dinitrophenyl-pepstatins at pH 5.0 or pH 7.4. After washing briefly, the cells were incubated at the appropriate pH value with anti-dinitrophenyl antibody labelled with fluorescein. When examined by fluorescence microscopy the cells stained at pH 5.0 showed fluorescent perinuclear granules, which were not seen in the cells treated at pH 7.4. The distribution of cathepsin D, determined by indirect immuno-fluorescence at pH 7.4, closely resembled that revealed by the dinitrophenyl-pepstatins at pH 5.0. 7. NN'-(3-Pepstatinylaminopropyl-3'-dinitrophenylaminopropyl)piperazine gave the most intense lysosomal staining and showed no non-specific binding. We conclude that this reagent is suitable for the subcellular localization of the active conformation of cathepsin D.     

Distinct cleavage specificity of human cathepsin E at neutral pH with special preference for Arg-Arg bonds

In order to clarify the potential role of cathepsin E at neutral pH, the cleavage specificity of human cathepsin E was examined at pH 7.4 toward reduced-carboxymethylated(RCm-)ribonuclease A and various bioactive and related peptides. The specificity of the enzyme at pH 7.4 was found to be considerably different from that at acidic pH; preferential cleavages were observed with Arg-X and Glu-X bonds, which are not the major cleavage sites at acidic pH. Moreover, the Arg-Arg bond was found to be the most preferential site of cleavage. This unique specificity observed at pH 7.4 suggests the possibility that cathepsin E might be involved in processing and/or degradation of certain proteins and/or peptides at or near neutral pH in vivo.     

Endosomal proteolysis of insulin-like growth factor-I at its C-terminal D-domain by cathepsin B

IGF-I is degraded within the endosomal apparatus as a consequence of receptor-mediated endocytosis. However, the nature of the responsible protease and the position of the cleavage sites in the IGF-I molecule remain undefined. In vitro proteolysis of IGF-I using an endosomal lysate required an acidic pH and was sensitive to CA074, an inhibitor of the cathepsin B enzyme. By nondenaturing immunoprecipitation, the acidic IGF-I-degrading activity was attributed to the luminal species of endosomal cathepsin B with apparent molecular masses of 32- and 28-kDa. The cathepsin B precursor, procathepsin B, was processed in vitro within isolated endosomes at pH 5 or at 7 in the presence of ATP, the substrate of the vacuolar H(+)-ATPase. The rate of IGF-I hydrolysis using an endosomal lysate or pure cathepsin B was found to be optimal at pH 5-6 and moderate at pH 4 and 7. Competition studies revealed that EGF and IGF-I share a common binding site on the cathepsin B enzyme, with native IGF-I displaying the lowest affinity for the protease (IC50 approximately 1.5 microM). Hydrolysates of IGF-I generated at low pH by endosomal IGF-I-degrading activity and analyzed by reverse-phase HPLC and mass spectrometry revealed cleavage sites at Lys68-Ser69, Ala67-Lys68, Pro66-Ala67 and Lys65-Pro66 within the C-terminal D-domain of IGF-I. Treatment of human HepG2 hepatoma cells with the cathepsin B proinhibitor CA074-Me reduced, in vivo, the intracellular degradation of internalized [125I]IGF-I and, in vitro, the degradation of exogenous [125I]IGF-I incubated with the cell-lysates at pH 5. Inhibitors of cathepsin B and pro-cathepsin B processing, which abolish endosomal proteolysis of IGF-I and alter tumor cell growth and IGF-I receptor signalling, merit investigation as antimetastatic drugs.     

Effective activation of the proenzyme form of the urokinase-type plasminogen activator (pro-uPA) by the cysteine protease cathepsin L.

Increased levels of both the cysteine protease, cathepsin L, and the serine protease, uPA (urokinase-type plasminogen activator), are present in solid tumors and are correlated with malignancy. uPA is released by tumor cells as an inactive single-chain proenzyme (pro-uPA) which has to be activated by proteolytic cleavage. We analyzed in detail the action of the cysteine protease, cathepsin L, on recombinant human pro-uPA. Enzymatic assays, SDS-PAGE and Western blot analysis revealed that cathepsin L is a potent activator of pro-uPA. As determined by N-terminal amino acid sequence analysis, activation of pro-uPA by cathepsin L is achieved by cleavage of the Lys158-Ile159 peptide bond, a common activation site of serine proteases such as plasmin and kallikrein. Similar to cathepsin B (Kobayashi et al., J. Biol. Chem. (1991) 266, 5147-5152) cleavage of pro-uPA by cathepsin L was most effective at acidic pH (molar ratio of cathepsin L to pro-uPA of 1:2,000). Nevertheless, even at pH 7.0, pro-uPA was activated by cathepsin L, although a 10-fold higher concentration of cathepsin L was required. As tumor cells may produce both pro-uPA and cathepsin L, implications for the activation of tumor cell-derived pro-uPA by cathepsin L may be considered. Different pathways of activation of pro-uPA in tumor tissues may coexist: (i) autocatalytic intrinsic activation of pro-uPA; (ii) activation by serine proteases (plasmin, kallikrein, Factor XIIa); and (iii) activation by cysteine proteases (cathepsin B and L).     

On the substrate specificity of cathepsins B1 and B2 including a new fluorogenic substrate for cathepsin B1.

Cathepsin B1 from bovine spleen exhibited its greatest rates of hydrolysis on peptide β-naphthylamide (βNA) derivatives containing paired basic residues, i.e., Cbz-Arg-Arg-βNA, t-Boc-Lys-Lys-βNA, and t-Boc-Lys-Arg-βNA. Internal peptide bonds were not attacked. At its pH 6.5 optimum, cathepsin B1 hydrolyzed Cbz-Arg-Arg-βNA (K_m 0.18 mM) 64 times faster than Bz-DL-Arg-βNA (K_m 3.3 mM or 1.6 mM for the L isomer) and was therefore chosen to replace the latter as a more soluble and sensitive substrate for the assay of cathepsin B1. Although cathepsin B2 had no action on the β-naphthylamide substrates, it did manifest carboxypeptidase activity by attacking COOH-terminal residues exposed by the action of cathepsin B1. At its pH 5.0 optimum, cathepsin B2 behaved as a SH-dependent, non-specific carboxypeptidase by releasing COOH-terminal amino acids from a variety of Cbz-Gly-X substrates and polypeptides such as glucagon, Val-Leu-Ser-Glu-Gly, and penta-lysine.     

Crystal structure of cathepsin A,a novel target for the treatment of cardiovascular diseases

The lysosomal serine carboxypeptidase cathepsin A is involved in the breakdown of peptide hormones like endothelin and bradykinin. Recent pharmacological studies with cathepsin A inhibitors in rodents showed a remarkable reduction in cardiac hypertrophy and atrial fibrillation, making cathepsin A a promising target for the treatment of heart failure. Here we describe the crystal structures of activated cathepsin A without inhibitor and with two compounds that mimic the tetrahedral intermediate and the reaction product, respectively. The structure of activated cathepsin A turned out to be very similar to the structure of the inactive precursor. The only difference was the removal of a 40 residue activation domain, partially due to proteolytic removal of the activation peptide, and partially by an order–disorder transition of the peptides flanking the removed activation peptide. The termini of the catalytic core are held together by the Cys253–Cys303 disulfide bond, just before and after the activation domain. One of the compounds we soaked in our crystals reacted covalently with the catalytic Ser150 and formed a tetrahedral intermediate. The other compound got cleaved by the enzyme and a fragment, resembling one of the natural reaction products, was found in the active site. These studies establish cathepsin A as a classical serine proteinase with a well-defined oxyanion hole. The carboxylate group of the cleavage product is bound by a hydrogen-bonding network involving one aspartate and two glutamate side chains. This network can only form if at least half of the carboxylate groups involved are protonated, which explains the acidic pH optimum of the enzyme.     

Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules

Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH(2)-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in secretory vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in 'model' neuropeptide-containing secretory vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.     

Cathepsin B cleavage of Ii from class II MHC alpha- and beta-chains

Class II MHC-associated invariant chain (Ii) might regulate binding of digested peptides to the Ag binding site (desetope) of class II MHC proteins by directly or allosterically blocking that site until cleavage and release of Ii from MHC alpha- and beta-chains at the time of peptide charging. We examined the cleavage and release of Ii from class II MHC alpha/beta Ii trimers by cathepsin B, which has been shown by others to colocalize with class II MHC molecules in intracellular compartments and to generate antigenic peptide fragments. Cathepsin B at pH 5.0 cleaved and released Ii from class II MHC alpha- and beta-chains. Cathepsin B digested Ii from alpha- and beta-chains in a dose-dependent fashion, yielding 23-, 21-, and 10-kDa fragments. Blockage of cathepsin B activity with leupeptin restored the 2D(nonequilibrium pH gradient gel electrophoresis/SDS) PAGE patterns of Ii and sialic acid-derivatized forms of Ii seen without the protease. The fragmentation pattern of cathepsin D treatment was different from that of cathepsin B, yielding 25-kDa intermediates.     

Characterization of new fluorogenic substrates for the rapid and sensitive assay of cathepsin E and cathepsin D.

Cathepsin E and cathepsin D are two major intracellular aspartic proteinases implicated in the physiological and pathological degradation of intra- and extracellular proteins. In this study, we designed and constructed highly sensitive synthetic decapeptide substrates for assays of cathepsins E and D based on the known sequence specificities of their cleavage sites. These substrates contain a highly fluorescent (7-methoxycoumarin-4-yl)acetyl (MOCAc) moiety and a quenching 2,4-dinitrophenyl (Dnp) group. When the Phe-Phe bond is cleaved, the fluorescence at an excitation wavelength of 328 nm and emission wavelength of 393 increases due to diminished quenching resulting from the separation of the fluorescent and quenching moieties. The first substrate, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Le u-Lys(Dnp)gamma-NH2, in which the Lys-Pro combination at positions P5 and P4 was designed for specific interaction with cathepsin E, is hydrolyzed equally well by cathepsins E and D (kcat/Km = 10.9 microM(-1) x s(-1) for cathepsin E and 15.6 microM(-1) x s(-1) for cathepsin D). A very acidic pH optimum o was obtained for both enzymes. The second substrate, MOCAc-Gly-Lys-Pro-Ile-Ile-Phe-Phe-Arg-Le u-Lys(Dnp)gamma-NH2, in which the isoleucine residue at position P2 was meant to increase the specificity for cathepsin E, is also hydrolyzed equally by both enzymes (kcat/Km = 12.2 microM(-1) x s(-1) for cathepsin E and 16.3 microM(-1) x s(-1) for cathepsin D). The kcat/Km values for both substrates are greater than those for the best substrates for cathepsins E and D described so far. Unfortunately, each substrate shows little discrimination between cathepsin E and cathepsin D, suggesting that amino acids at positions far from the cleavage site are important for discrimination between the two enzymes. However, in combination with aspartic proteinase inhibitors, such as pepstatin A and Ascaris pepsin inhibitor, these substrates enable a rapid and sensitive determination of the precise levels of cathepsins E and D in crude cell extracts of various tissues and cells. Thus these substrates represent a potentially valuable tool for routine assays and for mechanistic studies on cathepsins E and D.     

Metabolism of a synthetic L-isoaspartyl-containing hexapeptide in erythrocyte extracts. Enzymatic methyl esterification is followed by nonenzymatic succinimide formation

The synthetic peptide, L-Val-L-Tyr-L-Pro-L-isoAsp-Gly-L-Ala, is a substrate for the erythrocyte and brain protein carboxyl methyltransferases. These enzymes catalyze the methyl esterification of the free alpha-carboxyl group of the isoaspartyl residue, to which the glycyl residue is linked through the side chain beta-carboxyl group. In this work, we show that the alpha-methyl ester of this peptide was rapidly demethylated (t1/2 = 4 min at 37 degrees C, pH 7.4) in erythrocyte cytosolic extracts and that the product of this reaction appears to be the succinimide ring derivative of the peptide. The rate of demethylation, measured at either pH 6.0 or 7.4, was the same in buffer and erythrocyte extracts, suggesting that succinimide formation was a nonenzymatic reaction. The L-succinimide is more stable than the ester, but can be hydrolyzed in buffer at pH 7.4 (t1/2 = 180 min at 37 degrees C) to give a mixture of about 75% isoaspartyl peptide and 25% normal aspartyl peptide. The metabolism of the succinimide hexapeptide in erythrocyte extracts appears to be more complex, however. The implications of this work for the methylation and demethylation of cellular proteins containing structurally altered aspartyl residues are discussed.     

Cleavage of peptide bonds bearing ionizable amino acids at P1 by serine proteases with hydrophobic S1 pocket

Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx = Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3-11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5-8.0, 5.5-6.0, and ∼10, respectively. At the normally reported pH optimum (pH 7-8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The pK values of the side chains of Asp and Lys in the hydrophobic S₁ pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.     

Cleavage C-terminal to Asp leads to covalent crosslinking of long-lived human proteins

With age, long-lived proteins in the human body deteriorate, which can have consequences both for aging and disease. The aging process is often associated with the formation of covalently crosslinked proteins. Currently our knowledge of the mechanism of formation of these crosslinks is limited. In this study, proteomics was used to characterize sites of covalent protein-protein crosslinking and identify a novel mechanism of protein-protein crosslinking in the adult human lens. In this mechanism, Lys residues are crosslinked to C-terminal Asp residues that are formed by non-enzymatic protein truncation. Ten different crosslinks were identified in major lens proteins such as αA-crystallin, αB-crystallin and AQP0. Crosslinking in AQP0 increased significantly with age and also increased significantly in cataract lenses compared with normal lenses. Using model peptides, a mechanism of formation of the Lys-Asp crosslink was elucidated. The mechanism involves spontaneous peptide cleavage on the C-terminal side of Asp residues which can take place in the pH range 5–7.4. Cleavage appears to involve attack by the side chain carboxyl group on the adjacent peptide bond, resulting in the formation of a C-terminal Asp anhydride. This anhydride intermediate can then either react with water to form Asp, or with a nucleophile, such as a free amine group to form a crosslink. If an ε-amino group of Lys or an N-terminal amine group attacks the anhydride, a covalent protein-protein crosslink will be formed. This bi-phasic mechanism represents the first report to link two spontaneous events: protein cleavage and crosslinking that are characteristic of long-lived proteins.     

The effects of polar and/or ionizable residues in the core and flanking regions of hydrophobic helices on transmembrane conformation and oligomerization

To explore the influence of amino acid composition on the behavior of membrane-inserted alpha-helices, we examined the behavior of Lys-flanked polyleucyl (pLeu) helices containing a single polar/ionizable residue within their hydrophobic core. To evaluate the location of the helices within the membrane by fluorescence, each contained a Trp residue at the center of the sequence. When incorporated into dioleoylphosphatidylcholine (DOPC) model membrane vesicles, pLeu helices with or without a single Ser, Asn, Lys, or Asp residue in the hydrophobic core maintained a transmembrane state (named the N state) at neutral and acidic pH. In this state, the central Trp exhibited highly blue-shifted fluorescence, and fluorescence quenching by nitroxide-labeled lipids showed it located at the bilayer center. A state in which Trp fluorescence red-shifted by several nanometers (named the B state) was observed above pH 10-11. B state formation appears to result from deprotonation of the flanking Lys residues. Despite the red shift in Trp emission, fluorescence quenching showed that in the B state the Trp at most is only slightly shallower than in the N state, suggesting the B state also is a transmembrane or near-transmembrane structure. The B state is characterized by increased helix oligomerization, as shown by the dependence of Trp lambda(max) on the concentration of the peptide within the bilayer at high pH. The pLeu peptide with a Asp residue in the core underwent a pH-dependent transition at a lower pH than the other peptides (pH 8-9). At high pH, it exhibited both a more highly red-shifted fluorescence and shallower Trp location than the other peptides. This state (named the S state) did not exhibit a concentration-dependent Trp lambda(max). We attribute S state behavior to the formation of a charged Asp residue at high pH, and a consequent movement of the Asp toward the membrane surface, resulting in the formation of a nontransmembrane state. We conclude that a polar or ionizable residue can readily be tolerated in a single transmembrane helix, but that the charges on ionizable residues in the core and regions flanking the helix significantly modulate the stability of transmembrane insertion and/or helix-helix association.     

Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.     

Chromophoric and fluorophoric peptide substrates cleaved through the dipeptidyl carboxypeptidase activity of cathepsin B

The action of bovine spleen cathepsin B as a dipeptidyl carboxypeptidase on newly synthesized substrates of the type peptidyl-X-p-nitrophenylalanyl (Phe(NO2))-Y (X,Y = amino acid residue) or 5-dimethylaminonaphthalene-1-sulfonyl (Dns)-peptidyl-X-Phe(NO2)-Y was investigated. The kinetic parameters of hydrolysis of the X-Phe(NO2) bond were determined by difference spectrophotometry (delta epsilon 310 = 1600 M-1 cm-1) or by spectrofluorometry by following the five- to eightfold increase of Dns-group fluorescence with excitation at 350 nm and emission at 535 nm. The substrates were moderately sensitive to cathepsin B; kcat varied from 0.7 to 4 s-1 at pH 5 and 25 degrees C; Km varied from 6 to 240 microM. The very acidic optima of pH 4-5 are characteristic for dipeptidyl carboxypeptidase activity of cathepsin B. Bovine spleen cathepsins S and H had little and no activity, respectively, when assayed with Pro-Glu-Ala-Phe(NO2)-Gly. These peptides should be a valuable tool for routine assays and for mechanistic studies on cathepsin B.     

Cathepsin B1. A lysosomal enzyme that degrades native collagen

1. Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid-soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. 2. At acid pH values cathepsin B1 released (14)C-labelled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4-chloromercuribenzoate, by the chloromethyl ketones derived from tosyl-lysine and acetyltetra-alanine and by human alpha(2)-macroglobulin. 3. Cathepsin B1 degraded collagen in solution, the pH optimum being pH4.5-5.0. The initial action was cleavage of the non-helical region containing the cross-link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment-long-spacing crystalloids (measured as native collagen molecules aligned with N-termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an alpha-chain was attacked it was rapidly degraded to low-molecular-weight peptides. 4. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. 5. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH3.0. Neither cathepsin B1 nor D cleaved Pz-Pro-Leu-Gly-Pro-d-Arg. 6. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. 7. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.     

The influence of pH on the degradation of bovine myelin basic protein by bovine brain cathepsin

The degradation of bovine myelin basic protein by bovine brain cathepsin D (ED 3.4.23.5) was studied over a pH range of 2.75 - 6.0. Throughout this pH range pepstatin, an inhibitor of cathepsin D, prevented the degradation. The degradation at a pH away from the optimum of pH 3.5 was predictably slower, but also resulted in more restricted cleavage. Above pH 4.5 bovine basic protein peptide 1 - 42 was not degraded further to peptide 1 - 36 as occurs at pH 3.5. Additionally, at pH 5.5 another fragment of basic protein, peptide 1 - 91, persisted indicating that under certain basic protein as well as basic protein peptide 43 - 169 may be cleaved in the molecular region of basic protein around the phenylalanyl-phenylalanine residues at position 88 - 89. The small amount of peptides 1 - 91 and 92 - 169 detected at pH 5.5 suggests that the bond between residues 91 and 92 in intact basic protein is a minor cleavage site. The options and variation in cleavage around residues 88 - 92 of basic protein presumably result from pH-dependent changes in conformation in the is region but could also be due to changes in conformation of cathepsin D. These results indicate that local tissue changes such a pH amy affect not only the velocity of the reaction but also the nature of th product formed by the degradation of basic protein by brain cathepsin D     

Purification and characterization of the yeast glycosylphosphatidylinositol-anchored, monobasic-specific aspartyl protease yapsin 2 (Mkc7p).

The Saccharomyces cerevisiae YPS2 (formerly MKC7) gene product is a glycosylphosphatidylinositol-linked aspartyl protease that functions as a yeast secretase. Here, the glycosylphosphatidylinositol-linked form of yapsin 2 (Mkc7p) was purified to homogeneity from the membrane fraction of an overexpressing yeast strain. Purified yapsin 2 migrated diffusely in SDS-polyacrylamide gel electrophoresis (molecular mass approximately 200 kDa), suggesting extensive, heterogeneous glycosylation. Studies using internally quenched fluorogenic peptide substrates revealed cleavage by the enzyme carboxyl to Lys or Arg. No cleavage was seen when both Lys and Arg were absent. No significant enhancement was seen with multiple basic residues. However, cleavage always occurred carboxyl to the most COOH-terminal basic residue. V(max)/K(m) was insensitive to P(2) and P(3) residues except that Pro at P(2) blocked cleavage entirely. These results suggest that yapsin 2 is a monobasic amino acid-specific protease that requires a basic residue at P(1) and excludes basic residues from P(1)'. The pH dependence of V(max)/K(m) for a substrate containing a pro-alpha factor cleavage site was bell-shaped, with a maximum near pH 4.0. However, V(max)/K(m) for a substrate mimicking the alpha-secretase site in human beta amyloid precursor protein was optimal near pH 6.0, consistent with cleavage of beta amyloid precursor protein by yapsin 2 when expressed in yeast.