Cathepsin L--a latent proteinase in guinea pig sperm

Guinea pig spermatozoa were found to contain a fully-latent cysteine proteinase that could be unmasked by incubating epididymal sperm for 2 hr at pH 3.5 and 37 degrees C. The proteinase was identified as cathepsin L (EC 3.4.22.15) on the basis of its optimal hydrolysis of benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide (Z-Phe-Arg-NMec) at pH 5.5; lack of action on Z-Arg-Arg-NMec and Arg-NMec; urea-enhanced digestion of azocasein; marked sensitivity to thiol reagents, leupeptin, Z-Phe-Phe-CHN2, and L-trans-epoxy-succinylleucylamido(3-methyl)butane (Ep-475 or E-64-c); and insensitivity to pepstatin and serine proteinase inhibitors. Gossypol, a male antifertility agent, was inhibitory. The unmasking phenomenon was reversibly inhibited by HgCl2 and mersalyl acid, and prevented by leupeptin and Ep-475, but not by pepstatin.     

Cathepsin D inactivates cysteine proteinase inhibitors, cystatins

The formation of inactive complexes in excess molar amounts of human cathepsins H and L with their protein inhibitors human stefin A, human stefin B and chicken cystatin at pH 5.6 has been shown by measurement of enzyme activity coupled with reverse-phase HPLC not to involve covalent cleavage of the inhibitors. Inhibition must be the direct result of binding. On the contrary the interaction of cystatins with aspartic proteinase cathepsin D at pH 3.5 for 60 min followed by HPLC resulted in their inactivation accompanied by peptide bond cleavage at several sites, preferentially those involving hydrophobic amino acid residues. The released peptides do not inhibit papain and cathepsin L. These results explain reported elevated levels of cysteine proteinases and lead to the proposal that cathepsin D exerts an important function, through inactivation of cystatins, in the increased activities of cysteine proteinases in human diseases including muscular distrophy.     

Two-step affinity-chromatographic purification of cathepsin D from pig myometrium with high yield.

Cathepsin D was purified by two-step affinity chromatography on concanavalin A-- and pepstatin--Sepharose. The main purification was achieved by washing the enzyme bound to the pepstatin--Sepharose column with buffered 6 M-urea. This step separated cathepsin D from all low- and high-molecular-weight impurities. Although the 1700-fold purified acid proteinase was homogeneous on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it still showed microheterogeneity.     

Cathepsin L. A new proteinase from rat-liver lysosomes.

1. Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondrial pellet, gel filtration of the lysosomal extract and chromatography on CM-Sephadex. 2. Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate its molecular weight was found to be 23000-24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8-6.1 ascertained by analytical isoelectric focusing. 3. Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6-7. In contrast to cathepsin B1, its capability of hydrolyzing N-substituted derivatives of arginine is low and it does not split esters. 4. Greatest activity is obtained close to pH 5.0 with 70-90% of maximal activity at pH 4.0 and pH 6.0 and 30-40% at pH 7.0. 5. The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl-lysine. Leupeptin acts as a pseudo-irreversible inhibitor. 6. The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep-frozen state.     

Purification and Characterization of Cathepsin D from Normal Human Breast Tissue

The lysosomal aspartyl protease cathepsin D is present in most mammalian cells and is active in the catabolism of intracellular and endocytosed proteins. It appears to be overexpressed and abnormally secreted in breast cancer cells, and may contribute to the process of tumor metastasis. In the present study, cathepsin D was purified 4500-fold from normal human breast tissue using pepstatin-agarose, DEAE Sephadex, and Sephadex G-75 chromatography. The resulting enzyme on SDS-PAGE contained five protein bands (47, 31, 29, 13, and 12kDa) which were all immunoreactive on western blot analysis using anti-cathepsin D polyclonal antibodies. The isoform profile of purified cathepsin D consisted of three major peaks at approximate pI 7.3, 6.8, and 6.3, and a broad area of lower activity between pI of 5.0 and 2.0. The purified enzyme had a broad pH optimum centered around pH 3.3. Lectin blotting indicated that cathepsin D is a glycoprotein which is recognized by Galanthus nivalis agglutinin and concanavalin A, suggesting the presence of mannose residues. However, Sambucus nigra agglutinin, Tetragonolobus purpureas agglutinin, Triticum vulgaris agglutinin, and Erythrina cristagalli agglutinin failed to recognize cathepsin D, suggesting a lack of lectin-available sialic acid, fucose, N-acetylglucosamine, and galactose residues, respectively.     

Cathepsin D generates gamma-endorphin from beta-endorphin.

A crude extract of porcine anterior pituitary was found to contain endopeptidase activity that splits the Leu⁷⁷-Phe⁷⁸ peptide bond of β-lipotropin in a pH range 3.0–7.0. The specificity and susceptibility to pepstatin of pituitary extract were the same as those of cathepsin D (E.C. 3.4.23.5) isolated from calf brain by affinity chromatography. Cathepsin D was shown to split the same Leu-Phe peptide bond of β-endorphin, leading to the formation of γ-endorphin. Based on the above data it is suggested that cathepsin D is a major factor in the generation of endorphins of intermediate size.     

Heat stable proteinase from Thermomonospora fusca. Characterization as a serine proteinase

An extracellular proteinase secreted by the thermophilic bacteria Thermomonospora fusca YX (YX-proteinase) is a serine proteinase as shown by its inactivation by the site specific reagents, phenylmethanesulfonyl fluoride, dansyl fluoride, and carbobenzoxy-L-phenylalanine chloromethyl ketone. This conclusion is further supported by the effect of various proteinase inhibitors on its activity. The activity of the proteinase toward small synthetic ester substrates shows that the enzyme has a primary specificity for the aromatic and hydrophobic amino acids. The amino acid composition and NH2-terminal sequence, as well as its size, suggest that the enzyme is related to the chymotrypsin-like microbial proteinase, alpha-lytic protease from Myxobacter 495 and protease A and B from Streptomyces griseus.     

Purification of a neutral proteinase, associated with the actomyosin complex, from uterine myometrium.

We have purified and characterized a neutral proteinase activity from pig uterine myometrium. The proteinase co-purified with the actomyosin complex and could only be separated from it by a high concentration of a chaotropic ion, 3M-NaBr. The proteinase was further purified by gel filtration and affinity chromatography. The purified protein showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to an Mr of 28 000. Gel filtration on Sephadex G-100 in a buffer containing 3M-NaBr gave an Mr of 27 500. Without the addition of the chaotropic Br- ion, the proteinase aggregates to high-Mr forms of more than 10(6)Da. The proteinase has optimum hydrolytic activity with casein as substrate at pH 7.5-8.0. The thiol-group-blocking reagents p-chloromercuribenzoate, p-chloromercuribenzenesulphonate and Hg2+, as well as soya-bean trypsin inhibitor and 4-aminobenzamidine, inhibited the proteinase. Other bivalent cations, chelating agents and the serine-specific reagents 7-amino-1-chloro-3-tosylamido-L-heptan-2-one and phenylmethanesulphonyl fluoride were without any effect on proteinase activity. The proteinase degraded myosin very rapidly at a molar ratio of proteinase to myosin of 1:50, concomitant with the rate of loss of the ATPase activity. Compared with myosin, actin was only a poor substrate and was degraded at a much lower rate, even at a high molar ratio of the proteinase to actin.     

Cathepsin D. Purification of isoenzymes from human and chicken liver

1. The Barrett (1967) assay for cathepsin D was slightly modified. 2. The enzyme was purified from liver of man and chicken by a procedure involving autolysis, acetone fractionation, ion-exchange chromatography and isoelectric focusing. 3. Several isoenzymes of cathepsin D were resolved in the isoelectric-focusing step, and three major forms, alpha,beta and gamma, were distinguished for each species. 4. A modified analytical method of isoelectric focusing in polyacrylamide gel indicated a high degree of homogeneity of the purified beta and gamma isoenzymes from each species, and this was supported by their constant high specific activities. 5. Gel filtration of the isoenzymes in a calibrated column of Sephadex G-100 showed that each had a molecular weight of 45000. 6. Human cathepsin D had a pH optimum of 3.5, and that of chicken enzyme was 3.0, haemoglobin being used as substrate. In each species, the three isoenzymes have the same pH-dependence curve. 7. The purified cathepsin D samples showed very little action on acid-denatured albumin.     

Cathepsin B, the lysosomal thiol proteinase of calf liver

Cathepsin B from calf liver was obtained by a method involving preparation of a lysosomal-mitochondrial pellet and treatment of this pellet with acetone. The material was extracted with an acid buffer, pH4.0, and then precipitated from the extract with acetone. The precipitate was dissolved in phosphate buffer, pH7.4, and subjected to gel filtration on Sephadex G-200 and G-100. The cathepsin B emerged in a range of molecular weight much lower than 50000 as a well-defined component. The purity of this material was checked by electrophoresis. To obtain maximum activity the enzyme had to be activated with a chelating agent and a reducing agent (i.e. EDTA and cysteine). A number of different substrates were used. The enzyme was active for the hydrolysis of both peptide bonds and ester bonds and had approximately equal reactivity in the two cases. The pH-dependence of the hydrolysis was the same with both substrates. The binding of the substrates was half-maximal at pH4.5 and at pH6.8. A thiol group occurred in the active centre but this group ought to have a much higher pK than that found in this enzyme.     

Characterization of the proteinase inhibitors from bull seminal plasma and spermatozoa.

Two acid stable proteinase inhibitors are present in bull seminal plasma and washed ejaculated bull spermatozoa. Inhibitor I with a molecular weight of about 8700 (estimated by gel filtration) is a very strong inhibitor of bull sperm acrosin but also inhibits bovine trypsin and chymotrypsin and porcine plasmin; inhibition of porcine pancreatic and urinary kallikrein was not observed. In this respect inhibitor I resembles the well known cow colostrum trypsin inhibitor. Inhibitor II with a molecular weight near 6800 (estimated by gel filtration) inhibits bovine trypsin and chymotrypsin, porcine plasmin and pancreatic and urinary kallikrein as well as bull acrosin. The inhibition specificity of inhibitor II is thus very similar to that of the basic inhibitor from bovine organs (Kunitz-type). In view of the inhibition strength and other characteristics, however, the acid stable bull seminal inhibitors are not identical with the inhibitor from cow colostrum or bovine lung (organs).     

Characterization of a chemotactic and cytotoxic proteinase from human skin.

A proteinase (EC 3.4.-.-) active at physiological pH has been isolated from human skin utilizing gel filtration and affinity chromatography techniques. The proteinase has a molecular weight of approx. 28 000 and it is inhibited by alpha 2-macroglobulin, alpha 1-antitrypsin, C-1 inactivatory, soybean trypsin inhibitor and diisopropyl fluorophosphate. 2njection of 1 ng of purified proteinase into rabbit skin induces polymorphonuclear leukocyte infiltration of the cutis. Inhibition of enzyme activity with diisopropyl fluorophosphate inhibits the chemotactic effect. Addition of 0.2 microgram/ml of purified proteinase to fibroblast cultures kills the cells within minutes. This proteinase may play a significant role in modulating the inflammatory response after cellular injury.     

Characterization of the Epstein-Barr virus proteinase and comparison with the human cytomegalovirus proteinase.

The BVRF2 gene of Epstein-Barr virus (EBV) shows homology to the UL26 and UL80 genes of herpes simplex virus type 1 (HSV-1) and cytomegalovirus (CMV), respectively. These genes are believed to provide a scaffold protein for the assembly of capsids leading to the formation of infectious viral particles. We have cloned the BVRF2 gene from the B95.8 strain of EBV and shown that the BVRF2 gene product is a polyprotein capable of autoproteolytic cleavage. Two Ala-Ser-containing recognition sequences were identified in the BVRF2 polyprotein at amino acid positions 568/569 and 570/571 where this cleavage was expected to occur. Here, we show that EBV proteinase is capable of cleaving at the first Ala-Ser bond but not the second. Comparison of the processing of the EBV and human CMV assembly domains in vitro by either EBV or human CMV proteinase revealed that, while both proteinases could cleave their native assembly domain, only EBV proteinase was able to cleave the assembly domain of the other virus.     

Cathepsin S. The cysteine proteinase from bovine lymphoid tissue is distinct from cathepsin L (EC 3.4.22.15).

Cathepsin S was purified from bovine spleen by acid autolysis, (NH4)2SO4 fractionation and chromatography on CM-Sephadex C-50, CM-cellulose and activated-thiol-Sepharose. Cathepsin L was isolated from lysosomal fractions of rat liver, rat kidney and bovine liver. Generally, cathepsin L was bound tightly to CM-Sephadex C-50. Preparations of cathepsin L from rat liver, rat kidney and bovine liver were shown to have kinetic constants for the substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide in the same range (Km 2-3 microM). Benzyloxycarbonyl-Phe-Phe-diazomethane proved to be a sensitive irreversible inhibitor of cathepsin L from different species. Cathepsin S differed in all these characteristics from cathepsin L. A polyclonal antibody to cathepsin L from rat reacted with bovine cathepsin L but not with bovine cathepsin S.     

Characterization of a proteinase inhibitor isolated from the fungal pathogen Coccidioides immitis.

1. Human erythrocytes were incubated in autologous plasma containing [32P]Pi, and sampled by a method which avoids washing the cells. 2. In experiments of up to 3 h duration, the specific radioactivity of cellular Pi stabilized at a value below that of extracellular Pi. This can be explained on the basis of a single cellular Pi pool exchanging with a large unlabelled pool of cellular organic phosphates. 3. However, a rapid initial phase of labelling, occurring within 30 s, was inconsistent with the situation described in point 2. A possible explanation is that about 1/4 of cellular Pi occurs in a separate, fast-labelling pool. 4. When the extracellular Pi concentration was doubled, most of the corresponding increase in the steady-state cellular Pi concentration was accounted for by the apparent fast-labelling Pi pool, which also doubled. 5. The observed initial rate of labelling of cellular organic phosphates [which probably occurs through the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12)] was considerably lower than that predicted from the flux through the Embden-Meyerhof pathway. This implies that the enzyme is exposed to Pi whose specific radioactivity is lower than the mean specific radioactivity of cellular Pi, and fails to support earlier suggestions that this enzyme uses extracellular Pi. 6. In 3 h incubations, the rate of organic phosphate labelling was roughly constant throughout, even though the specific radioactivity of cellular Pi had risen slowly to a plateau. Viewed in conjunction with point 5, this again suggests some inhomogeneity in cellular Pi. 7. Cellular Pi and extracellular Pi only reached isotopic steady state after 2 days. At this stage some organic phosphates were probably still incompletely labelled. 8. We conclude that, whatever their physical or technical reasons, such labelling inhomogeneities and slow attainment of isotopic steady state may cause serious misinterpretation of results if ignored during 32P-labelling of intact cells.     

New Functional Aspects of Cathepsin D and Cathepsin E

Cathepsin D (CD) and cathepsin E are representative lysosomal and nonlysosomal aspartic proteinases, respectively, and play an important role in the degradation of proteins, the generation of bioactive proteins, antigen processing, etc. Recenty, several lines of evidence have suggested the involvement of these two enzymes in the execution of neuronal death pathways induced by aging, transient forebrain ischemia, and excessive stimulation of glutamate receptors with excitotoxins. CD has also been shown to mediate apoptosis induced by various stimuli and p53-dependent tumor suppression. To gain more insight into in vivo functions of CD, mice deficient in this enzyme were generated. The mutant animals showed a progressive atrophy of the intestinal mucosa, a massive destruction of lymphoid organs, and a profound accumulation of ceroid lipofuscin, and developed a phenotype resembling neuronal ceroid lipofucinosis, suggesting that CD is essential for proteolysis of proteins regulating cell growth and tissue homeostasis. It has also been shown that CD molecules secreted from human prostate carcinoma cells are responsible for the generation of angiostatin, a potent endogenous inhibitor of angiogenesis, suggesting its contribution to the prevention of tumor growth and angiogenesis-dependent growth of metastases. Interestingly, pro-CD from human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that from prostate carcinoma cells. Since deglycosylated CD molecules from both carcinoma cells showed a low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in the carbohydrate structures of CD molecules between the two cell types and to contribute to their potency to prevent tumor growth and metastases.     

Cathepsin D from horse spleen. II. Study of certain enzymatic properties]

This work reports some enzymatic properties of highly purified horse spleen cathepsin D. Hydrolysis rate of several proteins are compared. The Kinetic constants (Km = 4.95 10(-5) M and Vm = 1,76 delta DO/mn/mug) have been determined in the presence of a denatured haemoglobin substrate. Stability of the enzymatic preparation is discussed according to the pH, concentration and time of storage. Some investigations concerning the active site are described. Enzymatic and chemical results show that dicarboxylic and tryptophanyl residues seem to be involved in the hydrolytic process. Catalysis does not depend on sulfhydryl or seryl residues. Different salts, particularly nitrate, nitrite and polyphosphate are potent inhibitors of enzymatic activity.     

Characterization of a maize root proteinase.

The major proteinase in maize (Zea mays) roots behaves as a serine endopeptidase. A possible physiological role of this enzyme could be in the turnover of nitrate reductase (NR) and, as such, it could be of great importance in regulating the assimilation of nitrate. The objective of this research was to elucidate the specificity and uniqueness of maize root proteinase. When bovine serum albumin and an NR purified from Chlorella vulgaris were used as substrates, the maize root proteinase exhibited a preference for cleavages such that the amino acid on the amino side of the scissile bond was alanine. This information was established by microsequence analysis of the N termini of proteolytic fragments, and carboxypeptidase Y analysis of the C termini of proteolytic fragments of substrates hydrolyzed by the proteinase. Cleavage occurred at the sequence Ala/Ala-Ala-Ala-Pro-Glu in Chlorella NR, and at the sequence Ala-Asp-Glu-Ser-His-Ala-Gln in bovine serum albumin. When bovine serum albumin was the substrate, the maize root proteinase yielded a peptide map that is unique relative to those created with the other serine endopeptidases elastase, trypsin, or chymotrypsin. Based on our data, the maize root proteinase appears to cleave peptide bonds at the carboxy side of alanine. Because of its specificity, it should have useful applications in protein chemistry.     

New Functional Aspects of Cathepsin D and Cathepsin E

Cathepsin D (CD) and cathepsin E are representative lysosomal and nonlysosomal aspartic proteinases, respectively, and play an important role in the degradation of proteins, the generation of bioactive proteins, antigen processing, etc. Recenty, several lines of evidence have suggested the involvement of these two enzymes in the execution of neuronal death pathways induced by aging, transient forebrain ischemia, and excessive stimulation of glutamate receptors with excitotoxins. CD has also been shown to mediate apoptosis induced by various stimuli and p53-dependent tumor suppression. To gain more insight into in vivo functions of CD, mice deficient in this enzyme were generated. The mutant animals showed a progressive atrophy of the intestinal mucosa, a massive destruction of lymphoid organs, and a profound accumulation of ceroid lipofuscin, and developed a phenotype resembling neuronal ceroid lipofucinosis, suggesting that CD is essential for proteolysis of proteins regulating cell growth and tissue homeostasis. It has also been shown that CD molecules secreted from human prostate carcinoma cells are responsible for the generation of angiostatin, a potent endogenous inhibitor of angiogenesis, suggesting its contribution to the prevention of tumor growth and angiogenesis-dependent growth of metastases. Interestingly, pro-CD from human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that from prostate carcinoma cells. Since deglycosylated CD molecules from both carcinoma cells showed a low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in the carbohydrate structures of CD molecules between the two cell types and to contribute to their potency to prevent tumor growth and metastases.