Acid proteinase activity in fish II. Purification and characterization of cathepsins B and D from Mujil auratus muscle

Two cathepsins were detected in Mujil auratus muscle extracts. They were classified as a thiol- and aspartyl-proteinase (cathepsins B and D, respectively) on the basis of their catalytic behaviour in presence of specific inhibitors. Following extraction in 1% KCl, the proteinases were purified by autolysis, acetone fractionation, affinity chromatography, and gel permeation chromatography. The haemoglobin-agarose column chromatography allowed us to separate the two activities. Sephadex G-75 column chromatography resulted in apparent molecular weights of 25,000 (cathepsin B) and 35,000 (cathepsin D). The molecular size, together with pH-activity profiles and kinetic parameters are similar to those reported for mammalian cathepsins B and D. This was not the case with the temperature-activity profiles, the optimum temperature as well as the heat stability being higher for fish cathepsins than for those obtained from other sources. Cathepsin B was characterized by its ability to inactivate aldolase. Fluorescence quenching experiments showed that tryptophyl residues of cathepsin B were less occluded and located in a more electronegative microenvironment that those pertaining to cathepsin D.     

Degradation of myofibrillar proteins by cathepsins B and D

1. The procedure of Barrett [(1973) Biochem. J.131, 809-822] for isolating cathepsins B and D from human liver was modified for use with rat liver and skeletal muscle. The purified enzymes appeared to be similar to those reported in other species. 2. Sephadex G-75 chromatography of concentrated muscle extract resolved two peaks of cathepsin B inhibitory activity, corresponding to molecular weights of 12500 and 62000. 3. The degradation of purified myofibrillar proteins by cathepsins B and D was clearly demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After incubation with enzyme, the polypeptide bands representing the substrates decreased in intensity and lower molecular weight products appeared. 4. Cathepsins B and D, purified from either rat liver or skeletal muscle, were shown to degrade myosin, purified from either rabbit or rat muscle. Soluble denatured myosin was degraded more extensively than insoluble native myosin. Degradation by cathepsin B was inhibited by lack of reducing agent, or by myoglobin, iodoacetic acid and leupeptin, but not by pepstatin. The same potential modifiers were applied to cathepsin D, and only pepstatin produced inhibition. 5. Rat liver cathepsin B had a pH optimum of 5.2 on native rabbit myosin. The pH optimum of cathepsin D was 4.0, with a shoulder of activity about 1pH unit above the optimum. 6. Rat liver cathepsins B and D were demonstrated to degrade rabbit F-actin at pH5.0, and were inhibited by leupeptin and pepstain, respectively. 7. The degradation of myosin and actin by cathepsin D was more extensive than that by cathepsin B.     

三个三倍体鲫鱼品系及野鲫mtDNA的比较研究

用16种限制性内切酶研究了银鲫(3N=156～162)、彭泽鲫(3N=162)和缩骨鲫(3N=150)3个三倍体鲫鱼品系及野鲫(2N=100)的线粒体DNA。有6种酶在种系间和种系内产生限制性片段长度多态性(RFLPs),银鲫共存在4种单倍型,彭泽鲫2种,野鲫3种,缩骨鲫1种。彭泽鲫和银鲫拥有相同的常见单倍型,缩骨鲫的单倍型属于野鲫的常见型。根据限制性位点的变异数据,计算了单倍型间的相似性、核苷酸多样性、品系内核苷酸多样性和品系间的遗传距离,确定彭泽鲫属于银鲫的一个地方品系,缩骨鲫属于野鲫的一个地方品系。根据核苷酸的差异,推算出银鲫和野鲫两个亚种的分化大约在11万年前完成。     

Expression of cathepsins B, D and L in mouse corneas infected with Pseudomonas aeruginosa.

C57BL/6J na?ve and immunized mice were intracorneally infected with Pseudomonas aeruginosa. Semi-quantitative RT-PCR was performed to detect cathepsin gene expression and the results were further confirmed by immunoblot analysis. The enzymatic activities of cathepsins B, D and L were measured by peptidase assays. Immunohistochemical staining was carried out to localize the expression of the cathepsins. Cathepsins B, D and L were detected in the normal cornea by RT-PCR. A peptidase assay revealed activities of all three cathepsins under normal physiological conditions. In na?ve mice, enzymatic activities of cathepsins B, D and L were all significantly enhanced when the corneas were infected with P. aeruginosa and the peak of the induction appeared around day 6 postinfection. Immunoblot analysis showed increased expression of cathepsins B, D and L. The infected corneal samples from immunized mice exhibited much lower induction of enzymatic activities compared to those from na?ve mice. Immunohistochemistry showed that the expression of cathepsins in the normal cornea was restricted to the epithelial tissue while the induced expression of cathepsins was predominantly in the substantia propria. Our data revealed up-regulated enzymatic activities of cathepsins B, D and L in the na?ve corneas infected with P. aeruginosa, which correlated well with the inflammatory response. Immunization of mice against P. aeruginosa attenuated the inducing effect on cathepsin expression caused by infection. The time sequence for induction of cathepsin proteins and enzymatic activities suggests a mechanism of host proteolytic degradation of the extracellular matrix resulting in corneal destruction after P. aeruginosa infection.     

Immunohistochemical localization of cathepsins B,D and L in the rat osteoclast

Immunohistochemical localization of cathepsins B, D and L in the osteoclasts of rat alveolar and femoral bones was investigated by using the avidin-biotin-peroxidase complex method for semithin, 1-m-thick cryosections. Extracellular immunoreactivity for cathepsins B and L was clearly demonstrated along the bone resorption lacunae; the intensity of the extracellular immunoreactivity of cathepsin L was stronger than that of cathepsin B. However, the intracellular immunoreactivity of both cathepsins was weak compared with that of cathepsin D. The intracellular immunoreactivity of cathespin D in the osteoclasts was clearly observed in the granules and/or vacuoles, but extracellular cathepsin D immunoreactivity was either negligible or not detected along the resorption lacunae. In the adjacent sections stained with anti-cathepsin L or D, extensive extracellular deposition of cathepsin L was found along the bone resorption lacunae, with or without osteoclasts, although the intracellular reactivity of cathepsin L was weak. This is the first morphological study in which cathepsins B and L have been demonstrated to be produced in the osteoclasts and extensively secreted into resorption lacunae, and in which cathepsin D was found to be present in the cells but scantily secreted into the lacunae. These findings suggest that cathepsins B and L directly and effectively participate in the degradation of the bone matrix.     

The selective role of cathepsins B and D in the lysosomal degradation of endogenous and exogenous proteins.

A selective inhibitor of cathepsin B, a derivative of E-64 (compound CA-074), and pepstatin-asialofetuin, a potent inhibitor of cathepsin D, were used for an in vivo study of the selective role of these proteinases in lysosomal proteolysis. Administration of compound CA-074 or pepstatinasialofetuin to rats caused only a slight shift of the lysosomal density and no increase in sequestered enzymes in the autolysosomal fraction, although cathepsin B or D activity in the liver was markedly inhibited. These treatments also had little effect on the inhibition of the degradation of endocytosed FITC-labeled asialofetuin. In contrast, leupeptin treatment caused marked inhibition of lysosomal degradation of endogenous and exogenous proteins. These results suggest a small contribution of cathepsins B and D to the initiation of lysosomal proteolysis.     

Cathepsin D from pig myometrium. Characterization of the proteinase.

The purification of cathepsin D from pig uterus by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose was described previously [Afting & Becker (1981) Biochem. J. 197, 519-522]. In this paper, chemical and physical properties of the proteinase are presented. The purified enzyme showed three bands on SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis, one main band corresponding to an Mr of 31 000 and two minor bands with Mr values of 43 000 and 15 000 respectively. Gel filtration on Bio-gel P-150 and sedimentation-diffusion equilibrium studies give an Mr for the main band of about 35 000. The pI of the enzyme was determined to be 7.2. Haemoglobin was the best substrate, with a Km value of 6.4 X 10(-6)M. It was hydrolysed with a pH optimum between 3.0 and 3.3 for a substrate concentration of 100 microM. The proteinase was stable over the pH range of 3.5-6.5. At pH 6 the enzyme showed stability up to a temperature of 50 degrees C; at pH 3 the activity was already decreased below 40 degrees C. Carbohydrate studies resulted in the staining of all three bands on an SDS/polyacrylamide gel by thymol/H2SO4. After treatment with endo-beta-N-acetylglucosaminidase H, all three bands were shifted to a region of lower Mr. Of various inhibitors tested, only pepstatin was strongly inhibiting, with a Ki of 2.1 X 10(-9)M.     

A comparison of four cathepsins (B, L, N and S) with collagenolytic activity from rabbit spleen.

We have separated four cathepsins (B, L, N and S) from rabbit spleen. They are all collagen-degrading cysteine proteinases, with Mr values of 25,250, 23,500, 34,000 and 30,000 for cathepsin B, L, N and S respectively. Cathepsins B, N and S have isoelectric points of 5.4, 6.2 and 6.8 respectively, whereas cathepsin L exhibited multiple charge forms in the range 5.0-5.7. A comparison of their specific activity against a variety of protein and synthetic substrates shows many differences. These differences can be visually illustrated through isoelectric focusing and detection of enzymic activity with protein and synthetic-substrate overlays. By using an enzyme-linked immunosorbent assay based on the binding to chicken cystatin and detection with polyclonal and monoclonal antibodies to native cathepsins B and L, no cross-reactivity of the four native enzymes was observed. Studies on the co-operative or synergistic effect in degrading collagen indicated that, of the different combinations tested, only the combination of cathepsin B and N exhibited enhanced collagenolysis.     

In utero and in vitro proteinase activity during the Mesocricetus auratus embryo zona escape time window

The goal of the present study was to investigate proteinase activity in uterine flushates collected during the zona loss time window (68-80 h post-egg activation) in both pregnant and pseudopregnant hamsters and in culture medium conditioned by hatching blastocysts. Several prominent enzyme activities appeared in all pregnant and pseudopregnant uterine flushates. However, only a 45, 43 x 10(-3) M:(r) doublet coincided with the zona loss time window; these bands were absent outside of this time window and were not found in conditioned medium. In medium conditioned by hatching blastocysts, enzyme activity was represented by a 70, 65 x 10(-3) M:(r) doublet identical to a doublet seen in all uterine flushates collected and in serum. There were 12 pregnant and 8 pseudopregnant uterine flushates that were capable of zona lytic activity in vitro (positive bioassays). Of these positive bioassays, five pregnant and four pseudopregnant uterine flushates exhibited the 45, 43 x 10(-3) M:(r) doublet (correlative positive bioassays). These data suggest that there is an important uterine contribution to blastocyst escape from the zona pellucida, consisting of proteinases secreted during a finite time window prior to blastocyst attachment that are different from the proteinases responsible for the zona lytic activity in vitro.     

Lysosomal cathepsins B, L, and D in the development of murine experimental leukemias

Lysosomal proteases are actively involved into pathogenesis of malignant tumors. Impairments in the interaction between proteases and their inhibitors are implicated in the processes of tumor invasion and metastasis. Among proteases associated with malignant growth, cysteine cathepsins B and L and aspartic cathepsin D are considered to play the major role in the tumor development. The present study was designed to investigate the activity of cathepsins B, L, and D during the development and treatment of murine experimental leukemias and to determine correlation between these proteases and course of pathological process as well as efficiency of the chemotherapeutic treatment. P-388 leukemia was characterized by a more aggressive development and unfavorable prognosis than L1210/1 leukemia. In mice with P-388 leukemia the activity of lysosomal cathepsins B, D, and L in the tumor tissue, liver and spleen, as well as the activity of cathepsins B and L in serum were lower than activities of these enzymes in mice with L1210/1 leukemia. Changes in the activity of cathepsins in liver and spleen of leukemic mice reflected a level of aggressiveness of the tumor development and invasion of these organs with tumor cells. Treatment of these experimental leukemias resulted in the increase of cathepsin B, L and D activity in the tumor tissue, liver, spleen and the increase in cathepsin B and L activity in serum. The highest protease activity was detected in the groups of mice characterized by the highest inhibition of the tumor growth. These data demonstrate that lysosomal proteases are involved in the progression of murine experimental leukemias and elimination of tumor cells in the result of treatment. Thus, determination of the activity of cysteine and aspartic proteases can be used for evaluation of cancer malignancy, tumor sensitivity for chemotherapy and efficiency of treatment.     

Enzyme-substrate interactions in the hydrolysis of peptides by cathepsins B and H from rat liver.

Free Ca2+ in the cytosol ([Ca2+]i) of individual rat ventricle cells injected with aequorin was measured under anoxia. In glucose-free medium myocytes spontaneously shortened after about 60 min, although [Ca2+]i was still at or near resting levels. However, within minutes a net inward movement of Ca2+ across the sarcolemma developed and [Ca2+]i began to rise. Provided oxygen was readmitted before [Ca2+]i exceeded 2-3 microM, cells were able to restore [Ca2+]i to resting levels through caffeine-sensitive sequestration of Ca2+ in the sarcoplasmic reticulum. We suggest that Ca2+-independent shortening of anoxic cardiomyocytes reflects onset of rigor which triggers loss of [Ca2+]i homoeostasis.     

The roles of cathepsins B1 and D in the digestion of cytoplasmic protiens in vitro by lysosomal extracts.

By means of the inhibitors leupeptin and pepstatin it is shown that both thiol proteinases (such as cathepsin Bl) and the acid proteinase cathepsin D play substantial roles in the degradation in vitro of cytoplasmic proteins by mixtures of lysosomal enzymes.     

The purification and properties of yeast proteinase B from Candida albicans.

A serine proteinase (ycaB) from the yeast Candida albicans A.T.C.C. 10261 was purified to near homogeneity. The enzyme was almost indistinguishable from yeast proteinase B (EC 3.4.21.48), and an Mr of 30,000 for the proteinase was determined by SDS/polyacrylamide-gel electrophoresis. The initial site of hydrolysis of the oxidized B-chain of insulin, by the purified proteinase, was the Leu-Tyr peptide bond. The preferential degradation at this site, analysed further with N-blocked amino acid ester and amide substrates, demonstrated that the specificity of the proteinase is determined by an extended substrate-binding site, consisting of at least three subsites (S1, S2 and S'1). The best p-nitrophenyl ester substrates were benzyloxycarbonyl-Tyr p-nitrophenyl ester (kcat./Km 3,536,000 M-1 X S-1), benzyloxycarbonyl-Leu p-nitrophenyl ester (kcat./Km 2,250,000 M-1 X S-1) and benzyloxycarbonyl-Phe p-nitrophenyl ester (kcat./Km 1,000,000 M-1 X S-1) consistent with a preference for aliphatic or aromatic amino acids at subsite S1. The specificity for benzyloxycarbonyl-Tyr p-nitrophenyl ester probably reflects the binding of the p-nitrophenyl group in subsite S'1. The presence of S2 was demonstrated by comparison of the proteolytic coefficients (kcat./Km) for benzyloxycarbonyl-Ala p-nitrophenyl ester (825,000 M-1 X S-1) and t-butyloxycarbonyl-Ala p-nitrophenyl ester (333,000 M-1 X S-1). Cell-free extracts contain a heat-stable inhibitor of the proteinase.     

Thyroglobulin processing by thyroidal proteases. Major sites of cleavage by cathepsins B, D, and L

The normal provision of thyroid hormones to the body requires their release from the prohormone, thyroglobulin (Tg). Previous work established the importance of cathepsins B, D, and L (formerly designated cysteine proteinase I) to this process but had not defined the points of proteolytic attack for each enzyme. In the present study we labeled rabbit Tg in vivo with sodium 125I and performed limited digestions with cathepsins B, D, and L, purified from human thyroids. The resultant peptide fragments were analyzed by amino-terminal sequencing and located within the Tg molecule by comparison with the cDNA-derived sequences from human Tg. We identified three cleavage points for cathepsin B, corresponding to P'1 residues 532, 795, and 2487; four cleavage points for cathepsin L, corresponding to P'1 residues 2389, 2452, 2490, and 2657; and four cleavage points for cathepsin D, corresponding to P'1 residues 551, 1835, 2468, and 2643. None of the cleavage points was near Tgs known hormonogenic sites, but these peptide fragments contained three of the four major hormonogenic sites in rabbit Tg, suggesting some preference for their early proteolytic processing. Cathespin B alone among the three endopeptidases had some exopeptidase activity toward Tg. The cleavage specificities for each of the endopeptidases resembled those described with other protein substrates. Thus, cathepsin D preferentially cleaved bonds between hydrophobic residues, and cathespin L cleaved bonds with hydrophobic residues at P2 and P3. Although cathepsin Bs specificity was less obvious, it produced a major cleavage between 2 leucine residues. The existence of three endopeptidases cleaving at different sites shows that Tg proteolysis is a complex process, suggests synergism among their enzyme activities, and provides a physiological mechanism for selective hormone release, including its regulation by TSH.     

Comparative study on proteinase R, T, and K from Tritirachiam album limber

Proteinase R and T purified from Tritirachiam album limber were characterized in comparison with proteinase K using circular dichroism (CD), enzyme activity, thermal melting, and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). CD analysis suggested that these three proteins possess some beta-sheet structure, with little alpha-helix except for proteinase R which showed about 14% alpha-helix. SDS-PAGE and gel filtration in 0.1% SDS indicated that proteinase T and K are resistant to SDS-induced unfolding similar to subtilisin. Thermal denaturation experiments showed the melting temperature for proteinase T to be 67 degrees and that for proteinase K to be 65 degrees in the absence of Ca2+, with higher melting temperatures in the presence of Ca2+. However, the enzyme activities of proteinase T and R were significantly lower than those of proteinase K.     

Comparative biochemistry of acid proteinase from animal origins

Acid proteinases from 17 tissues of 12 animal species were compared with respect to molecular weight, inhibition by pepstatin and activation by tripolyphosphate. Gel filtration of acid proteinases from protochordates and vertebrates showed a common elution profile and three peaks with mol. wts of -20,000, -45,000 and above 150,000 were detected with acid-denatured hemoglobin as substrate at pH 3.6. The main component of vertebrate acid proteinases was identified as cathepsin D. In the invertebrate acid proteinases, the elution profiles through gel filtration were characteristic to the tissues examined, and were not so distinct as those of vertebrates. Through a biochemical survey, the animal acid proteinase was discussed from a comparative point of view.