Specific Activity Of Cathepsin Research Articles

ACTIVITY OF CATHEPSIN H IN THE TUMOR AND CIRCUMFERENTIAL TISSUES IN WOMEN WITH INVASIVE LOBULAR FORM OF BREAST CANCER

Breast cancer takes the first place in the structure of oncologic morbidity of female population of the planet. Potential targets in elaboration of drugs are cysteine proteinases taking part in many processes, associated with cancer progression: Hyperproliferation of tumor cells, apoptosis, tumor-induced angiogenesis, invasion and matastasis.Cathepsin H cysteine proteinase possesses both proteinase and exopeptidase activity. High cathepsin H activity has been determined in the following tumors: glioblastoma, anaplastic astrocytoma, colorectal carcinoma, in head and neck tumors in blood plasma of patients with lung cancer, melanoma.In this connection the aim of the work consisted in analysis of cathepsin H activity in the tissue of invasive lobular form of breast cancer in order to elaborate additional biochemical test for presence of a neoplasm.Samples of mammary gland without neoplasms (circumferential tissue) and samples with invasive lobular form of breast cancer at I-II stages of cancer development were analysed. The patients were informed and gave the written agreement for the use of the anatomical material for biochemical research. In the supernatant the content of protein was determined by biuret method and cathepsin Н activity was determined by the modified Bradshaw method. Cathepsin activity was expressed in mmole of leucine, formed in the process of oxytocin hydrolysis, per a mg of protein per a min at 37 °С.It was found that in case of invasive lobular form of BC, the content of protein in tumour tissue at all stages of its development was 1.6-2.6 higher than the content of protein in the circumferential tissue. It was shown that cathepsin Н activity changed depending on the stage of invasive lobular BC development. The maximal relative and specific activity of cathepsin Н in tumour tissue was determined at I-II stage of tumor development. With tumor progression relative and specific activity of cathepsin Н in tumour tissue

Immunohistochemical localization of cathepsin D and a possible role of melanocytes during tail resorption in tadpoles of a tropical toad

Programmed cell death during anuran tail resorption is primarily brought about by apoptosis. Cathepsin D, a lysosomal aspartyl protease, is involved in the death of tail tissues. Thus, anuran tail resorption presents an ideal model to study cathepsin-mediated cell death during vertebrate development. Present study describes the trend of specific activity of cathepsin D in the tail of different developmental stages and immunohistochemical localization of cathepsin D in the tail tissues of the common Asian toad, Duttaphrynus melanostictus. Cathepsin D was involved in programmed cell death in epidermis, muscle, spinal cord, and blood cells in the resorbing tail. Interestingly, it was also involved in the pre-resorbing tail before visible tail resorption which indicates initiation of cell death even before actually the tail resorbs. Melanocytes were found to be one of the causative agents in degrading tail tissues and were associated with the degradation of muscle, epidermis and spinal cord of the resorbing tail.

Detection of cathepsin L in red cell membranes from fish blood

Cathepsin L, one of the cysteine proteases found in fish muscle, is considered to be the main cause of post-mortem autolysis of fish muscle. We have determined the presence of cathepsin L in the membranes of red blood cells of carp, amberjack, and red sea bream and measured its activity. Immunoblotting of an extract of the red blood cell membranes from these three fish species using human anti-cathepsin L antibody revealed the presence of cathepsin L of different molecular masses. The molecular masses of cathepsin L was estimated to be 120 and 85 kDa in the amberjack and 75 and 70 kDa in the carp. These proteins have higher molecular masses than the mature form of cathepsin L, suggesting that they are precursor forms. In contrast, the protein in the red sea bream was estimated to have a molecular mass of 30 kDa, suggesting that this cathepsin L is a mature form. The specific activity of cathepsin L was highest in the red blood cell membranes of the amberjack, followed by the carp and the red sea bream in descending order.

Reduced cystatin B activity correlates with enhanced cathepsin activity in progressive myoclonus epilepsy

BACKGROUND: Loss-of-function mutations in the gene encoding cystatin B (CSTB) underlie an inherited neurodegenerative disorder, progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1). CSTB is an inhibitor of several papain-family cysteine proteases, the lysosomal cathepsins. Its physiological function and the molecular pathways leading to the clinical EPM1 phenotype are unknown. AIM: To elucidate the role of CSTB and different cathepsins in pathogenesis of EPM1. METHOD: We determined the total papain inhibitory (cystatin) and papain-like (cathepsin) activity as well as specific activities of cathepsins B, H, L and S in lymphoblastoid cells of EPM1 patients, carriers and controls. RESULTS: In EPM1 patients, who express reduced levels of CSTB mRNA, the papain inhibitory activity was significantly decreased or absent. This reduction was correlated with significant increase in general cathepsin activity. The increase in cathepsin B, L and S activities was highly significant, whereas the increase in cathepsin H activity was not. CONCLUSIONS: This is the first demonstration of cysteine protease activity being regulated by CSTB activity in a biological context. The effects of decreased CSTB activity in EPM1 pathogenesis may, at least in part, be mediated by cathepsins through increased activity of cathepsins S and L.

Increased activity of cathepsin B in fibroblasts isolated from primary melanoma in comparison to fibroblasts from normal skin.

We determined activity of cathepsin B in early-passage fibroblasts isolated from primary melanoma and in fibroblasts from normal skin. Our results show an up to 5-fold increase in activity of cathepsin B in the tumor-derived fibroblasts in comparison to the fibroblasts from normal skin. We conclude that fibroblasts isolated from melanoma tissue are altered with regard to their specific activity of cathepsin B and preserve this elevated activity in early-passage cell culture. The data support the idea that stromal cells are not passive elements of the peritumoral environment but actively participate in the production of proteolytic enzymes.

RECOVERY OF PROTEINASE FROM PACIFIC WHITING SURIMI WASH WATER

A proteinase from Pacific whiting surimi wash water (SWW) was recovered by ohmic heating, ultrafiltration and freeze drying with overall yield of 0.83 g protein/L SWW and 78% recovery of activity. Ohmic heating conditions were optimized for the maximum recovery of the enzyme. Different applied voltages (50, 70, 90 V) showed no differences in efficiency for removing protein and retaining cathepsin L activity. Cathepsin L activity reached its maximum after ohmic heating to 55C whereas cathepsin B activity decreased constantly with increased temperature. A constant reduction in protein content was observed with the increase in temperatures from 45C to 60C and holding time up to 5 min. The highest retention of both total and specific activity of cathepsin L was obtained with the treatment at 55C for 3 min. Under these conditioner, 193% activity was recovered from SWW although a large amount of the activity was lost by the subsequent steps of ultrafiltration and freeze drying.

Characterization of the cathepsin-like cysteine proteinases of Schistosoma mansoni

Adult Schistosoma mansoni parasites synthesize and secrete both cathepsin L and cathepsin B cysteine proteinases. These cysteine proteinase activities, believed to be involved in hemoglobin digestion by adult schistosomes, were characterized by using specific fluorogenic peptide substrates and zymography. Both cathepsin L- and B-like activities with pH optima of 5.2 and 6.2, respectively, predominated in soluble extracts of worms, and both these activities were secreted by adult worms into the culture medium. The specific activity of cathepsin L was about double that of cathepsin B when each was assayed at its pH optimum, and moreover, the specific activities of cathepsins L and B in extracts of female schistosomes were 50 to 100% higher than in extracts of male schistosomes. Analysis of the primary structure of two cloned S. mansoni cathepsins L, here termed cathepsin L1 and cathepsin L2, revealed that they are only 44% similar and that cathepsin L2 showed more identity (52%) with human cathepsin L than with schistosome cathepsin L1. Moreover, differences in their active site, propeptide region, and potential for glycosylation suggest separate functions for schistosome cathepsin L1 and cathepsin L2.

Characterization and Cloning of the Cathepsin L Proteinases of Schistosoma japonicum

Adult Schistosoma japonicum parasites synthesize and secrete both cathepsin L and cathepsin B cysteine proteinases. The specific activities of cathepsin L were many-fold higher than that of cathepsin B. The cDNAs encoding two distinct cathepsin L proteinases, here termed cathepsin L1 and L2, were isolated. The deduced amino acid sequences of the mature cathepsin L1 and L2 were ∼ 41% identical, and moreover, S. japonicum cathepsin L2 showed more similarity with human cathepsin L than with schistosome cathepsin L1. Schistosome cathepsin L proteinases may be involved in the digestion of hemoglobin obtained from host erythrocytes. However, since we detected their presence in schistosome eggs, the release of these enzymes by eggs trapped in the liver and other organs may be associated with the granulomatous responses which characterize the pathology of human schistosomiasis.

Protein catabolism in fibroblasts cultured from patients with mucolipidosis II and other lysosomal disorders

Protein catabolism in fibroblasts cultured from the skin of normal individuals and of patients with mucolipidosis II (I-cell disease) and several other lysosomal storage diseases was examined by metabolic labelling with [3H]leucine and following the fate of radioactive proteins in pulse-chase experiments. In mucolipidosis II cells, overall protein degradative rates were found to be distinctly lower than in normal control cells. To distinguish lysosomal from non-lysosomal degradation, labelling experiments were carried out in the presence and absence of 10 mM NH4Cl, an inhibitor of lysosomal function. It was found that mucolipidosis II fibroblasts exhibited a markedly reduced rate of lysosomal protein degradation, whereas the rate of nonlysosomal degradation appeared normal. Serum and amino acid starvation led to a marked increase in lysosomal protein degradation in normal cells, but had only a minimal effect on that in mucolipidosis II fibroblasts. The specific activities of cathepsins B, H and L were profoundly diminished in all mucolipidosis II cell lines tested. Lysosomal protein degradation in a mucolipidosis III cell line was impaired to a similar degree as in mucolipidosis II cells, whereas it was decreased to a lesser extent in fibroblasts from patients with mucopolysaccharidoses I and VI, galactosialidosis and GM1-gangliosidosis. We conclude that fibroblasts from patients with mucolipidosis II and III have a severely compromised capacity for endogenous lysosomal protein degradation that appears to result from multiple cathepsin deficiency. This lysosomal defect is likely to have pathophysiological consequences.

Cathepsin D as a vitality marker in human skin wounds.

This paper shows the results obtained by studying the lysosomal enzyme Cathepsin D as a potential marker for the vitality of wounds in human specimens. We have analyzed 53 samples using enzymological and histological techniques. Our results show the ability of Cathepsin D to establish the vital origin of wounds inflicted 5 minutes or less before death, where the specific activity of cathepsin D reached 0.055 units at the wound edge and 0.01 units in their respective controls (P < 0.001). As previously demonstrated in an experimental series, Cathepsin D seems to be a very useful marker of high forensic interest in especially difficult cases. Further studies are in progress to check the influence of different factors such as drugs intake and clinical conditions on Cathepsin D activity.

Proteolytic enzyme activity in rat hindlimb muscles in fetus and during post-natal development.

The enzymatic activity of two lysosomal enzymes, acid phosphatase and cathepsin D, was determined in fetus and during post-natal development of the rat gastrocnemius muscle in comparison to the histological differentiation of this muscle. The specific activity of cathepsin D and acid phosphatase was 7 and 2.5 fold higher in the muscle during development until 20 days after birth, than that of mature muscle, respectively. A trend of gradual decrease in the activity of these enzymes was observed concomitantly with the differentiation and maturation of the muscle from mononucleated cells in the fetus to myotubes formation at day 1 after birth, followed by the formation of "young" and then striated myofibers in 10- and 20-day old neonates, respectively. However, no correlation could be found between the lysosomal enzyme activity and the developmental stages of the muscle until 20 days after birth. It is suggested that the elevated activity of lysosomal acid hydrolases may be associated with late developmental processes from young to mature myofibers in normal skeletal muscle and not only in various pathological conditions.

Postnatal changes of cathepsin D activity in rat liver and brain

Total and specific activity of cathepsin D (EC. 3.4.23.5) were measured in rat liver and brain from 1 to 98 days of age. The activity of cathepsin D in the liver of adult and newborn rats was the same while in the rat brain it was higher in adult than in newborn rats. In the liver maximum specific activity of cathepsin D occurred on the 10th postnatal day and minimum on the fourth day of age. In the brain maximum specific activity of the enzyme occurred on the 14th postnatal day. Total activity of cathepsin D increased after birth in rat liver and brain. These results are discussed in relation to the functional role of cathepsin D in the rat liver and the brain.

Role of cathepsin B and L in anti-glomerular basement membrane nephritis in rats.

We have examined the potential role of the cysteine proteinases, cathepsin B and L, in renal tubular protein degradation and increased permeability of the glomerular basement membrane (GBM) which occurs in a neutrophil- and complement-independent model of anti-GBM antibody disease. The specific activity of cathepsin L, but not cathepsin B, was significantly increased (157%, p greater than 0.01) in cortical homogenates (85-90% tubules) prepared from anti-GBM-treated rats compared to saline-treated controls. Using highly purified cathepsin B and L, we documented the ability of these proteinases to degrade albumin in vitro (Km 5.92 and 0.22 microM for B and L, respectively). In two separate studies, treatment of rats with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, (E-64), a specific and irreversible inhibitor of cysteine proteinases, significantly reduced proteinuria (-45 and -41%, p less than 0.01) in the 24-hour period following injection of the anti-GBM IgG. Taken together, these data suggest an important role for cysteine proteinases in the increased tubular protein degradation which occurs in response to increased filtered protein loads and in the increased GBM permeability (proteinuria) characteristic of glomerular disease.

Effects of botulinum toxin induced muscle paralysis on endocytosis and lysosomal enzyme activities in mouse skeletal muscle.

The effects of botulinum toxin (type A) induced muscle paralysis on endocytosis and lysosomal enzyme activities in skeletal muscle were compared with the effects of surgical denervation. Muscle atrophy, measured as decrease in total muscle protein content, was as large or larger after botulinum toxin treatment as after denervation. Endocytic activity, measured as the in vitro uptake of horseradish peroxidase, and the specific activities of the lysosomal enzymes N-acetyl-beta-D-glucosaminidase and cathepsin D were all increased six days after denervation. Only the specific activity of cathepsin D was increased six days after botulinum toxin poisoning. The uptake of horseradish peroxidase and the specific activity of N-acetyl-beta-D-glucosaminidase were also increased eleven days after poisoning. Transverse sections of eleven days botulinum poisoned muscles from animals injected with horseradish peroxidase showed fibres with dense peroxidase staining similar to those seen in denervated muscle although they seemed to occur less frequently. The results show that increases in endocytic activity and lysosomal enzyme activities may occur in skeletal muscle without the presence of degenerating axons. The differences in effects of surgical denervation and botulinum toxin induced paralysis are discussed in terms of what is known about the mechanism of action of botulinum toxin and the possible functional roles of the two lysosomal enzymes studied.

Elastinolytic activity of human cathepsin L.

The hydrolysis of a tritiated elastin substrate by the human cysteine proteinases cathepsins B and L has been studied. Cathepsin L was found to be at least 100-fold more active on this substrate than cathepsin B. The specific activity of cathepsin L at pH 5.5 for hydrolysis of elastin was about the same as that of pig pancreatic elastase at its optimum pH of 8.8.

Intracellular distribution and properties of cathepsin B in the rat retina and its inhibition by a cytosolic fraction

The intracellular distribution and properties of cathepsin b in the rat retina are similar to those of cathepsin b in other mammalian tissues. The specific activity of cathepsin b was highest in the mitochondrial-lysosomal fraction [1650±450 pmole mg −1 protein hr −1 (mean± s.d.)]. Inhibition of cathepsin b activity by a cytosol fraction of retina is also described.

Endogenous cathepsin B inhibitor activity in normal and myopathic red and white skeletal muscle.

Despite extensive biochemical and morphological studied on the degenerative muscle diseases, the primary chemical lesions are still obscure, both in humans and animals. In this report we examine the activities of the lysosomal endoproteinase cathepsin B and its endogenous inhibitor(s) in the red and white skeletal muscles of guinea pigs with nutritional muscular myopathy induced by vitamin E deficiency. We observed a twofold increase (P less than 0.005) in the activity of cathepsin B in the white skeletal muscles of the vitamin E-deficient (E-) animals over that of the normal (N) and control (E+) groups. Assessment of the activity of endogenous cathepsin B inhibitor revealed a one and a half times greater amount of inhibitor in N when compared to E-; this difference in inhibitor activity applied to both red (masseter) and white (medial head, gastrocnemius) muscle. When the specific activity of cathepsin B in the E-tissue was corrected for inhibitor activity, the corrected value was not significantly different from either the E+ or the N tissue.

Inhibition of basal protein degradation in rat embryo fibroblasts by cycloheximide: correlation with activities of lysosomal proteases.

Rat embryo fibroblasts were grown in medium containing 14C-leucine and 3H-thymidine. After a 24-hour chase in nonlabeled medium, cultures were placed in either fresh growth medium or medium containing 10-20 microgram/ml cycloheximide. Cell monolayers were processed at daily intervals for three days. Four hours prior to processing, cultures were placed in fresh medium and the accumulation rate of trichloroacetic acid soluble 14C in the media assayed. Cycloheximide effects a progressive decrease in the fractional degradation rate of the labeled cell protein, primarily during the first 24 hours. The specific activities of cathepsin D, cathepsin B, and neutral protease correlate closely with the fractional degradation rate. Other lysosomal hydrolases show little change during this period. The activities of the lysosomal proteases approach a new steady state which is correlated with the new steady state level of protein synthesis. A model is proposed which relates the rate of protein breakdown in the cell to the level of protein synthesis. The data also suggests the possibility that subpopulations of high turnover and low turnover cells exist in these cultures.

Control of cell protein degradation: Changes in activities of lysosomal proteases

In cycloheximide-treated fibroblasts, the rate of cell proteolysis and the specific activity of cathepsin D show a rapid, concurrent, and proportional decrease over a 48 h period. Cathepsin B 1 also decreases, but other lysosomal enzymes show little or no change in specific activity. These findings are consistent with the hypothesis that the rate of proteolysis in the cell is in part controlled by the specific activities of lysosomal proteases.

Clinical, enzymatic and histological study of synovium in coxarthrosis

A comparison was made between the specific activity of cathepsin D and a histologic score for inflammation on synovial tissue obtained from 36 patients undergoing total hip replacement and an excellent correlation was found between these parameters (correlation coefficient 0.82). In addition it was found that 5 patients with the clinical diagnosis of rheumatoid arthritis had high cathepsin-specific activity (0.92±0.07) and high histologic scores (27.0±7.0). Patients with other diagnoses (failed prosthesis, idiopathic avascular necrosis, former resected hip) had low enzyme levels (0.44±0.23) as well as low histologic scores for inflammation (11.4±5.5). In cases with degenerative hip disease two groups could be definitively separated. Group 1 (20%) was characterized by high enzyme levels (0.89±0.19) and high histological scores (20.6±5.4). Group II (80%) was characterized by low enzyme levels (0.31±0.15) and low histologic scores (8.6±4.7) (Table 1).