Catheptic Activity Research Articles

A simple purification procedure of buffalo lung cathepsin H, its properties and influence of buffer constituents on the enzyme activity

BackgroundCathepsin H (E.C.3.4.22.16) belongs to a family of lysosomal cysteine protease which regulates diverse normal biological processes mainly in intracellular proteolysis. MethodsPurification of cathepsin H from an unstudied system i.e. buffalo lung has been achieved by a simple process developed after incorporating appropriate alteration in the available methods for isolation of the enzyme from other sources. The use of DEAE-Cellulose and SP-Sephadex C-50 helped in better and simultaneous separation of cathepsin B and H up to homogeneity. ResultsThe SDS-PAGE result showed buffalo cathepsin H to be a single-chain molecule having MW, NH2- and COOH- terminal residues of 25.4 kDa, Lys and Val respectively. The enzyme was a glycoprotein with pI of 6.2; it hydrolyzed Leu-NA (Vmax/Km = 301.6) as the most efficient substrate followed by Arg-NA, Arg-Arg-NA and BANA. Buffalo enzyme showed maximum activity at 36 °C, pH 6.75 and at a buffer concentration of 2 × 10−3 M. ConclusionCatheptic activity was found to be quite stable at least for 20–30 min between pH 4.5–7.0, buffer concentration of 1 × 10−2 to 4 × 10−2 M and the temperature resistance up to 36 °C. The effects of various substances present in the buffers routinely used for the assay of catheptic activity revealed that the activity of buffalo lung cathepsin H depends not only qualitatively but also quantitatively on the constituents of assay buffer. General significanceThis study seems to provide valuable information regarding the biochemistry of cathepsin H in general as well as influence of buffer constituents on enzyme activity and physiological role in particular.

Changes in protein fractions of rainbow trout ( Oncorhynchus mykiss) gravads during production and storage

Gravad fish products belong to a group of low-processed products obtained from fresh fish by sprinkling fillets with a mixture of sugar and salt and then placing them in cold storage. Little is known about changes in the tissue during the production and storage of gravads. The purpose of the present study was to investigate the type and scope of changes in rainbow trout ( Oncorhynchus mykiss) gravad proteins during processing and vacuum storage at 3 °C and −30 °C. The results showed that the solubility of protein was barely affected by the gravading process, but did change during the cold storage and freezing of the gravad. Catheptic activity decreased with gravading and further storage. A SDS-PAGE analysis revealed that the intensity of the M-protein and troponin bands was reduced, while the bands of α-tropomyosin and the myosin heavy chain increased when the trout muscle was subjected to the gravading process. Two bands with molecular weights of 255 and 135 kDa disappeared in the gravad, and new bands with molecular weights of 163 and 117 kDa appeared. An HPLC analysis revealed that gravading had a limited effect on the relative areas of peaks corresponding to low molecular weight substances of protein origin, while more significant changes were observed during gravad storage. Moreover, the gravading process caused a lowering of the actin and myosin denaturation temperature, which was proved using the DSC method. In conclusion, changes in rainbow trout muscle protein appeared to have a crucial effect on product quality.

Changes in lysosomal enzyme activities and shear values of high pressure treated meat during ageing

The effects of pressure treatment (520 MPa for 260 s at 10°C) on lysosomal enzyme activities, Warner–Bratzler (WB) shear values and physico-chemical parameters of post-rigor (day 2) beef ( Biceps femoris and Longissimus dorsi muscles) were investigated immediately after treatment and during ageing. Activities of cathepsin D and acid phosphatase in pressurised meat samples were higher than in controls at 2 days post-mortem and throughout storage. This increase in activities was related to the breakdown of the lysosomal membrane and/or to enzyme activation. The WB peak forces were higher for pressurised samples of both muscles at 2 days than in the control and remained higher throughout ageing. The WB shear force deformation curves from pressurised samples showed that the increase in toughness was due to the increase in shear force of the myofibrillar component and not the collagen component. Pressurised muscle led to a significant decrease in sarcomere length and higher cooking loss. Post-rigor application of high pressure induced higher catheptic activity but no conclusive effect on the post-mortem rate of tenderization or tenderness of meat was observed.

Characterization of the proteases involved in gel weakening of beef heart surimi.

The goal of this study was to elucidate the nature and characteristics of the proteases involved in gel weakening of beef heart surimi. Acidic (E1) and neutral (E2) protease extracts were prepared from the surimi. The major active components in E1 were found to be cathepsins B and L. E1 exhibited optimum activity to hydrolyze substrates specific to cathepsins B and B+L at 50 degrees C and pH 5.5. At pH 6.0, it retained approximately 50% of its maximum activity. The catheptic activity of E1 was inhibited almost completely by E-64 and leupeptin. The active component in E2 was unidentified and was not inhibited by cysteine or serine protease inhibitors. However, beef plasma powder effectively inhibited the hydrolysis of FITC-casein and myosin heavy chain by E2.

Altered Major Histocompatibility Complex Proteins and Peptides for the Induction of Tolerance After Organ Transplantation

Transplantation tolerance is defined as permanent acceptance of an allograft without the need for nonspecific immunosuppressants, which predispose patients to infectious and neoplastic complications. Our approach uses chemically modified antigenic proteins to modify the first signal that triggers allorecognition. The first signal, which is generated by the trimolecular interaction between the T cell receptor, the antigen-presenting major histocompatibility complex (MHC) protein and the antigenic peptide, is distinguished from a second signal that results from the co-stimulatory interactions between T lymphocytes and antigen-presenting cells and the third signal that results from the stimulatory effects of cytokines. Previous studies in animal models have utilised pretransplant inoculation of the recipient with various types of donor-type cells, such as erythrocyte, bone marrow, transfectant or transgenic cells, or extracted transplantation antigens, which have been prepared by sonication, autolysis/proteolysis, detergent treatment or salt (3 mol/L KCl) extraction, which appears to be the most efficient method. Antigens extracted from natural cells induce tolerance in animal models when administered in pretreatment regimens via the intrathymic route (in conjunction with T cell depletion) or via the intravenous route (after preconditioning by total lymphoid irradiation). Exposure to synthetic allopeptides representing sequences from the hypervariable or the constant regions of either class I or class II MHC molecules produces variable effects on in vivo and in vitro alloimmune reactions. In addition to the interactions between peptides and MHC proteins and/or T cell receptor sites, at least some peptides act by binding to receptors of the heat shock protein family, thereby increasing intracellular calcium concentrations without inducing co-stimulatory signals, and/or by interacting directly with unique surface receptors on natural killer cells. However, several factors intrinsic to peptides may limit their use for tolerance induction in vivo. First, peptides are rapidly cleared by non-immunological mechanisms from the circulation, requiring the use of large quantities. Second, peptides are unable to contact the host system in a fashion that reflects the immunogenicity/tolerogenicity of the epitope on the native molecules. Third, peptides are unable to be processed by antigen-presenting cells. Our approach to tolerance induction uses allochimaeric MHC proteins, which are constructed by engrafting selected donor-type tolerogenic epitopes onto host-type MHC molecular backbones. When delivered in rat models via the intrathymic, intraportal or oral gavage route, allochimaeric class I MHC molecules induce immunodominant responses, namely they overwhelm all other responses toward foreign epitopes. Although most peritransplant antigen treatment regimens require concomitant administration of subtherapeutic doses of nonspecific immunosuppressants, some constructs induce tolerance without adjunctive immunosuppressive therapy. One critical requirement for tolerance induction by allochimaeric MHC antigens is the presence of host rather than third-party flanking amino acid sequences. The allochimaeric sequences seem to steer the host response toward tolerance by directly binding to immune cell receptors that deliver a 'self' signal (T cell receptor hypothesis), by directing endosomal catheptic activity to yield tolerogenic rather than immunogenic peptides (peptide hypothesis), or by altering the interactions in the tri-molecular complex, thereby interfering with the usual participation of the MHC molecule on the antigen-presenting cell (supertolerogen hypothesis).

Seasonal changes and preliminary characterization of cathepsin D‐like activity in sardine (Sardina pilchardus) muscle

The seasonal changes of cathepsin D‐like activity in white and dark muscle of sardine were followed and maximum activity was found in April and October, which could be related to the spawning period of this species. Higher catheptic activity was measured in dark muscle and females. The crude extract of this muscle enzyme had an optimum pH and temperature of 3.2 and 55 °C, respectively, and the activity was strongly inhibited by low levels of NaCl (6%). The thermal stability was lower at pH 3.2 than at pH 6.5 and the enzyme was stable in the pH range 3–4.5. The activity of this protease was detected during the ripening process of salted sardine which suggests its participation in this process.

Immunohistochemical localization of cathepsin B and L in the white muscle of chum salmon (Oncorhynchus keta) in spawning migration: probable participation of phagocytes rich in cathepsins in extensive muscle softening of the mature salmon

The study was undertaken to elucidate the cause of the enhancement of catheptic activity in the white muscle of chum salmon during spawing migration The study may provide a new concept that makes clear the mechanisms of protein turnover taking place in the muscle of the salmon and the extensive muscle softening, i.e., the muscle tissue degradation occurring after the death of the fish

アユの成熟に伴う筋肉および肝臓のカテプシン活性の上昇

アユの成熟に伴う筋肉および肝臓のカテプシン活性の上昇

Purification and some properties of buffalo liver cathepsin B.

Buffalo liver cathepsin B was isolated by acid extraction, ammonium sulfate fractionation, Sephadex gel filtration, DEAE-Sephadex chromatography and Sephacryl S-300 chromatography. The enzyme preparation was found to be homogeneous by gel filtration and SDS-polyacrylamide gel electrophoresis but could be resolved into two major and four minor protein bands on polyacrylamide gel electrophoresis in the absence of SDS. The enzyme showed catheptic activity against synthetic substrates such as BANA and BAPNA as well as against denatured hemoglobin. Various physico-chemical and enzymatic properties of the enzyme, such as molecular weight, Stokes radius, frictional coefficient, pH optimum, Michaelis constant, and Vmax, were determined. The values of these parameters were 27,500, 2.41 nm, 1.2, 6.5, 2.08 mM, and 42.4 units/mg, respectively. The hydrodynamic properties suggest a compact and globular conformation for this enzyme. Various compounds were tested for their influence on the activity of cathepsin B. Of these compounds, membrane phospholipids were found to increase significantly the activity of this enzyme. This increase in activity could be of physiological importance since the concentration of phospholipids is increased after endocytosis and autophagy.

Metabolic activity of lysosomes in tin-intoxicated regenerating rat liver

The metabolic activity of lysosomes was monitored by assaying the lysosomal enzymes. DNase, RNase, acid phosphatase and cathepsin in rat liver regenerating after partial hepatectomy at various intervals after the operation. Results of the present study demonstrate that the activities of lysosomal enzymes are increased significantly during the process of rapid regeneration, especially at 24 h and 48 h. A small rise in enzymatic activities was noticed at 36 and 72 h of regeneration, leading towards normalisation at 168 h. Administration of tin(II) tartrate ( 25 μ mol Sn 2+ 100 gm body wt. ) significantly increased the DNAse and RNAse activity at 24 h of the regeneration, while a decrease was noticed in acid phosphatase activity. An increase was also found in the catheptic activity of tin-treated hepatectomized rats. It can thus be concluded that an increased metabolic activity of lysosomes is reflected by the increased activity of its hydrolytic enzymes during the regeneration, and tin at low concentrations is helpful to this process. Further work is in progress to gain insight into the other metabolic activity of the lysosomes during the process of regeneration.

Acid proteinase activity in fish—I. Comparative study of extraction of cathepsins B and D from Mujil auratus

1.1. Acid catheptic activity was measured in crude extracts of muscle, liver, heart, spleen and gonads from the fishes Mujil auratus, Sparus aurata and Lightonatus mormyrus. The spleen was the organ which showed the highest activity.2.2. A comparative study of the seven most commonly used extraction methods was made. Some were modified to account for the characteristics of the fish organs and the activity extracted from them. The Siebert method resulted as the best extraction method only if 1 mM EDTA was present in the medium.3.3. The activity from Mujil auratus muscle was strongly inhibited by iodoacetate, N-ethylmaleimide, p-hydroxy mercuribenzoate, and diazo-acetyl-dl-norleucine methyl ester. The results indicated the presence of a carboxyl-proteinase and a thiol-proteinase.4.4. According to inhibition studies, the levels of proteinase and amidase activities shown by different organs of Mujil auratus were re-examined. The spleen extract showed the maximum activity for both cathepsins, but muscle extract accounted for more than 95% of total catheptic activity.

The Effects of Dietary Selenium and Vitamin E on Avian White Muscle Disease as Measured by Both Chemical and Physical Parameters

Commercial chicken broilers were fed a semipurified diet deficient in vitamin E and selenium from day 1 to day 13 ex ova and subsequently fed varying levels of dietary selenium and vitamin E. All birds were sacrificed on the 28th day, stored for 36 hr at 2 C to allow the onset and resolution of rigor, and frozen at −32 C until needed. Total cathepsin content of the Pectoralis major depended upon dietary vitamin E for birds receiving 0 to 12 IU/kg, whereas selenium administered at .05 to .16 ppm in the diet showed no statistically significant effect. Similary, total protein content of P. major increased with increasing level of dietary vitamin E, but the level of dietary selenium had no effect. Muscle break strength was significantly affected by dietary selenium and vitamin E (P = .0092) interacting together. Catheptic activity and muscle protein explained 6.36% and 3.58% of the variability in muscle break strength. Birds with more advanced avian hite muscle disease showed higher break strength values. Ultrastructural deterioration of the myopathic muscle included disintegration of blood vessel walls, transverse tubules, and mitochondrial membranes as well as the obvious disruption of the myofibrillar components. Myelin figures were present in diseased, but not in normal, muscle. Accumulation of adipocytes both extracellularly and intracellularly occurred in selenium and vitamin E-deficient birds.

CHARACTERIZATION OF FISH MUSCLE PROTEASES USING RADIO‐LABELED PROTEIN SUBSTRATES

ABSTRACTCathepsin D and alkaline protease in the kidney and skeletal muscle tissues of Atlantic croaker were studied using either 14C‐casein or 14C‐hemoglobin. In general, the alkaline protease is more stable than cathepsin D upon heat treatment and the alkaline protease from muscle is more heat stable than that of kidney tissue. Both cathepsin D and alkaline protease showed specificity toward the different protein substrates. Under the defined conditions, it is possible to use a single substrate (casein or hemoglobin) for, the study of both proteases. The pH activity curves using both substrates suggest that at physiological pH, both acid and alkaline proteases may contribute to the proteolytic activity observed during holding or processing of fish products at lower temperatures (45–60°C). It was demonstrated that pepstatin may be effectively used to selectively inhibit the catheptic activity so that the relative contribution of alkaline protease to the degradation of the fish protein may be evaluated.

Effect of Trypsin Inhibitor on the Degradation and Transfer of Immunoglobulin across the Small Intestine of the Suckling Rat

The effects of certain chemical additives on, and the molecular characteristics of, the intestinal proteases of the suckling rat have been investigated. The catheptic activity of the intestinal wall was thiol dependent, whereas the luminal protease was markedly inhibited by trypsin inhibitor. There was no significant improvement in immunoglobulin transport across the gut wall on inhibition of luminal proteolytic activity.

Polyphosphate anions increase the activity of bovine spleen cathepsin D

Bovine spleen cathepsin D is activated by polyphosphate anions when bovine serum albumin is used as substrate at pH 4.6. In the presence of ATP at 10 mM, the catheptic activity at this pH is enhanced as high as 17 times over the control. Similar activating effects were observed, though to varying degrees, with sodium tripolyphosphate, nucleotides, nucleotide analogues, CoA, polyU and yeast RNA. The possible mechanism and biological significance of the activation were discussed with regard to the intralysosomal polyanionic substance.

Characterization of cathepsin D in the regressing tadpole tail of bullfrog, Rana catesbeiana

1. 1. Catheptic activity, measured by the hydrolysis of hemoglobin, increased remarkably during metamorphosis in the tadpole tail of Rana catesbeiana. 2. 2. Pepstatin inhibited about 75% of the catheptic activity, and the combination of pepstatin and monoiodoacetate inhibited it completely. Leupeptin slightly inhibited catheptic activity. 3. 3. By gel chromatography on Sephadex G-100, three molecular forms of catheptic activity were obtained. Their molecular weights were: Fraction I—larger than 100,000, Fraction II—50,000 and Fraction III—32,000. 4. 4. Fraction II, showing the highest activity was strongly inhibited by pepstatin and assumed to be cathepsin D. Fraction III was strongly inhibited by monoiodoacetate, indicating the existence of SH-proteinase.

Extraction and purification of squid myosin.

Isolation of myosin from the obliquely striated muscle of squid mantle was studied by comparing the various conventional isolation methods used for the vertebrate and invertebrate muscles. A method suitable to the squid myosin is proposed. The purity of each obtained preparation was checked by SDS polyacrylamide gel electrophoresis. The myosin preparations obtained by the conventional methods did not give any band corresponding to the myosin heavy chain, but those less than 14×104 in molecular weight were not free from contaminating proteins such as paramyosin and actin. The myosin preparation obtained by the proposed method showed an electrophoretic profile with a heavy chain band (18-20×104) and two light chain bands (1.2 and 1.5×104) of myosin with a faint band of paramyosin (10×104) in 7.5% gel. The tryptic digestion of the squid myosin shows that it is less resistant than fish myosins, and much less than rabbit myosin. The difficulties in obtaining an intact heavy chain during the isolation procedures were attributed to the fragility of the squid myosin molecule and to the possibility of high catheptic activity in the squid muscle.

Delayed degeneration of dystrophic and normal muscle cell cultures treated with pepstatin, leupeptin, and antipain

The effect of the proteinase inhibitors, pepstatin, leupeptin, and antipain, on dystrophic and normal embryonic chicken muscle cells growing in tissue culture was determined. The three inhibitors are effective against lysosomal cathepsins as well as other proteinases. The inhibitors appeared to delay atrophy and degeneration of dystrophic muscle fibers markedly; the effect on the normal muscle fibers was less striking. Catheptic activity and acidic autolysis are known to increase in the dystrophic chicken. These results support the suggestion that lysosomal proteases are involved, by an unknown mechanism, in the degradative process in dystrophic tissue. Delay in the process of degradation of muscle tissue suggests that these low molecular weight, nontoxic inhibitors offer some prospects as therapeutic agents for treatment of muscular dystrophy and other degenerative muscle diseases.

Age-related changes in the proteolytic enzymes of mammalian lens.

A longitudinal study has been conducted on three exopeptidases which include an esterase, leucine aminopeptidase, and a triglycinopeptidase and two endopeptidases, a neutral protease and a proteinase or catheptic activity in bovine lenses. The esterase and leucine aminopeptidase behave similarily showing increased activities with aging. Triglycinopeptidase has a somewhat lower order of magnitude when compared to the other exopeptidases. The neutral protease shows initially high activity early in the developmental life period, whereas, the proteinase or catheptic activity indicates a continuous increase with aging. Data whereas, the proteinase or catheptic activity indicates a continuous increase with aging. Data is also provided on enzymic activity in the different sections of the lens which include the combined anterior cortex and epithelium, lens nucleus, posterior cortex, and equatorial ring.

Studies on Proteolytic Activity in Bovine Lenses with Ageing

A longitudinal study of two endopeptidases including a neutral protease and a proteinase (catheptic activity) in bovine lenses was evaluated. The neutral protease shows an increase in activity during the 1st year of life, followed by a slower rate of activity up to 5 years of age. Enzyme activity picks up momentum throughout the remaining life span. The proteinase shows an almost continuous rise in activity over the entire life span. Activities for both enzymes are highest in the equatorial sections of the lens in the 5- and 7-year-old ge groups.