Assay of Cathepsin D activity in fresh pork muscle and dry-cured ham

Different assays of cathepsin D activity in both porcine muscle and dry-cured ham extracts have been tested in order to find the best conditions for a reliable detection of cathepsin D in dry-cured meat products. The enzyme was effectively extracted with 0·2% (v/v) of Triton X-100.

Nucleases if present were not observed to interfere with the assays. The best conditions for a reliable detection of cathepsin D in dry-cured ham were found to be the incubation of the enzyme extract for 1 h at 45°C in a reaction mixture containing 0·60% (w/v) of haemoglobin in 0·2 sodium citrate buffer, pH = 3·7.     

Lactose absorption by postweaning rats from yogurt, quarg, and quarg whey

Lactose-intolerant postweaning rats were fed experimental diets including yogurt, quargs prepared from yogurt culture and buttermilk culture, and two types of whey obtained from quarg processing. After feeding each diet for a period of 7 d, absence of blood glucose elevation and occurrence of diarrhea were used as indicators of lactose malabsorption. Blood glucose assays and absence of diarrhea indicated that yogurt and quargs prepared from yogurt and buttermilk culture were well tolerated by the rats. Wheys containing the same levels of viable organisms and lactose as the quargs caused severe symptoms of diarrhea and poor lactose absorption as indicated by no changes in blood glucose levels. Plate counts and enzyme assays of gastrointestinal contents confirmed presence of viable culture organisms and beta-galactosidase activity after feeding the two types of quarg. The availability of viable organisms, the exogenous lactase activity, and especially the slow gastric emptying may all have contributed to more efficient hydrolysis and digestion of lactose from quargs and yogurt than from the wheys.     

Thermal Stability of Cathepsin D

The effect of pH and temperature on Cathepsin D stability was examined using a hemoglobin assay following preincubation of the enzyme at various pH and temperature combinations. The results of the study showed that the enzyme retained 87% of its activity at 45°C (pH 3.5) when compared to a control at 37°C (pH 3.5). Further increases in temperature and pH resulted in decreased enzyme activity and approximately 43% remained at 55°C (pH 5.5). The amount of activity remaining at higher temperatures decreased but suggests that the enzyme could contribute to textural changes in meat at temperatures up to 60°C.     

Production of quarg by ultrafiltration

The increased protein concentration in UF concentrate caused some problems in achieving the desired pH for quarg making when yogurt and mixed lactic cultures were used. Yogurt culture could ferment concentrated milk to a lower pH than the mixed culture. With the increasing concentration during UF, levels of total ash, calcium and phosphorus in the concentrate increased, but these increases were much lower at pH 4.6. Quarg obtained from UF concentrated sour milk was rated close to conventional quarg and had no bitter taste. A high heat treatment of milk before lactic fermentation and subsequent UF concentration resulted in a quarg with a smoother texture. Diafiltration of UF concentrated milk did not result in significant elimination of excessive calcium. The quality of the quarg was also poor with respect to taste, body and texture. Thus diafiltration would be of little use in quarg making.     

Purification and Properties of Carp Muscle Cathepsin D

A carp muscle cathepsin was purified as an electrophoretically homogeneous preparation. The preparation represented about 2,000-fold purification and about 4% yield against the crude extract. The activity against hemoglobin was maximal at pH 2.6—2.8 with 0.6M buffer and near 3.2 with 0.12M buffer and at 50°C. The molecular weight was found to be 41,000 and the isoelectric point to be pH 5.4. From the effect of various inhibitors on the enzyme activity, the enzyme was identified as cathepsin D under the classification of Barret. Carp muscle cathepsin D hydrolyzed myofibrils optimally at pH 3–4, but did not above pH 6.0. The participation of the enzyme in autolysis is very doubtful.     

Cathepsin B and D activity in the mucous membrane of the small intestine of rats on a high-saccharose diet and added selenium

Selenium added to diets (700-750 micrograms per kg bw) enhanced the activity of cathepsins B and D; in this case, the protein content in the rat small intestine mucosa was found to be considerably reduced. High-sucrose diet did not directly influence the lysosomal proteinase activity, but enhanced selenium action on the proteolysis.     

The Effective Methods in Refolding and Activation of Cathepsin L-like Soybean Protease D3

A novel cysteine protease D3, which was purified from germinating soybean cotyledons, showed high homology with cathepsin L and cathepsin K. In our previous study, because of the specificity of the enzyme, hydroly‐sates treated with D3 treatment showed a prominent property of less bitterness than other hydrolysates treated with commercially available proteases. However, active recombinant D3 prepared from Escherichia coli inclusion bodies was so intricate and less productive that it made further studies on this protease and hydrolysates difficult. In the concrete, the refolding process of the immature proD3 from inclusion bodies takes more than a day, and autocatalytic activation of refolded immature proD3 at low pH was difficult to control. In this study, we aimed to establish an efficient refolding and activating method of protease D3. In the refolding step, the procedures could be simplified by using a size‐exclusive column‐based method. In the activation step from immature proD3, we utilized another protease, subtilisin, rather than autocatalytic activation by D3 itself. After subtilisin treatment, the peptide having 12 amino acids‐length of N‐terminal pro sequence was initially cleaved, and residual proD3 showed only a half proteolytic activity of active D3. However, when the pH was shifted lower (pH4.5), D3 automatically changed to have the same proteolytic activity as active one, and this activated recombinant had the same N‐terminal sequence as purified D3 from germinating soybean cotyledons. By using this method, all preparation processes of D3 from inclusion bodies to active D3 could be completed within a few hours, and it became possible to carry out the investigation on hydrolysates on a large scale.     

Cathepsin B,D, H and L activities in the processing of dry-cured ham

The activities of cathepsins B, D, H and L have been assayed at various stages in the slow processing (15 months) of dry-cured ham. Cathepsins B, H and L showed low recovered activity (5–10% of the initial activity) at the end of the process. However, cathepsin D almost disappeared after 5–10 months. Water-soluble protein extractability decreased throughout the process while myofibrillar protein extractability was constant. There was a progressive disappearance of myosin heavy chain, myosin light chains 1 and 2, and troponins I and C, a marked increase of three breakdown products with molecular weights of about 150, 95 and 16 kDa and some minor products in the ranges 50–100 kDa and 20–45 kDa. Cathepsins B and L could be particularly active in the observed proteolysis which had a special relevance when the drying started. Actin, actinin, troponin T and tropomyosin did not seem to change.     

骨骼肌中组织蛋白酶(英文)

<正>Introduction Although cathepsin and its main functions have beenfound in animal muscle tissue,since man foundthat calcium-activated enzyme and calcium alone candirectly induce the degradation of     

骨骼肌中组织蛋白酶

组织蛋白酶是动物组织细胞内位于溶酶体中的一类重要蛋白酶。在骨骼肌中的主要是B、D、E、H、L型组织蛋白酶,它们对宰后肉的食用品质具有非常重要的作用;骨骼肌组织蛋白酶降解蛋白质的性质在时空上各有差异,很多因素可以影响其活性。     

Effect of Cathepsin D on Bovine Myofibrils under Different Conditions of pH and Temperature

Purified cathepsin D was incubated with bovine skeletal muscle myofibrils under in virro conditions resembling those found in postmortem muscle. SDS-PAGE analysis of myofibrils treated at pH 5.5 and 37°C and the sedimented, showed degradation of myosin heavy chains and titin. A small amount of actin, tropomyosin, troponins T and I, and myosin light chains also were degraded. The cathepsin D treated myofibrils were not fragmented to any greater extend than untreated myofibrils. Raising the pH and/or lowering the temperature greatly reduced the effectiveness of cathepsin D suggesting that the enzyme does not play a principal role in the tenderization process occurring in muscle postmortem.     

Distribution of Cathepsin D Activity between Lysosomes and a Soluble Fraction of Marinating Brine

This paper is the first ever to describe the phenomenon of bimodal distribution of cathepsin D in the lysosomal and soluble fractions of brine left after herring marinating. Up to 2 times higher cathepsin D activity was observed in the lysosome fraction. Activity of cathepsin D in brine increased according to the logarithmic function during low frequency‐high power ultrasounds treatment or according to the linear function after multiple freezing‐thawing of brine. Activity enhancement was achieved only in the brine devoid of lipids and suspension. Study results show also that measurement of lysosomal cathepsin D activity in the marinating brine requires also determining cathepsin E activity. Decreasing pore size of microfilter from 2.7 to 0.3 μm significantly reduced the lysosome content in the brine. The presence of lysosomes and the possibility of their separation as well as the likely release of cathepsins shall be considered during industrial application of the marinating brine, as new cathepsins preparations in fish and meat technology.     

Electrical stimulation of pigs-effect on pH fall, meat quality and Cathepsin B+L activity

This work was performed to evaluate the effect of electrical stimulation (ES) on pH fall and meat quality of LD and BF in Danish halothane free pigs stunned with CO(2). ES resulted in a significant drop in pH of 0.3 units in both LD and BF and 3 h lairage resulted in 0.1 units lower pH at 20 min post mortem. Lairage time did not affect and did not interact with the effect of ES on any of the measured meat quality parameters. ES did not affect the ultimate pH in LD, BF, SM and SC or internal reflection value in LD. However, ES caused higher internal reflection and drip loss in BF and increased the PSE frequency in LD (2 to 7%) and in BF (2 to 49%). The frequency of PSE areas in the centre of SM was 70% for ES pigs compared to 9% for control pigs. ES and ageing improved the tenderness and reduced the hardness in LD as well as shear force of unaged BF. The effect of ES on tenderness and hardness was approximately half the effect of ageing, but the effect of ES and ageing were additive. ES significantly increased the activities of cathepsin B+L in the myofibrillar fraction, but there were no differences in proteolytic activity in the other fractions. It is concluded that ES improves tenderness in LD and BF, but has a negative effect on the quality of BF and SM. Therefore ES is not an economically attractive alternative for improvement of tenderness in LD compared to ageing in Danish pigs. ?     

天然抑制组织蛋白酶B的生物活性肽

组织蛋白酶B是一种细胞溶酶体巯基蛋白酶，参与细胞内水解以及多种病理过程。组织蛋白酶B抑制剂具有潜在抗癌作用。介绍了组织蛋白酶B活性的测定方法，综述了天然存在的具有抑制组织蛋白酶B活性的肽的特性。     

Cathepsin Degradation of Pacific Whiting Surimi Proteins

Cathepsin B was the most active cysteine protease in Pacific whiting fish fillets; cathepsin L was predominant in surimi. Cathepsin L showed highest activity at 55°C in both fish fillets and surimi, indicating its function in myosin degradation during conventional heating of fillets and surimi, gels. Washing during surimi processing removed cathepsin B and H but not cathepsin L. Myosin heavy chain was the primary substrate during autolysis of surimi paste and actin and myosin light chain showed limited hydrolysis during 2 hr incubation. Purified Pacific whiting cathepsin L hydrolyzed myofibrils, myosin and native and heat-denatured collagen. The degradation pattern of myofibrils by the protease was the same as the autolytic pattern of surimi.     

Bovine cathepsin D activity under high pressure

The stability and catalytic activity of bovine cathepsin D in Bis-Tris buffer (pH 6.0) in different pressure–temperature domains (0.1–650 MPa, 20–75 °C) were investigated and described with mathematical models. Cathepsin D inactivation followed first-order kinetics at all pressure–temperature conditions tested. The protease was largely pressure stable at room temperature and heat stable at ambient pressure up to 300 MPa and 55 °C, respectively, causing less than 10% inactivation after 10 min treatment. Pressure and temperature act synergistically on the enzyme inactivation under most conditions. However, at 100 MPa a significant stabilisation of the enzyme against temperature-induced inactivation was observed. Pressure drastically inhibited the cleavage of a synthetic substrate by cathepsin D in Bis-Tris buffer (pH 6.0) causing a reduction of the catalytic rate of more than 50% at 100–400 MPa. Maximal substrate cleavage by cathepsin D was identified at 60 °C and ambient pressure conditions after 20 min treatment.     

Proteinase (Cathepsin B, D, L and Calpains) levels and conditioning rates in normal, electrically stimulated and high-ultimate-pH chicken muscle

Control, electrically stimulated (ES) and glycogen-depleted (GD) chicken muscles were conditioned at 15°C with continuous mechanical testing for extensibility. The ES and GD muscles went into rigor 3·6 and 2·8 h earlier, respectively, than control muscle. At 24h post-rigor the extensibility of control muscle (11·2%) was markedly less than ES (19·2%) and GD (27·3%) muscles indicating that these latter two treatments should provide more tender meat. Measurement of sarcomere lengths showed no significant differences between control and GD muscle and thus, the greater extensibility in the high pH condition may be restricted to a wider separation of myofibriller fragments at the intermittent fracture zones when under load. Examination of muscle proteinase (cathepsins B, D and L, calpains I and II) and glycosidase (β-d-glucuronidase, N-acetyl-β-d-glucosaminidase) levels at 0 and 48h post-slaughter revealed changes in some key enzymes between the different treatments. Calpain I activity declined markedly during 48h storage of ES muscle (83%) compared to control (58%) and GD (63%) muscles. Cathepsin B and L activities did not decline during storage of ES muscle but there was a slight fall in control and GD muscles. Dosing of chicken shortly before slaughter with inhibitors of cysteine proteinases had a negligible effect on conditioning rate, apparently due to lack of inhibition of these proteinases during this short time period in the intact muscle.     

牛胰脏组织蛋白酶L的纯化和酶学性质

新鲜牛胰脏匀浆物经酸化粗提后,再通过盐析、柱层析等步骤纯化,制备了0.58 mg纯酶,纯化倍数达530.54。经凝胶电泳分析,该酶有2 个亚基,分子质量为29.1 kD和18.9 kD。牛胰脏组织蛋白酶L的最适反应温度为50 ℃,最适反应pH值为6.5。巯基还原剂二硫苏糖醇、L-半胱氨酸均明显激活了该酶活性,10 μmol/L的N-（反式-环氧丁二酰基）-L-亮氨酸-4-胍基丁基酰胺（E-64）可完全抑制其活性。1 mmol/L的Zn2＋对酶活性有明显抑制作用。该纯化酶可水解苄氧羰基-苯丙氨酰-精氨酰-甲基香豆素（Z-Phe-Arg-MCA）,其Km值为3.52 μmol/L。     

Effect of Pressure-Heat Treatments on Cathepsin B1 Activity

The substrate Nα-benzoyl-DL-arginine-2-naphthylamide was used to measure the activity of crude cathepsin Bl in reaction mixtures that had been subjected to various combined pressure and heat treatments. Pressures of 100 KPa (atmospheric), 100 MPa and 150 MPa and temperatures in the range 30–30°C were used. High ressure appeared to protect the enzyme against heat inactivation. Maximum activity was observed at 60°C and 150 MPa and was 12 times that observed at 60°C and atmospheric pressure. An increase in activity of cathepsin B1 may account, at least in part, for the tenderization of meat by pressure-heat treatments.     

Inhibition of lysophospholipase D activity by fish egg extracts

Lysophospholipase D (lysoPLD) is known to convert lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). In this study, we examined the inhibitory effect of fish egg extracts, containing lipids, on bovine lysoPLD activity. Fish eggs extracts were tested for the inhibition of lysoPLD activity, and the inhibitory action was expressed as 50% inhibitory concentration (IC₅₀). Among fish egg extracts of 20 fish species, the most potent inhibition was expressed by Hairtail egg extract (IC₅₀, 0.07 ± 0.01 mg egg weight/mL), followed by extract of Spanish mackerel egg extract (0.11 ± 0.02 mg egg weight/mL) and extract of Pacific saury egg (0.48 ± 0.03 mg egg weight/mL). In ESI/MS analysis, major lysoPLD-inhibitory lipid components in egg extracts were identified to be species of LPC, LPA and fatty acid. From these results, it is suggested that the strong inhibition of lysoPLD activity by fish egg extracts might be ascribed to the presence of lysophospholipids. In a separate study, enzymatic oxidation using lipoxygenase or non-enzymatic oxidations such as HOCl oxidation or Cu²⁺-catalyzed oxidation enhanced the inhibitory activity to some extent, suggesting that the oxidation of polyunsaturated lysophospholipids might contribute to the increase of lysoPLD-inhibitory action. Taken together, it is suggested that fish eggs may contain potent lipid inhibitors of lysoPLD, and that the inhibitory action of lipid inhibitors was enhanced by oxidative process.