Participation of cysteine protease cathepsin L in the gel disintegration of red bulleye (Priacanthus macracanthus) surimi gel paste

BACKGROUND: Endogenous proteases, among them cysteine‐type proteases, are reported to contribute to gel disintegration, resulting in kamaboko of poor quality. Severe gel disintegration occurs in red bulleye surimi gel paste. The objective of this study was to clarify the participation of cysteine protease cathepsin L in the gel disintegration of red bulleye surimi. The surimi was made into kamaboko with and without cathepsin L inhibitors. To confirm its hydrolysis action, crude cathepsin L was also extracted and added to the surimi to make kamaboko. RESULTS: The gel strength of kamaboko obtained by both one‐step (50 °C, 2 h) and two‐step (50 °C, 2 h + 80 °C, 20 min) heating was very low in the absence of inhibitors. Protease inhibitors E‐64 and leupeptin were found to enhance the gel strength considerably. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the hydrolysis of kamaboko was promoted by crude cathepsin L and inhibited by E‐64 and leupeptin. The gel strength of two‐step heated kamaboko was increased from 12 to 110 and 130 g cm^?2 by E‐64 and leupeptin respectively at a concentration of 0.2 g kg^?1 surimi. CONCLUSION: Endogenous cathepsin L of red bulleye surimi participates in gel disintegration during kamaboko processing. It does so by degrading the myosin heavy chain of actomyosin and consequently hindering the gelation of red bulleye surimi. Copyright © 2009 Society of Chemical Industry     

COMPARISON OF CATHEPSIN B, D, H AND L ACTIVITY IN FOUR SPECIES OF PACIFIC FISH

The activities of cathepsins B, D, H and L were compared in crude muscle extracts from four species of fish: Pacific whiting (PW), (Merluccius productus); arrowtooth flounder (ATF), (Atheresthes stomias); Alaska pollock (AP), (Theragra chalcogramma); and Pacific cod (PC), (Gadus macrocephalus). Both PW and ATF are known to undergo softening during post-mortem handling and cooking while AP and PC do not. Cathepsin B and L activities were both higher in extracts of PW and ATF than in AP and PC. Cathepsins B and L activities were both much higher in PW than in ATF. Cathepsin H activity was highest in AP followed by ATF with PC and PW having the lowest activities. Cathepsin D activity was extremely low in all four species. The heat stability of the various cathepsin activities showed cathepsin B to be the most heat labile and was inactivated by 50C heating for 15 min. Cathepsin H activity was inactivated at 60C, while cathepsin L required 70C for inactivation. Cathepsins B and L are the most likely responsible for softening in PW and ATF during holding and subsequent processing. However, during cooking, cathepsin L likely causes the most damage since it requires 70C for inactivation. The difference in heat stability of cathepsins B and L can be used to differentiate between their activities.     

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.     

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.     

Effect of Chemical and Physical Dry-curing Parameters on Cathepsins B, H and L from Ostrich Muscle

The effects of curing agents (NaCl, nitrate, ascorbic acid and glucose) and processing parameters (pH, temperature and cooking temperatures) on cathepsins B, H and L activities were investigated. NaCl, nitrate, ascorbic acid and glucose exhibited different influences on ostrich cathepsin B, B+L and H activities. In the range 20-60gl(-1), NaCl inhibited cathepsin B+L and H activities. All three cysteine proteinase activities were inhibited by up to 8g ascorbic acid l(-1). With the exception of cathepsin B activity, which was inhibited by glucose, nitrate and glucose had very little effect on cathepsin B, B+L and H activities. Cathepsins B and D were active at 65 and 69°C and might play an important degradative role during the cooking of meat and meat products. Cathepsins B, B+L and H were optimally active at temperatures of 40-45°C and 50°C, and were still quite active at the low temperatures used in the dry-curing process; they showed maximum activity in the pH range 5·5-7. A simulation of the three stages of the dry-curing process of hams revealed that cathepsins B and B+L might play an important role throughout the complete process, whereas cathepsin H could only participate in the middle and at the end of the dry-curing process. Although ostrich cathepsins show many properties similar to those from other species, the present study also revealed some interesting distinguishing features.     

Effects of high pressure treatment on the proteolytic enzymes in meat

This paper describes the effects of high-pressure treatment on proteolytic enzymes in muscle, especially catheptic enzymes which influence meat tenderization, and on acid phosphatase, used as an index of disruption of lysosomal membranes. Acid phosphatase activity in the extract from pressurized muscle increased with increasing pressure applied to the muscle up to 500 MPa. Activity of cathepsin B, D and L increased up to 400 MPa, then tended to decrease at 500 MPa. Cathepsin H and aminopeptidase B decreased with the increasing pressure. Measurements of enzymic activity in the pressurized crude extract, to investigate the pressure effect on the enzymes themselves, showed that all enzymes studied in this paper lost their enzymic activity as applied pressure increased. When the pressurized extracts were subjected to the gel-filtration chromatography, a decrease in the activities of cathepsin H and aminopeptidase B and an increase in the activities of cathepsins B and L and acid phosphatase were observed. It seems that the decrease in activity of the enzymes eluted early from the column (cathepsin H and aminopeptidase B) is due to decrease in the amount of protein eluted by the pressure treatment, whereas the increase in activity of the enzymes eluted late (cathepsin B, L and acid phosphatase) is due to an increase in the amount of protein eluted. From the results, it was concluded that the pressure-induced increase in the amount of protease activity in the muscle was due to the release of the enzymes from lysosomes.     

Purification and Characterization of Low Molecular Weight Kininogen from Pig Plasma

A cysteine proteinase inhibitor from pig plasma with a molecular weight (MW) of 55 kDa, purified to electrophoretical homogeneity, inhibited μ- and m -calpains, cathepsins B, L, and L-like, and papain, but did not inhibit trypsin, β-chymotrypsin, and cathepsin D. The purified inhibitor was stable at pH 3.0 to 10.5. The amounts for 50% inactivation (ID₅₀) of papain, cathepsins B, L, and L-like, μ- and m-calpains were 10.55, 12.91, 2.18, 2.18, 30.91, and 29.27 nM, while the inhibition constant (K_i) for cathepsins B, L, L-like, and X, and μ- and m-calpains were 1.1, 0.64, 63.33, 8.19, 26, and 23.57 nM, respectively. It could inhibit the proteolysis of mackerel myosin heavy chain caused by purified cathepsin L-like at 55 °C. Based on the MW, stability and specificity, it was identified as L-kininogen.     

Effect of dry-curing process parameters on pork muscle cathepsin B, H and L activity

Summary The effect of chemical agents (salt, nitrate, ascorbic acid and glucose), and process parameters (water activity, pH and temperature) on porcine muscle cathepsins B, H and L was studied. Salt strongly inhibited cathepsin H activity. Only 40–50% of the original activity was recovered in the presence of 4–5% salt, typical of dry-cured meat products, while cathepsins B and L were less affected, recovering around 65–75% of their original activity. Ascorbic acid (above 1 g/L) strongly inhibited cathepsin H whereas glucose (up to 2 g/L) activated (around 30%) cathepsin B. Nitrate did not affect cathepsin activity. A decrease in water activity affected cathepsin B and H activity and cathepsin H activity was almost negligible below pH 5.5, which is usual in dry-cured sausage processes. All three enzymes were very active in the temperature range, 20–30° C, typical of dry-curing processes. A study in vitro, representing three stages of the dry-curing process of hams, revealed that cathepsins B and L might play an important role throughout the complete process whereas cathepsin H only participated in the middle and at the end of the dry-curing.

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Einfluß des Trockenpökelns auf die Aktivität der Cathepsine B, H und L

Zusammenfassung Es wird die Wirkung von Salz, Nitrat, Ascorbinsäure und Glucose sowie die Wasseraktivität, pH und Temperatur auf die Wirksamkeit der Kathepsine B, H und L des Schweinefleisches untersucht. Das Salz hemmt das Kathepsin H sehr stark, denn nur 40–50% der ursprünglichen Aktivität wurde in Anwesenheit von 4–5% Salz erreicht, was typisch für ein Pökelmilieu ist, während die Kathepsine B und L weniger beeinflußt werden und ca. 65–75% der ursprünglichen Wirksamkeit wieder erlangen. Die Ascorbinsäure (1 g/L Lösung) hindert sehr stark das Kathepsin H, während die Glucose (über 2 g/L Lösung) zu ca. 30% das Kathepsin B fördert. Die Enzymtätigkeit wird vom Nitrat nicht beeinflußt. Bei abnehmen der Wasseraktivität werden die Aktivitäten der Kathepsine B und H reduziert, wobei letztere jegliche Aktivität bei einem pH-Wert unter 5,5 verliert, ein beim Reifen der Wurstwaren üblicher Wert. Die drei Enzyme waren sehr aktiv bei Temperaturen zwischen 20 °C–30 °C. Eine Untersuchung in vitro mit den drei Phasen des Reifens offenbart, daß die Kathepsine B und L insgesamt eine wichtige Rolle spielen, wohingegen das Kathepsin H nur ab Mitte des Pökelvorganges wirksam ist.

     

Mackerel Cathepsins B and L Effects on Thermal Degradation of Surimi

During surimi processing, cathepsins B and L activities in minced, leached and NaCl-ground meats were 6.02, 5.23, and 4.07 units/g, respectively. About 80% activity remained in surimi after 8 wk storage at ?40°C suggesting that these proteinases were stable and difficult to remove. At 40°～ 55°C, pH 6.5 ～ 7.5, cathepsins B and L and purified cathepsin B had high hydrolytic activity on myosin heavy chain (MHC). The strength of surimi gel with cathepsins B and L or with purified B decreased (p < 0.05) after 2 hr incubation at 55°C. This suggested that the residual cathepsins B and L had MHC-degrading activity and consequently caused gel softening.     

Effect of proteolytic enzyme activity and heating on the mechanical properties of bovine single muscle fibres

The effect of proteolysis and heating on the mechanical properties of single muscle fibres isolated from bovine M. semitendinosus was investigated. Short incubations with μ-calpain (pH 7.5) and cathepsin B (pH 5.6) at room temperature reduced (P<0.01) the raw fibre strength by 50%. After subsequent heating, μ-calpain-incubated fibres were 20% weaker and cathepsin B-incubated fibres 50% weaker than heated controls. Fibres were incubated for 8 days at 2?°C under post-mortem like pH conditions (pH 5.6) or pH conditions optimal for the calpains (pH 7.5). At pH 5.6 no significant weakening of the fibres was observed when incubations were performed in 0.1, 1 or 10 mM Ca(2+). In contrast, incubation in 1 or 10 mM Ca(2+) at pH 7.5 decreased the strength of the fibres. Addition of the protease inhibitors E-64 or PD150,606 prevented this weakening, suggesting that the weakening is caused by proteolyses and not directly by Ca(2+).     

ROLE OF MUSCLE PROTEINASES IN MAINTENANCE OF MUSCLE INTEGRITY AND MASS

Current evidence indicates that, of the thirteen known lysosomal peptide hydrolases, only seven, cathepsins A, B, C, D, H, L, and lysosomal carboxypeptidase B are located inside skeletal muscle cells. Only one of the reported neutral and alkaline proteases is located inside skeletal muscle cells', this neutral protease is the Ca²⁺-dependent proteinase, CAF. With the possible exception of cathepsin N, which can degrade collagen, it seems probable that any protease that contributes to postmortem tenderization needs to be located inside muscle cells. Because very little degradation of myosin or actin occurs in postmortem muscle, most of the small amount of proteolytic degradation of the myofibrillar proteins that occurs during postmortem storage must be due to CAF, which is unique in being unable to degrade myosin and actin. It is not certain that postmortem proteolysis by CAF causes increased tenderness; some recently discovered actin-fragmenting proteins could be involved.     

Recovery of cathepsins from marinating brine waste

In the fish industry, brine left after herring marinating is discarded to waste. This study presents laboratory‐scale findings regarding recovery of aspartyl (D + E) and cysteine cathepsins (B + L) from waste brine. Ultrafiltration enabled cathepsin recovery from both the soluble and lysosomal fraction of the marinating brine. Retentate 10 kDa had the highest cathepsin activity 140% compared to crude brine. The amount of recovered cathepsins using membranes 30 and 100 kDa was less than 10 kDa, because part of cathepsins passes to permeate. In turn, the salting‐out with ammonium sulphate enabled activity recovery of only 80%, but ensured over twofold higher purity of the enzymes than ultrafiltration. The highest precipitation of D + E cathepsins was noted at salt concentration of 40–60%, whereas that of B + L cathepsins at 50–80%. It is recommended to dissolve the salted‐out aspartyl cathepsins at pH 4.0 or 8.0, whereas the cysteine ones at pH 5.0. The appropriate selection of parameters enables controlling the aspartyl to cysteine cathepsins ratio in the preparation in the range from 1:2 to 2:1. The new cathepsin preparations showed high activity in a wide range of pH values and ionic strength and can be used in food production.     

CHANGES IN THE CALPAIN-CALPASTATIN AND CATHEPSIN (B, B+L, H AND D) DURING POSTMORTEM STORAGE OF GOAT MUSCLES

The objective of this study was to elucidate a relationship between some endogenous proteinases and their inhibitors in four different goat muscles during postmortem storage. Samples were taken from the longissimus dorsi (LD), biceps femoris (BF), semimembranosus (SM) and semitendinosus (ST) muscles stored up to 20 days postmortem at 5C. Activities of calpain-I, calpain-II, calpastatin, cathepsins (B, B+L, H and D) and cystatin(s) were determined. Decreases in calpain-I and calpastatin activities were significantly more than that of calpain-II activity. The cathepsin B, B+L, H and cystatin level were found to fall by 9–35 % after 20 days, whereas the cathepsin D showed 11–17% decline in all the muscles. Thus changes in muscle proteinases and their inhibitors during postmortem storage differ and the results may shed light on their role in myofibrillar proteolysis and goat meat tenderization.     

The effects of Ca ions, EGTA and storage time on myofibrillar protein degradation, levels of Ca(2+)-dependent proteases and cathepsins B, H, L and D of ostrich skeletal muscle

The effect of Ca ions and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) on myofibrillar protein degradation showed that when ostrich iliotibialis lateralis muscle was incubated with 10 mM EGTA at 2–4 °C for 24 hr, the activity of extracted cathepsin H was unchanged compared with a buffer-incubated sample. Ca⁺⁺ had no effect on extracted cathepsin H activity, while that of Ca²⁺-dependent protease (CDP) decreased significantly (p < 0.05). Ca²⁺-treatment enhanced post-mortem changes observed in myofibrillar protein patterns (production of fragments around 30 K) that were not observed in EGTA-incubated myofibrils. The effect of storage time on shear force, CDP activity, cathepsin B, D, H and L activities and the SDS-PAGE pattern of myofibrils showed a time-dependent reduction in CDP activity. Of the cathepsins studied only cathepsin H showed a reduction (40%) in activity. The most prominent component appearing on storage at 2–4 °C had a M_r of 27 K. The incubation of myofibrils with CDP mimicked the post-mortem changes. CDP may be responsible for some of the post-mortem changes observed, although shear force measurements suggest these changes do not lead to significant tenderisation.     

鱿鱼肝脏蛋白酶的鉴定及组织蛋白酶L的分离纯化

鱿鱼肝脏含有丰富的蛋白酶,为利用其内源蛋白酶进行可控的酶解,本研究以鲤鱼肌原纤维蛋白为底物对鱿鱼肝脏内源蛋白酶的种类和性质进行了研究。反应体系中添加E-64、1,10-菲啰啉和苯甲基磺酰氟(phenylmethylsulfonyl?fluoride,PMSF)后,肌球蛋白重链(myosin?heavy?chain,MHC)的降解得到了显著抑制,确定了鱿鱼肝脏含有金属类、半胱氨酸类、丝氨酸类3类蛋白酶。半胱氨酸类蛋白酶热稳定性最好,在50℃以上仍然具有较大活性,可将肌原纤维蛋白酶解成小分子质量的降解产物。利用特异性底物对半胱氨酸蛋白酶种类进行鉴定发现,该酶只酶解Z-Phe-Arg-MCA,添加亮抑酶肽后相对酶活性为0%,添加E-64相对酶活性仅存0.6%,初步确定鱿鱼肝脏中的半胱氨酸蛋白酶主要为组织蛋白酶L。最后,通过硫酸铵沉淀、离子交换层析、凝胶过滤对组织蛋白酶L进行分离纯化,在电泳上得到了分子质量约为25?kD单一条带。     

Comparative action of cathepsins D, B, H, L and of a new lysosomal cysteine proteinase on rabbit myofibrils

Specific action of cathespins D, B, H, L, and of a new high Mr (molecular weight relative to hydrogen) cysteine proteinase, on rabbit muscle myofibrils was studied at pH 5·7 by following changes affecting their ATPase activities, their calcium sensitivity, their effect on the ultrastructure, as well as the electrophoretic pattern of the contractile proteins in the presence of SDS. With regard to the MgCa-enhanced ATPase activity, all these proteinases had a very similar effect. A decrease in this activity was thus noted concomitantly with a shift of the straight-line graph obtained when plotting the present acto-myosin ATPase activity versus KCl concentrations. Cathepsins D, B, L and the high Mr cysteine proteinase induced a decrease in both the calcium ATPase activity of myosin and the calcium sensitivity of myofibrils. On the contrary, the Mg-EGTA-dependent ATpase activity was increased. Except for cathepsin H, extensive hydrolysis of proteins occurred in myofibrils treated with each of the lysosomal proteinases tested. However, different specificities could be distinguished. On the one hand, cathepsins D and B affected mainly myofibrillar protein running above and below actin, respectively, on SDS-polyacrylamide gel electrophoresis; on the other hand, the high Mr cysteine proteinase exhibited broader specificity since most of the proteins were hydrolyzed irrespective of their Mr. Myofibrils incubated with cathepsins B and the high Mr cysteine proteinase showed ultrastructural modifications at the level of Z-line, M-bands and A-bands. Myofibrils treated with cathepsin D and cathepsin H appeared almost unaltered. On the basis of these characteristics, cathepsin H hardly affected myofibrils. These results provide evidence for the involvement of the lysosomal proteinases in the meat ageing process and are discussed in regard to the changes occurring at the myofibrillar level during conversion of muscle into meat.     

Cysteine protease activity in bovine milk

Proteolytic activity of native cysteine proteases was studied in bovine milk. Five fractions (fI–fV) with cysteine protease activity were separated from acid whey prepared from raw bovine milk by ion-exchange chromatography on Q-Sepharose. The hydrolytic action of the most active fractions (fIII and fV), after further purification using gel permeation chromatography on Superdex S75, was studied against individual caseins. The two fractions contained different cysteine protease activities capable of hydrolyzing both α_s1- and β-casein. Studies of the effects of different reagents on the activity of partially purified fIII showed that the activity in this fraction was unaffected by aprotinin, slightly inhibited by p-chloromercuribenzoate and o-phenanthroline and completely inhibited by -trans-epoxysuccinyl- -leucylamido (4-guanidino) butane, consistent with identification as a cysteine protease. Phenylmethylsulphonyl fluoride and pepstatin A reduced activity of fIII by 40% and 50%, respectively. The partially purified fIII retained 20% of its cysteine protease activity after heating at 55°C, 60°C, 65°C and 72°C for 40 min, 20 min, 10 min and 30 s, respectively. Immunoblotting of fIII with antibodies to the bovine lysosomal cysteine protease, cathepsin B, clearly indicated the presence of immunoreactive cathepsin B in this fraction. This study presents strong evidence for the presence of a heterogeneous group of cysteine proteases in bovine milk, with one of these enzymes probably being cathepsin B.     

Purification process to obtain cathepsin D,B and L suitable to be used in food industry

The use of enzymes in the food industry allows the improvement of the manufacture and quality of the products. To date, enzymes have not been widely used in the meat industry. To permit a further study of the role and application of cathepsins, a purification process of cathepsins B, D and L from porcine cardiac muscle has been developed. The protocol applied was: extraction of enzyme from porcine cardiac muscle, using 3% NaCl, 15 mM HCl; and ammonium sulfate fractionation and a subsequent purification by gel filtration, ultrafiltration and ion exchange. This easy to scale‐up procedure, which may permit the application of the purified enzymes as food additives, made it possible to obtain cathepsins B, L and D separately, with a recovery of 21.60%, 18.24% and 3.68% respectively. The degree of purity was 29.84 and 8.32 for cathepsin B and cathepsin L respectively.     

An Immunological Assessment of Myosin Degradation in Pressurized Chicken Muscle

ABSTRACT: The factors affecting myosin degradation that occurred during aging following high-pressure treatment over a pressure range from 200 to 600 MPa were investigated by using SDS-PAGE and immunoblotting analysis. The immunoblot pattern of myosin in muscle stored at 37°C for 48 h after pressure treatment at 0. 1 MPa (atmospheric pressure) or 200 MPa for 5 min was similar to that of native myosin incubated with cathepsin D, whereas at 400 or 600 MPa the pattern was close to that of native myosin incubated with cathepsin B. This phenomenon was reflected in the pressure-susceptibilities of cathepsins B and D as reported in the literature (Homma and others 1994). However, these catheptic enzymes released by pressure treatment are unlikely to play a role in pressure-induced tenderization of meat.     

Effects of the terminal sire type and sex on pork muscle cathepsins (B, B+L and H), cysteine proteinase inhibitors and lipolytic enzyme activities

Pork muscle cathepsins (B, B+L, and H), cysteine proteinase inhibitors and lipolytic enzyme activities were measured in the offspring of five different genetic sire types: Danish Duroc (DU), Dutch Large White (LW(D)), English Large White (LW(E)), Belgian Landrace × Landrace (BL×LR) and Belgian Landrace (BL). Cathepsin B and B+L activities were higher for LW(E) and LW(D) sires than for BL×LR and BL. Cathepsin H activity showed an opposite evolution, being higher for BL and BL×LR sires than for DU, LW(D) and LW(E). Cysteine proteinase inhibitor activity was higher for LW(E) sires than for DU and BL. In lipolytic enzymes, BL sires had a lower acid lipase activity than DU and LW(E) sires and also a lower neutral esterase activity than LW(E) and LW(D) sires. Significant differences between sexes were found for cathepsin H activity only, being higher for females.