Identification of calpastatin, μ-calpain and m-calpain in Atlantic salmon (Salmo salar L.) muscle

A soft fish muscle is generally considered as a poor quality trait among consumers and producers. This degradation and softening of post mortem muscle is thought to be partly caused by proteolytic enzymes such as the calpain system. Separation and identification of μ-calpain and m-calpain and their inhibitor – calpastatin, from Atlantic salmon (Salmo salar) muscle were for the first time assessed in this study. A two-step chromatography approach was used, starting with a hydrophobic interaction column and followed by an anion exchange column. Calpastatin was successfully separated from calpain by hydrophobic interaction chromatography, and following the anion exchange chromatography, two forms of calpastatin (I and II) and two forms of calpain (micro (μ) and milli (m)) were revealed. The proteolytic activity of μ-calpain was detectable with column chromatography, but not consistently detected with casein zymography, and m-calpain was detected with both chromatography and casein zymogram. The proteolytic activity of m-calpain per g muscle was 15 times higher than that of μ-calpain. μ-Calpain had a temperature optimum of 15 °C and a maximum calcium requirement at 0.2 mM, while m-calpain had temperature optimum at 25 °C and a maximum calcium requirement of 0.6 mM. The two forms of calpastatin differed in inhibitory activity with calpastatin II having the highest activity. Both calpastatins tolerated heat treatment, as previously seen for mammals, and they kept their activity when stored at −80 °C, but not at −20 °C. The calpain to calpastatin ratio was 1:4.5 as observed for beef muscle. This study provides evidence that two calpain isoforms, likely to be μ- and m-calpain, in addition to two forms of calpastatin exist in Atlantic salmon muscle.     

Modelling post-mortem tenderisation-IV: Role of calpains and calpastatin in conditioning

A generalised model, based on published data, was developed quantifying the mechanism by which the activities of calpains I and II are responsible for post-mortem tenderisation. Tenderisation is proposed to result from the activities of 'free' activated-calpains, the activities of which are controlled by the changes in the calcium ion concentration, the binding of calpains to calpastatin, the inactivations of 'free' activated-calpains and their proteolysis of calpastatin. At the low myoplasmic ('free') calcium ion concentrations prevailing soon after slaughter, calpains are largely 'inert' and little tenderisation occurs. As the pH declines, the 'free' calcium ion concentration rises and activates calpain I: however, most of this activated-calpain I is then bound to calpastatin. With a futher decline in pH, the binding of activated-calpain I to calpastatin is reduced and the level of 'free' activated-calpain I rises and tenderisation increases. A comparable process occurs with calpain II at higher 'free' calcium ion concentrations which occur as the pH declines further. As the level of 'free' activated-calpains rises, their proteolysis of calpastatin also increases, causing a lowering of levels of calpastatin and reducing the inhibition of calphins. Concurrently, 'free' activated-calpains are inactivated. This balance between inhibition, inactivation and activity of calpains and their decrease as the pH declines maintains the proteolytic activity of calpains and produces the gradual process of tenderisation, initially by calpain I and at the later stages mainly by calpain II. Eventually, the activities of calpains decline to zero and tenderisatin stops. Equations were derived to describe the changes from stunning to the completion of conditioning, and parameters were calculated to determine the activities of calpains and tenderisation in beef M. longissimus dorsi.     

Levels of calpain and calpastatin in meat subjected to high pressure

The levels of μ-, m-calpain and calpastatin were assayed in pressurized rabbit muscle.

The crude calpain level from the pressurized muscle at 100 MPa was almost the same with that of control. Above 100 MPa, the level of calpain decreased rapidly with increased pressure. At 300 MPa, the calpain level was almost inactivated. When the crude extract was pressurized, the calpain level followed the same tendency as that in the pressurized muscle.

When the extracts from control or pressurized muscle were subjected to DEAE-Sephacel column chromatography, μ- and m-calpains and calpastatin lost their activity with increasing pressure, but the degree of loss was different for each.

Calpains resisted changes in pressurization at 200 MPa and were inactivated over 200 MPa. Inactivation of calpastatin at 100 MPa was faster than that of calpains.

From the results, it was concluded that calpain levels remained in muscle pressurized up to 200 MPa, whereas calpastatin levels were decreased by the pressurization. Thus the total activities of calpains in pressurized muscle appear to have been increased by the pressure treatment and this may result in tenderization of meat.     

In vitro study to evaluate the degradation of bovine muscle proteins post-mortem by proteasome and μ-calpain

The degradation of bovine muscle proteins by proteasome and ubiquitous calpains was explored via 2D gel proteome analysis by inhibition of the physiological level of the proteases by specific inhibitors. The inhibition of the proteasome chymotrypsin- and trypsin-like activity results in the lack of degradation of several fragments of structural proteins such as actin, troponin T, myosin light chain and nebulin. In addition the degradation of several sarcoplasmatic proteins was eliminated when proteasome was inhibited. The inhibition of the ubiquitous calpain only resulted in minor changes in the degradation pattern, which might indicate that p94, which is not inhibited by calpastatin, is involved in the degradation post-mortem. The results of the present study indicate a sequential degradation of the structural proteins post-mortem, where calpain initiates the disruption and destabilisation of the myofibrillar structure, and thereby allows the proteasome to act.     

宰后牦牛肉成熟过程中钙激活酶与嫩度指标的相关性分析

以10头甘南牦牛为研究对象,对宰后8d成熟期间肌原纤维小片化指数、剪切力、肌纤维直径、μ-钙蛋白酶(μ-calpain)、m-钙蛋白酶(m-calpain)、钙蛋白酶抑素(calpastatin)的活力进行了测定,同时研究了3种酶活力与肌原纤维小片化指数、剪切力、肌纤维直径3个嫩度指标之间的相关性.结果表明:μ-calpain、m-calpain、calpastatin 3种酶与剪切力值及肌纤维直径均呈正相关;3种酶与肌原纤维小片化指数呈负相关,其中μ-calpain与calpastatin呈显著负相关(P＜0.05).因此,钙激活酶活力的变化可能导致了肌原纤维小片化指数的增加,肌原纤维的弱化和肉的嫩化,μ-calpain可能是牦牛肉嫩化的主要贡献者.     

Changes in calpain and calpastatin activities of osmotically dehydrated bovine muscle during storage after treatment with calcium

Calpain and calpastatin activities were investigated in calcium-treated beef after osmotic dehydration. Dehydrated beef was soaked in 150 mM calcium chloride solution for 3 h, and then stored for 48 h at 3-4 °C. The untreated sample (control) was soaked in deionized water for 3 h instead of calcium chloride solution, after osmotic dehydration. The increase and decrease in the relative activity of crude calpain were observed in the untreated and the calcium-treated meat, respectively, during the storage. When the crude calpains were subjected to DEAE-Sephacel column chromatography, it was found that μ-calpain activity decreased rapidly during the storage in the untreated meat, whereas there was almost no change in the activity of m-calpain during the storage. The decrease of calpastatin activity was moderate compared with the decrease of μ-calpain activity. In the calcium chloride-treated meat, however, no μ-calpain nor calpastatin activities was detectable after 48 h at cold-room temperature, and m-calpain activity after 48 h had decreased to 6.1% of its activity immediately after thawing. It was concluded that 150 mM calcium chloride treatment after osmotic dehydration was sufficient to introduce calcium ions into the meat. In the presence of sufficient calcium, autolysis of calpains and proteolytic degradation of calpastatin, which eventually related to the rate of decrease in calpain and calpastatin activities, clearly seem to be related to a decrease in meat toughness.     

Effect of high pressure on the calpain-calpastatin system in fish muscle

ABSTRACT:  Calpains (calcium-activated neutral proteases) of sea bass ( Dicentrarchus labrax L.) muscle may participate in the degradation of muscle tissue during postmortem storage. These enzymes are regulated by calpastatin, their endogenous specific inhibitor. The objective of this study was to evaluate the changes encountered by the calpain system during the postmortem storage of fish muscle after high-pressure treatment. From 100 MPa, high-pressure treatment of purified calpains results in a loss of their activity as well as in the dissociation of the heterodimeric form. In muscle, the high-pressure processing decreases the initial activity of calpain. This loss in activity may be due to an inactivation by a change of structure. Initial calpastatin activity is not modified by the high-pressure treatment, but it decreases during the storage from the beginning for a treatment at 300 MPa after which calpastatin is stable during 2 d. Therefore, this study also suggests that high-pressure treatment could be a useful way to improve fish flesh quality.     

Calpains and calpastatin distribution in bovine, porcine and ovine skeletal muscles

The concentration of calpain II and calpastatin was determined in various beef, lamb and pork muscles showing very different metabolic and contractile types as assessed by measurement of lactic dehydrogenase (LDH), citrate synthetase (CS) and ATPase activities. The calpain II: calpastatin ratio, which is a good index of the efficiency of this proteolytic system, was also determined.

A species comparison revealed that while calpastatin level was lowest in pork, the ratio of calpain II to calpastatin was highest in this species. For both determinations, lamb was intermediate followed by beef. Conversely, the amount of calpain II was very similar in the three species.

In beef and pork, calpain II content decreased as muscle ATPase and LDH activities rose; and conversely increased with CS activity; whereas in lamb, the amount of this enzyme was highest in red muscles regardless of their speed of contraction. Except for masseter muscle, a comparable distribution was observed for calpastatin in beef and pork muscles. In lamb, the calplastatin concentration was highest in slow-twitch red muscles, intermediate in fast-twitch red muscles and lowest in fast-twitch white muscles. Variability of the calpain II: calpastatin ratio with muscle ATPase, LDH and CS activities appeared to be both muscle and species dependent. As results for masseter muscle are rather unexpected, especially in beef and lamb, this muscle was considered separately.

The present findings are discussed with regard to the conditioning rate of meat from different species and, within one species, from different muscles. It was concluded that the conditioning rate may be correlated positively to calpain II: calpastatin ratio and negatively to calpastatin content. In contrast, no relationship seems to exist between meat ageing rate and calpain concentrations.     

Expression and immunolocalization of the calpain-calpastatin system during parthenogenetic activation and fertilization in the rat egg

Calpastatin is an intrinsic intracellular inhibitor of calpain, a Ca(2+)-dependent thiol protease. The calpain-calpastatin system constitutes one functional proteolytic unit whose presence and function has already been investigated in various cell types, but not in the egg. We have previously shown that calpain is expressed in rat eggs and is activated upon egg activation. The present study was designed to investigate the calpain-calpastatin interplay throughout the process.Western blot analysis revealed two main calpastatin isoforms, the erythrocyte type (77 kDa) and the muscle tissue type (110 kDa). By immunohistochemistry and confocal laser scanning microscopy, we demonstrated that the 110 kDa calpastatin was localized at the membrane area and highly abundant at the meiotic spindle in eggs at the first and second meiotic divisions. The 77 kDa calpastatin isoform appeared to be localized as a cortical sphere of clusters. The 110 kDa calpastatin and beta-tubulin have both been localized to the spindle of metaphase II eggs, both being scattered all through the cytoplasm following spindle disruption by nocodazole treatment, implying a dynamic interaction between calpastatin and microtubule elements. Upon egg activation, membranous calpastatin translocated to the cortex whereas cortical millimolar (m)-calpain shifted towards the membrane. Spindle calpastatin and calpain remained static.We suggest that calpastatin serves as a regulator of m-calpain. The counter translocation of m-calpain and calpastatin could serve as a means of calpain escape from calpastatin inhibition and may reflect a step in the process of calpain activation, throughout egg activation, that is required for calpain to exert its proteolytic activity.     

Protease activity higher in postmortem water buffalo meat than Brahman beef

We previously demonstrated that postmortem water buffalo meat had higher tenderness than Brahman beef. In order to explain this difference in tenderness, the objective of the current study was to investigate the protease activity in these two meats. Five female crossbred water buffalo (Philippine Carabao×Bulgarian Murrah) and five female crossbred cattle (Brahman×Philippine Native) were slaughtered at 30months of age, followed by immediate sampling of Longissimus thoracis muscle for measurement of protease activity. Results showed that buffalo meat had significantly higher protease activity compared to beef (P<0.05). Furthermore, calpain inhibitor 1, a specific inhibitor of calpains 1 and 2, was the most effective inhibitor of protease activity. There was no difference in calpastatin activity, and no major differences were observed in calpains 1, 2, and calpastatin expression by Western blotting. This study suggests that higher calpain activity in early postmortem buffalo meat was responsible for the increased tenderness of water buffalo meat compared to beef.     

畜禽宰后肌肉嫩化相关酶研究进展

目前普遍认为参与宰后肌肉嫩化的相关蛋白酶主要有溶酶体组织蛋白酶、蛋白酶体、钙激活蛋白酶、钙激活酶抑制蛋白和半胱天冬酶5 种,然而对于成熟过程中肉的嫩化程度及机理颇具有争议。大量的研究表明,上述5 种嫩化酶在参与肉嫩化过程中其自身生理生化特性在宰后也发生变化。本文综述5 种参与宰后肉嫩化酶的分子质量、存在部位、作用底物、作用位点、激活条件、最适pH值以及对其活性具有一定影响的抑制剂,并就其影响宰后肉嫩度的作用机理及其自身状态的变化进行分析与阐述,旨在为后续研究嫩化酶在宰后改善肉嫩度方面的应用条件提供参考。     

Effect of extraction buffer on estimating calpain and calpastatin activity in postmortem ovine muscle

The effect of extraction buffer on extractable calpain and calpastatin activity in postmortem muscles was examined. Muscles were removed from ovine carcasses 24 h after slaughter and extracted with three volumes of two extraction buffers containing 20?(pH 7.5) and 100 (pH 8.3) mM Tris. There was a significant difference in pH of the muscle homogenates, having a pH of 5.84 and 7.58 for 20 and 100 mM Tris base, respectively. Calpastatin, μ-calpain and m-calpain all had significantly reduced activity in extracts made with 20 mM Tris (pH 7.5) buffer compared to 100 mM Tris (pH 8.3) buffer, showing a loss of, respectively, 30, 57 and 37%. These results indicate the impact of choice of buffer on the extractable calpains and calpastatin activity from postmortem muscle. To avoid loss of calpains due to isoelectric precipitation, the pH of the muscle homogenate (after homogenization and prior to the first centrifugation) must be above 6.2.     

Hind-limb protein metabolism and calpain system activity influence post-mortem change in meat quality in lamb

This study is concerned with the rate of protein turnover in the hind limb muscle bed of intact lambs, the activity of calpain proteolytic system in the M. biceps femoris, and subsequent rates of myofibre breakdown and tenderisation in the M. longissimus dorsi. Feed restriction increased protein degradation in hind-limb muscle of lambs (p<0.1), with a concominant decrease in the extractable activity of calpastatin (p<0.01), the endogenous inhibitor of calpain. IGF-1 analog treatment decreased both protein degradation and assayed μ-calpain activity (p<0.05) with no effect on the activity of calpastatin. β-Agonist treatment increased hind-limb protein synthesis (p<0.01), calpastatin activity (p<0.1) and decreased (p<0.01) μ-calpain activity, but did not effect protein degradation. Significant correlations were observed between Myofibril Fragmentation Index (MFI) values during post-mortem storage and initial post-slaughter calpastatin activity at days 3 (r=-0.34, p<0.1), 5 (r=-0.58, p<0.01) and 9 (r=-0.58, p<0.1), and μ-calpain activity at days 5 (r=0.35, p<0.1) and 9 (r=0.41, p<0.05). However, stronger correlations were observed between the ratio of μ-calpain to calpastatin, an estimate of potential μ-calpain proteolytic activity, and the rate of myofibril fragmentation (r=0.75, p<0.001) and tenderisation (r=-0.64, p<0.01) during aging. These results are consistent with the calpain system being the major proteolytic system involved in myofibril fragmentation and hence aging-related tenderisation of meat.     

Contribution of postmortem muscle biochemistry to the delivery of consistent meat quality with particular focus on the calpain system

Tenderness has been repeatedly reported as the most important quality aspect of meat. However, a number of studies have shown that a significant portion of retail meat can be considered tough. As a consequence, a significant consumer segment is willing to pay a premium for guaranteed tender meat. However, apart from measuring the shear force, there is no reliable method to predict tenderness. Most of the branded meat programs therefore attempt to ensure eating quality by controlling some of the factors that affect tenderness. Meat tenderness is determined by the amount and solubility of connective tissue, sarcomere shortening during rigor development, and postmortem proteolysis of myofibrillar and myofibrillar-associated proteins. Given the effect of postmortem proteolysis on the muscle ultrastructure, titin and desmin are likely key substrates that determine meat tenderness. A large number of studies have shown that the calpain proteolytic system plays a central role in postmortem proteolysis and tenderization. In skeletal muscle, the calpain system consists of at least three proteases, μ-calpain, m-calpain and calpain 3, and an inhibitor of μ- and m-calpain, calpastatin. When activated by calcium, the calpains not only degrade subtrates, but also autolyze, leading to loss of activity. m-Calpain does not autolyze in postmortem muscle and is therefore not involved in postmortem tenderization. Results from a number of studies, including a study on calpain 3 knockout mice, have shown that calpain 3 is also not involved in postmortem proteolysis. However, a large number of studies, including a study on μ-calpain knockout mice, have shown that μ-calpain is largely, if not solely, responsible for postmortem tenderization. Research efforts in this area should, therefore, focus on elucidation of regulation of μ-calpain activity in postmortem muscle. Discovering the mechanisms of μ-calpain activity regulation and methods to promote μ-calpain activity should have a dramatic effect on the ability of researchers to develop reliable methods to predict meat tenderness and on the meat industry to produce a consistently tender product.     

Postmortem changes in myofibrillar-bound calpain 3 revealed by immunofluorescence microscopy

An immunofluorescence microscopy method for following changes in myofibrillar-bound calpain 3 was developed. Afterward, proteolytic changes in calpain 3(p94), calpain 1, titin, and nebulin were examined in myofibrils prepared from ovine longissimusthoracis et lumborum (LTL) stored for 0, 1, 2, and 3 days postmortem. Western blot analysis revealed that the levels of intact calpain 3 (expressed as percentage of the level immediately postmortem) were 80%, 10% and not detectable in myofibrils prepared at 1, 2, and 3 days, respectively. Western blots for calpain 1 also indicated conversion of the intact protein (80 kDa) to a 76 kDa fragment during the same time period. Thus calpains 1 and 3 appear to be activated during postmortem storage. Immunofluorescence microscopy using an IS1 region specific antibody revealed that calpain 3 staining was most intense at the sarcomere Z- and M-lines. The fluorescence intensity declined significantly during storage, paralleling changes in the proteolytic breakdown of titin and nebulin associated with these structures.     

Early post-mortem conditions and the calpain/calpastatin system in relation to tenderness of double-muscled beef

Early post mortem temperature, pH, sarcomere length, colour, water holding capacity, calpain/calpastatin activities and myofibrillar protein concentrations (semi-quantative SDS-PAGE), measured at different times post mortem on 153 double-muscled Belgian Blue White bulls, were related to shear force (SF) measurements. The M. longissimus thoracis tenderised up to 8 days post mortem. SF was slightly correlated with temperature at 3 h post mortem (r = 0.20, p = 0.015), but not with pH early post mortem. Higher ultimate pH values measured at 24 h post mortem were related to darker meat, lower cooking losses and shorter sarcomere lengths. Sarcomere length was significantly related to SF, even after 12 days of ageing, suggesting that proteolytic activity was not able to overcome shortening. Up to 12 days post mortem, calpastatin and m-calpain activities were significantly correlated with SF suggesting a considerable role of calpains in meat tenderization of double-muscled Belgian Blue White bulls, although at 24 h post mortem, μ-calpain could no longer be detected. Titin, nebulin, filamin and troponin-T were degraded during the ageing period. Troponin-T was degraded by 80% between day 1 and 8. At 8 days post mortem its concentration was significantly correlated with 30 kDa (r = 0.63, p = 0.00), shear force (r = 0.39, p = 0.00), calpastatin activity at 1 day post mortem (r = 0.29, p = 0.01) and with the m-calpain/ calpastatin activity ratio at 1 day post mortem (r = -0.43, p = 0.00).     

钙蛋白酶系统与肌肉嫩度的关系

钙蛋白酶系统主要由钙蛋白酶(μ-calpain,m-calpain)及钙蛋白酶抑制蛋白(calpastatin)组成,calpain是存在于细胞质中的依赖于Ca2+的中性蛋白酶,calpastatin是钙蛋白酶的内源抑制蛋白。本文综述了钙蛋白酶系统各种酶的结构、作用、活性调节机能及其与肉质嫩度的关系。     

EFFECT OF FROZEN TEMPERATURE AND STORAGE TIME ON CALPAINS, CATHEPSINS (B, B + L, H AND D) AND THEIR ENDOGENOUS INHIBITORS IN GOAT MUSCLES

The effects of frozen storage on the biochemical properties of myofibrils, muscle proteinases (cathepsins and calpains) and their endogenous inhibitors were investigated. Longissimus dorsi, biceps femoris, semimembranosus and semitendinosus muscles from goat were frozen (?15C) and studied up to 120 days. The results showed that the percentage change in sarcomere length was 8.4–13.1. The calpain activity was determined after separation on a diethylaminoethyl–Sephacel column (Sigma, St. Louis, MO). Significantly greater percentage of calpain II activity was recovered when compared to calpain I. There was a 15–25% loss in calpastatin inhibitory activity, and the cystatin level fell by 11–16% after 80 days. Cathepsin B, B + L, H and D were very stable when compared to calpains. The calcium concentration may also be the factor for calpain activation. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis result showed the appearance of 55 kDa components. It was concluded that calpains, not cathepsins, play an important role in the proteolysis of myofibrillar proteins at the freezing temperature.     

钙激活酶-钙激活酶抑制蛋白系统对成肌细胞融合与分化的影响

钙激活酶－钙激活酶抑制蛋白系统在肌肉发育中起着重要的作用。在成肌细胞培养中,成肌细胞融合并分化形成多核肌细胞,其中钙激活酶抑制蛋白的含量与细胞膜的融合相关。成肌细胞融合需要钙激活酶的活性,融合率取决于细胞中钙激活酶与钙激活酶抑制蛋白的比例。本研究发现ＥＤＴＡ、３ＧＴＡ、ＧＳＴ－ｃａｌｐａｓｔａｔｉｎ可抑制成肌细胞的融合。ＧＳＴ－ｃａｌｐａｓｔａｔｉｎ是用ＩＰＴＧ诱导表达的分子量为４５ＫＤ的融合蛋白     

Low-voltage Electrical Stimulation Effects on Proteolysis and Lamb Tenderness

Lamb carcasses were subjected to low‐voltage electrical stimulation (ES; 28 V, 60 Hz) within 5 min postmortem (PM). After ES, the longissimus thoracis (LT) and semimembranosus (SM) muscles were removed to investigate subsequent biochemical changes. The pH values of the ES muscles declined rapidly until the 1^st 3 h PM (p < 0.05). The activities of μ‐calpain and calpastatin in ES muscles decreased significantly after 10 h PM (p < 0.05). Both ES LT and SM muscles tended to have lower shear‐force values, which suggested ES might accelerate activation of calpains as well as glycolytic rate, thereby improving muscle tenderness. Differences in thin and troponin‐T degradation between treatments were also observed with time PM.