Preliminary study on the effect of caspase-6 and calpain inhibitors on postmortem proteolysis of myofibrillar proteins in chicken breast muscle

The objective was to determine the effect of three different protease inhibitors, caspase-6 specific inhibitor VEID-CHO (N-Acetyl-Val-Glu-Ile-Asp-al), calpain inhibitor leupeptin or calpain inhibitor EGTA on protein degradation, ultrastructure of myofibrils and calpain activity during postmortem (PM) aging of chicken muscle. Results showed that proteolysis of nebulin, troponin-T and desmin during 14-days postmortem storage were inhibited significantly by leupeptin. Inhibitive effects of VEID-CHO and EGTA on these protein degradations were significant only during 1-day postmortem storage. The activities of calpains were inhibited noticeably by leupeptin and EGTA, but not by VEID-CHO. Samples treated with VEID-CHO, leupeptin and EGTA retarded structural disruption of chicken muscle fibers. These results demonstrate that calpain is a major contributor to PM tenderization; while caspase-6 plays, if any, a minimal role in the conversion of chicken muscle to meat.     

Post mortem changes in porcine longissimus muscle incubated with 0.1 M calcium lactate

The effects of calcium lactate incubation at 5 °C on post mortem changes in porcine longissimus muscle were studied. Myofibrils were purified from control (CON) and calcium lactate‐incubated (CL) muscles. Samples were taken at 0, 1, 3, 7 and 14 days post mortem. SDS‐PAGE results showed that the disappearance of titin, nebulin, tropomyosin and troponin‐T and the appearance of a ～30 kDa component were more pronounced in CL samples than in CON samples. Western blots labelled with a monoclonal antibody to desmin also demonstrated that desmin degraded more quickly in CL samples. Our data suggested that calcium lactate incubation might accelerate the post mortem changes in porcine longissimus muscle via activation of calpains. © 2001 Society of Chemical Industry     

Combination of low voltage electrical stimulation and early postmortem temperature conditioning on degradation of myofibrillar proteins in Korean native cattle (Hanwoo)

The combination effect of low voltage electrical stimulation (LVES) and early postmortem (PM) temperature conditioning (2, 16, and 30°C until 3 h PM) on degradation of myofibrillar proteins were determined from Korean native cattle (Hanwoo). Myofibrils were removed at 1, 2, 3, 7, and 14 days of PM storage (2°C) and analyzed for titin, nebulin, desmin, and troponin-T by SDS-PAGE and by Western blot analysis. Degradation rate of myofibrillar proteins was affected by the combination of LVES and temperature conditioning. LVES-30°C treatment resulted in faster degradation of titin, nebulin, desmin, and troponin-T during PM storage than the other treatments. Degradation of titin took place more slowly than nebulin, desmin or troponin-T.     

Quantitative determination of titin and nebulin in poultry meat by SDS-PAGE with an internal standard

The method of quantitative determination of titin and nebulin in chicken meat by SDS-PAGE electrophoresis technique was developed by application of β-galactosidase as the internal standard. The method was tested first on marker protein samples of known concentrations (myosin, transferrin, glutamic dehydrogenase) and then the method was used in the determination of titin and nebulin content in chicken meat. The method demonstrated high accuracy, confirmed by correlation coefficient 0.91÷0.99. Two gel analysis techniques, i.e. scanning and densitometry were also compared. By the use of marker proteins as well as titin and nebulin, higher accuracy and precision were achieved in scanning than in the densitometric technique. Recoveries of three marker proteins were between 93 and 102% for the technique of the scanning of the gel and between 99 and 116% for the densitometry.     

Effect of Postmortem Storage on Titin and Nebulin in Pork and Poultry Light and Dark Muscles

Purified myofibrils were prepared from samples of chicken breast and thigh muscles and from light and dark portions of pork semitendinosus muscle at death and after storage at 4° and 22°C for 1, 3, and 7 days postmortem. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine the effect of postmortem storage of muscle on titin and nebulin. Results indicated that titin and nebulin were more rapidly degraded in light than in dark chicken muscle. In contrast, titin and nebulin were more rapidly degraded in dark than in light pork muscle.     

Postmortem Degradation of Titin and Nebulin of Beef Steaks Varying in Tenderness

Purified myofibrils were isolated from “tender” and “less-tender” bovine longissimus muscle at death and at 1, 3, 7, and 14 days of postmortem storage (4^oC). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect changes in the myofibrillar/cytoskeletal proteins, titin and nebulin. Titin and nebulin bands were observed to be less intense on gels from “tender” than from “less-tender” steaks. These results suggest that titin and nebulin were more rapidly degraded in “tender” than in “less-tender” steaks, and that the extent of beef loin steak tenderness may be dependent upon the postmortem degradation of titin and nebulin.     

Up- and down-regulation of longissimus tenderness parallels changes in the myofibril-bound calpain 3 protein

The objective of this study was to utilize Ca(2+) and Zn(2+) treatments of meat to critically explore the possible role of calpain 3 in meat tenderisation. Calpains 1 and 2 were also examined for comparative purpose. Control animals plus animals infused with CaCl(2), ZnCl(2) or H(2)O were used (six lambs per treatment) to determine the temporal changes in muscle calpain 3 protein in the Longissimus thoracis et lumborum (LTL) during post-mortem storage. Concurrently, the temporal changes of; (1) shear force, (2) sarcomere length, (3) proteolysis of titin and nebulin and (4) calpains 1 and 2 proteins were also determined. Infusing LTL with Ca(2+) or Zn(2+) caused significant up- and down-regulation of LTL tenderisation, respectively, compared to water infusion and the control animals. Furthermore, the rate of breakdown of calpain 3, the rate of proteolysis of titin and nebulin and the rate of meat tenderisation during post-mortem storage of LTL in the various treatments were highly correlated. These studies suggest that calpain 3, like calpain 1, may be involved in the tenderisation of meat through limited proteolysis of specific muscle structural proteins such as titin and nebulin.     

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.     

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.     

EFFECTS OF MUSCLE PROTEASES, ENDOGENOUS PROTEASE INHIBITORS AND MYOFIBRIL FRAGMENTATION ON POSTMORTEM AGING OF GOAT MEAT

The present study was conducted to evaluate the extent of postmortem proteolysis in longissimus dorsi, biceps femoris, semimembranosus and semitendinosus goat muscles on postmortem aging at an ambient (27C) temperature. The activities of calpains and calpastatin were determined after separation on a (diethylamino)ethyl–Sephacel column (Sigma, St. Louis, MO) and cathepsin (B, B + L and H) by carboxymethyl–Sepharose column (Sigma). The results showed that the decrease in calpain I and calpastatin activities was significantly higher than that of calpain II. Cathepsin B, B + L, H and cystatin were found to fall by 30–80% after 12 h, whereas cathepsin D decreased significantly in all the muscles. The disappearance of titin 1 and nebulin, and the appearance of a 30‐kDa component were confirmed by Western blot analysis. The appearance of the 30‐kDa component reported here explains the time‐induced structural changes of myofibrils. The Z‐line degradation had occurred by 6 h postmortem. Cathepsins are not stable compared to calpains during postmortem aging, and both enzymes may play a significant role in the proteolysis of myofibrillar proteins at ambient temperature.     

极限pH对羊肉宰后成熟过程中肌原纤维蛋白特型的影响

为了研究羊肉宰后成熟过程中极限pH对肌原纤维蛋白特型即肌联蛋白、伴肌动蛋白、肌间线蛋白和肌钙蛋白-T降解及肌原纤维小片化指数的影响。本文选取50只羊的右侧背最长肌,贮存于4 ℃条件下,在宰后时间点分别为1 h、1、2、3、5 d和7 d时,测定其pH。按照宰后2 d的pH将肉样分成三组:高极限pH组(5.72±0.03),中极限pH组(5.54±0.01)和低极限pH组(5.40±0.02)。在每个宰后时间点,测定肌联蛋白、伴肌动蛋白、肌间线蛋白、肌钙蛋白-T降解程度和肌原纤维小片化指数(MFI)。结果表明:肌联蛋白在高极限pH组中宰后1 d开始降解;在宰后1 d时,高极限pH组肌间线蛋白相对灰度值显著低于中极限pH组和低极限pH组(p<0.05);肌钙蛋白-T在高极限pH组中,宰后1 d已出现降解条带。而伴肌动蛋白在中极限pH组中降解较快,在宰后1 d开始降解。另外在宰后1、2、3、5、7 d时,高极限pH组和中极限pH组的肌原纤维小片化指数显著高于低极限pH组的肌原纤维小片化指数(p<0.05)。极限pH通过影响这些肌原纤维蛋白降解来促进宰后肌肉成熟过程并且肌联蛋白、肌间线蛋白和肌钙蛋白-T的降解,加快了宰后前期嫩化过程。这为揭示宰后肉嫩度形成机理提供理论基础。     

Changes in Titin and Nebulin in Postmortem Bovine Muscle Revealed by Gel Electrophoresis, Western Blotting and Immunofluorescence Microscopy

Postmortem storage of bovine psoas major muscle resulted in almost complete degradation of nebulin by 48 hr; however, some intact titin was still observed after 2 wk storage at 4°C. The percentage of myofibrils having four anti-titin bands per sarcomere (immunofluorescence microscopy) was less than 1% at 45 min but increased to 65% at 48 hr after death and remained stable thereafter. A similar four band pattern was seen in frozen sections of whole muscle at 48 hr or more postmortem. The time course of nebulin degradation appeared to correlate with the titin two to four band transition.     

SDS-PAGE Conditions for Detection of Titin and Nebulin in Tender and Tough Bovine Muscles

Purified myofibrils were prepared from infraspinatus (tender) and rhomboideus (tough) muscles at 7 days postmortem and examined for myofibrillar/cytoskeleta1 protein degradation by using sodium dodecyl sulfate polyactylamide gel electrophoresis (SDS-PAGE). Four acrylamide/bisacrylamide ratios (37:1, 50:1, 75:l and 100:1) and two SDS-PAGE gel buffers (Tris-HCl, pH 8.0 and 8.9) were used to determine the optimum conditions for detection of titin and nebulin. Titin was degraded to a greater extent in myofibrils from the infraspinatus than in myofibrils from the rhomboideus. Very little nebulin was detected in either muscle. Use of acrylamide/bisacrylamide ratio of 37:1 and a gel buffer of pH 8.0 provided the most optimum conditions for detecting differences in the resolution of titin, nebulin and their apparent degradation products.     

The influence of calcium on the chromogenic Limulus Amoebocyte Lysate assay of lipopolysaccharide endotoxins in liquid milk

Calcium ions were shown to have a profound effect on the chromogenic Limulus Amoebocyte Lysate assay of lipopolysaccharides in liquid milk. There appeared to be two optima within the range of 0–1.0 mM added calcium chloride, with an inhibition of the assay at calcium ion concentrations between 0.2 and 0.4 mM, and above 0.6 mM. With products of this nature, standardization of calcium ion content is required for meaningful comparisons of results between samples. Further investigation is required to assess the possible influence of other serum constituents.     

Functions of Added Calcium in Acid Milk Coagulation

Changes in calcium solubilization and rennet reaction rate were investigated after the addition of calcium (6.25 mM) to reconstituted milk. Analysis of soluble and ionic calcium showed that distribution of added calcium was very fast (60 and 25 min, respectively), and that micelles of enriched milk remained more mineralized at pH 6.70 to 5.00. In the presence of sufficient amounts of rennet and hydrogen ions, K-CSSeh hydrolysis and aggregation rates were increased by addition of calcium. The calcium supplement led to extended maintenance of the micellar structure and to a more easily drained curd. Thus, it could be used advantageously for lactic acid cheese making.     

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.     

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.     

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.     

Calcium-Induced Destabilization of Oil-in-Water Emulsions Stabilized by Caseinate or by β-Lactoglobulin

The stability to aggregation of 20% soya oil-in-water emulsions stabilized by 0.3 to 2% sodium caseinate or β-lactoglobulin in the presence of calcium chloride solutions was studied using light scattering and electron microscopy. Stability increased with the amount of protein in the emulsion, and decreased with the concentration of added calcium. Growth of particle size with concentration of Ca²⁺ was more in emulsions containing lower concentrations of protein. Sodium chloride at 50 and 100 mM stabilized both systems to the presence of calcium ions. Microstructure and light scattering showed caseinate emulsions formed clusters even at low concentrations of Ca²⁺ while β-lactoglobulin emulsions formed extensive strands.     

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.