Effect of proteasome inhibitor on sarcoplasmic protein of bovine skeletal muscle during storage

Bovine skeletal muscle, immediately after slaughter (1.5 h; 0 d), was buffered, homogenized treated with proteasome inhibitor, and stored (2 d) for SDS-PAGE and Western blotting analysis. Ubiquitin antiserum (Sigma, St Louis) reacted with bands corresponding to purified ubiquitin and small amounts of other higher-molecular-mass proteins (about 30 and 40 kDa) which were considered to be ubiquitin-protein conjugates. These bands were faint in the control 2 d sample, suggesting that they had degraded. However, these tendencies of the ubiquitin positive bands to decrease were not clearly observed in the sample treated with proteasome inhibitors (MG132 and Lactacystin). These results suggest that both ubiquitin and the ubiquitin-protein conjugates were present in the skeletal muscle immediately after slaughter and they were then degraded during storage. This degradation was partially due to the action of proteasome.     

Purification and properties of rabbit muscle proteasome, and its effect on myofibrillar structure

This paper describes the purification and properties of a multicatalytic proteinase complex, proteasome, from rabbit skeletal muscle, and its effect on myofibrillar structure. The purified proteasome gave a single band on polyacrylamide gel electrophoresis under non-denaturing conditions and gave eight bands under denaturing conditions, indicating that this enzyme comprises multiple hetero-subunits with low molecular mass. The purified proteasome was not activated by ATP and ubiquitin, and was markedly inhibited by Z-Leu-Leu-Leu-H (aldehyde). These data indicate that the purified proteasome is not 26S, but 20S. The proteasome degraded synthetic peptides maximally at pH 8.0. Relative to pH 8.0, activities were gradually decreased with the lowering of pH, but the degree of decrease was substrate-dependent, and the activity at pH 5.0 still retained about 30˜60% of the activity at pH 8.0, indicating the possibility that the proteasome is active in the muscle during conditioning. When the proteasome was heated at 60 °C for 20 min and treated in the presence of 0.005% SDS, the activity increased over 1.5 and 4.5 times, respectively. SDS remarkably increased the V_max value of the enzyme at pH 8.0. The proteasome was also activated by high hydrostatic pressure up to 100˜150 M Pa and gradually decreased at 200 MPa or higher. Electron microscopic observation revealed that obvious gaps between filamentous structure, the complete loss of M-line and partial loss of Z-line structure were caused by proteasome.     

The action of a muscle proteinase on the myofibrillar proteins of bovine muscle

Myofibrillar proteins from bovine muscle have been treated with a Ca²⁺ activated muscle proteinase and the consequent changes in these proteins have been examined by various techniques. Tropomyosin, α-actinin and troponin were substrates for the enzyme, the last losing its property of inhibiting actomyosin ATPase in the absence of Ca²⁺ ions. Actin and actomyosin were apparently not digested but the Mg²⁺-activated ATPase activity of actomyosin was less after treatment whereas the Ca²⁺-activated ATPase was unaffected. It is suggested that the observed destruction of the Z-bands of the myofibrils by this proteinase is due to its digestion of the α-actinin, rather than the actin component.     

The effect of conditioning on the myofibrillar proteins of pork muscle

Storage of pork muscle caused changes in the troponin complex of myofibrillar proteins. The changes were temperature dependent and progressive as conditioning proceeded. An alteration in actin also occurred but this became apparent only when myofibrils had been extracted with 5 mM Tris pH 8.2. About 60% of the proteins in an extract of myofibrils in 5 mM Tris pH 8.2 were bound to the precipitate formed with added F-actin. After conditioning of the pork muscle, the amount of proteins in a Tris extract which were bound to added F-actin was considerably reduced. Some of these changes were also observed in myofibrils which had been treated with a Ca²⁺ activated proteinase.     

Chemical oxidation decreases proteolytic susceptibility of skeletal muscle myofibrillar proteins

The objective of this study was to investigate the effect of chemical oxidation on proteolysis susceptibility of myofibrillar proteins. Myofibrils were prepared from pig M. longissimus dorsi and oxidised by a hydroxyl radical generating system. Protein oxidation level was measured by the carbonyl content, free thiol group content and bityrosine formation. Oxidised or non-oxidised myofibrillar proteins were exposed to papain and proteolysis was estimated by fluorescence using fluorescamine. Oxidation of myofibrillar proteins was dependent upon the oxidising agent concentration. Disulfide bridge and bityrosine formation indicated that oxidation by OH° can induce protein polymerization. Electrophoretic study showed that myosin was the protein most sensitive to oxidation. Results showed a direct and quantitative relationship between protein damages by hydroxyl radical and decreased proteolytic susceptibility. Electrophoretic observations suggest that polymerization and aggregation may explain in part decreased susceptibility of myofibrillar proteins to proteolysis.     

Changes in proteasome activity during postmortem aging of bovine muscle

The effects of adding commercial-grade and eggshell calcium lactate on the microbiological and physicochemical properties of Nhams (Thai-style fermented pork sausage) were studied. The Nham calcium levels were 150, 300 and 450 mg/100 g. Compared to controls (no added calcium), calcium fortification did not affect the number of lactic acid bacteria or the colour value. The shear force of Nhams fortified with eggshell calcium lactate decreased (P<0.05) from 32.2 N in the controls to 19.5-22.8 N in Nhams fortified with eggshell calcium lactate. However, Nhams fortified with commercial calcium lactate had the same shear force as the controls. Sensory scores of sour taste, flavour and overall acceptance were not different between the control and calcium-fortified Nhams at a calcium level of 150 mg/100 g.     

The effect of acidification on myofibrillar proteins

Isolated myofibrillar proteins of mutton, beef and chicken were treated with an acidulent to give various pH values and stored at 5°C for 20 h before analysing the proteins using sodium dodecylsulphate polyacrylamide gel electrophoresis. It was found that protein degradation occurred below pH 4·5, with a decrease in band intensity of all major myofibrillar proteins, particularly myosin heavy chain, and the appearance of new bands at approximately 140 and 70 kd. The degradation had an optimum around pH 3·0 and was inhibited by a high temperature pre-treatment or the presence of the endopeptidase inhibitors pepstatin A and leupeptin. Results are discussed in terms of the action of acid proteinase enzymes.     

Differentially expressed proteins during fat accumulation in bovine skeletal muscle

The objective of this study was to identify the proteins involved in bovine intramuscular fat (IMF) development. Global proteins were monitored in bovine skeletal muscle at muscle-developing versus IMF-increasing stages and with higher versus lower IMF scores, respectively. We identified two differentially expressed (two-fold or more) proteins at the IMF-increasing stage, up-regulated heat shock protein beta 1 (HSPB1) and down-regulated ATP synthase D chain (ATP5H), and two down-regulated proteins with higher IMF scores, carbonic anhydrase 2 (CA2) and myosin light chain 3 (MYL3). In vitro, after adipogenic differentiation, the mRNA expression of HSPB1 and ATP5H did not be changed, but that of CA2 and MYL3 decreased significantly (P < 0.05). After myogenic differentiation, the mRNA expression of HSPB1 increased significantly (P < 0.05), but expression of other genes did not vary. We suggested that CA2 and MYL3, which expressed down during adipogenic differentiation, could be indicative markers for negative regulation of IMF development.     

Effect of frozen storage on the structure and enzymatic activities of myofibrillar proteins of rabbit skeletal muscle

The effect of frozen storage on the biochemical properties of myofibrils, and of their major constituents, actin and myosin, was investigated. Extractability of myofibrillar proteins increased slightly for 3 weeks during frozen storage of muscle, decreasing thereafter. The change in myofibrillar ATPase activity during frozen storage was consistent with that of a reconstituted acto-heavy meromyosin (HMM) complex prepared from frozen stored muscle at the same weight ratio of actin to myosin as in situ. However, myosin ATPase activity showed a different pattern of change when compared with myofibrillar ATPase activity. The maximum velocity of acto-HMM ATPase activity and the apparent dissociation constant of the acto-HMM complex decreased for 1 week during frozen storage, increasing thereafter, indicating that the affinity of actin for myosin was greatest in muscle which had been frozen for 1 week.     

Differences in properties of myofibrillar proteins from bovine semitendinosus muscle after hydrostatic pressure or heat treatment

Hydrostatic pressure (HP) and heat treatments of myofibrillar proteins have both been shown to induce protein denaturation, but different gel formation properties result from these treatments. To characterise differences in the properties of proteins resulting from HP or heat treatment, Ca‐ and Mg‐ATPase activities (ATP, adenosine triphosphate) and protein solubility in 0.1 and 0.6 mol L^?1 KCl buffers (pH 7) were evaluated in this study. The inactivation rate of Ca‐ATPase of myofibrillar proteins (Mf) induced by HP was slower than that of Mg‐ATPase at each of the tested pressures. However, the inactivation rate of Ca‐ATPase induced by heating was faster than that of Mg‐ATPase at each of the tested temperatures. The level of soluble proteins in Mf suspension induced by HP in 0.1 mol L^?1 KCl buffer increased with increasing pressure up to 400 MPa and then decreased slightly at 500 MPa. However, the level of soluble proteins in Mf suspension induced by heat treatment in 0.1 mol L^?1 KCl buffer increased with increasing temperature up to 55°C. According to the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis, the levels of soluble myosin heavy chain and actin in Mf suspension induced by HP in 0.6 mol L^?1 KCl buffer decreased simultaneously at pressures higher than 300 MPa. The level of soluble MHC in 0.6 mol L^?1 KCl buffer decreased gradually with increasing temperature, but there were no changes in the level of soluble actin in 0.6 mol L^?1 KCl buffer with increasing temperature up to 50°C. These results showed that the mechanism of HP‐induced protein denaturation was different from the mechanism underlying heat‐induced protein denaturation. Copyright © 2006 Society of Chemical Industry     

The effect of post-mortem conditions on the extractability and adenosine triphosphatase activity of myofibrillar proteins of rabbit muscle

Summary. 1. Washed myofibrils from rabbit muscle have been heated at pH values between 4.8 and 5.6 and temperatures between 35°C and 42°C. It has been found that, under these conditions, myofibrils lose their Ca²⁺ activated adenosine triphosphatase, their Mg²⁺ activated adenosine triphosphatase and also become less extractable in M KCl–30 mM sodium glycerophosphate, pH 6.2.
2. The reactions follow first-order kinetics and the rates are dependent on pH and temperature. The first order rate constants, enthalpies and entropies for the three reactions are sufficiently near each other to suggest that all three reactions are occurring simultaneously.
3. When a muscle is allowed to go into rigor at 37°C the extractability in M KCl–30 mM sodium glycerophosphate is reduced after 4 hr at 37°C when the pH of the muscle has reached 5.55. At the same time the Ca²⁺ adenosine triphosphatase activity falls but the Mg²⁺ adenosine triphosphatase does not. The latter is reduced by prolonging the period at 37°C to 6 hr.
4. It is suggested that there is present in muscle, undergoing rigor at 37°C, myosin which does not bind to actin and is readily denatured. When bound to actin, myosin in the myofibril is more resistant and denatures only after long exposure to a temperature of 37°C.     

The effect of myofibrillar/sarcoplasmic protein ratios on the properties of round scad muscle protein based film

The effect of the ratios of myofibrillar protein (MP) to sarcoplasmic protein (SP) from round scad (Decapterus maruadsi) muscle on the properties of the resulting films was investigated. Tensile strength (TS) of films decreased with increasing SP content (p < 0.05). Films prepared from MP/SP ratio of 10:0 (w/w) exhibited the highest TS (p < 0.05). Elongation at break (EAB) of films prepared with SP content greater than 30% had the decreased EAB (p < 0.05). Water vapor permeability (WVP) of films increased when SP content increased up to 20% and decreased with increasing SP content up to 30% (p < 0.05). Solubility of films decreased but protein solubility increased with increasing SP contents (p < 0.05). The a*-value and ΔE* of film increased with increasing SP content. Films with all MP/SP ratios exhibited the negligible transmission to the light in UV range. Therefore, it is suggested that the type and ratio of proteins in fish muscle, both SP and MP, influenced the properties of film from round scad muscle. Results suggested that the removal of sarcoplasmic protein from fish muscle by thorough washing was an effective means to improve the mechanical properties as well as color of the fish muscle protein-based film.     

高压处理对牛骨骼肌原肌球蛋白结构的影响

从牛骨骼肌中提取原肌球蛋白,施行0.1～400MPa的高压处理,解压后测定原肌球蛋白荧光光谱、光谱质量中心、芳香族表面疏水性、脂肪族表面疏水性、表面巯基含量的变化来探讨压力对原肌球蛋白结构的变化。荧光光谱强度及光谱质量中心与对照组(0.1MPa)相比,随着压力的增加有逐渐下降趋势,最大荧光波长发生蓝移。100MPa时芳香族、脂肪族表面疏水基团显示出最低荧光强度值,压力继续升高时又逐渐上升。表面巯基含量在100MPa时大幅增长,随着压力的增加有缓慢的增长趋势。从此结果可推测压力引起原肌球蛋白的结构变化,而这些变化可能是一定程度的可逆性变化。     

The effect of sex condition and growth implants on bovine muscle fiber characteristics

Steers, bulls and bulls implanted with Synovex-S or Ralgro were slaughtered at 12 and 16 months of age. With increasing animal age, the percentage of intermediate fibers decreased while the percentage of white fibers increased. The area of all fibers increased with increasing age. Bulls had a higher percentage of red fibers, larger red and intermediate fibers and a higher percentage area of red and intermediate fibers compared with steers. Implanted bulls were generally intermediate to intact bulls and steers in muscle fiber type characteristics. Carcass fat measurements and tenderness ratings were positively correlated to the percentage of white muscle fibers and negatively correlated to the percentage of intermediate fibers. Carcass characteristics and sensory properties of meat are discussed in relation to muscle fiber characteristics.     

Purification and characterisation of an alanine aminopeptidase from bovine skeletal muscle

A monomeric alanine aminopeptidase was purified to a single band in SDS–PAGE from bovine skeletal muscle by using a procedure including ammonium sulphate fractionation, adsorption on DEAE–cellulose, gel filtration on Ultrogel ACA 34, and adsorption on hydroxyapatite. Molecular weight determination by gel filtration and sodium dodecyl sulphate–polyacrylamide gel electrophoresis yielded a molecular size of 60 kDa. The aminopeptidase activity was optimal at pH 8.0 and 37 °C. It was totally abolished by Co²⁺ and Zn²⁺ ions, and almost completely inhibited by bestatin and Mn²⁺. The activity was strongly inactivated by phenylmethansulfonyl fluoride, Mg²⁺, and Fe³⁺ ions but stimulated by pepstatin and EDTA. However the activity was not affected by Ca²⁺, puromycin and iodoacetate. When compared with its activity toward Ala-β-naphthylamides (Ala-βNA), the enzyme exhibited 15–17% as much activity toward Pro- and Leu-βNA, 4–6% activity toward Met- and Arg-βNA, and negligible or no activity toward Glu- and Ser-βNA.     

牛血与肌原纤维蛋白混合物的凝胶特性

将不同量的牛血添加到肌原纤维蛋白溶液中,通过测定混合物的乳化能力、凝胶强度和保水性,研究不同血液添加量对牛血-肌原纤维蛋白混合物凝胶特性和乳化性的影响。结果表明:添加血液后,混合物凝胶强度与对照组相比显著提高(P0.05),在V(牛血)∶V(肌原纤维蛋白溶液)=1∶9~4∶6时,随着血液添加比例的增加混合物凝胶强度逐渐增加,但比例4∶6之后混合物凝胶强度增加不显著(P0.05);混合物凝胶的保水性随着血液添加量的增大出现先减小后增大的趋势;添加血液后,混合液与对照组相比,乳化能力得到显著提高(P0.05)且乳化活性指数(EAI)在比例为4∶6时达到最大值。     

Role of the ubiquitin‐proteasome pathway on proteolytic activity in postmortem proteolysis and tenderisation of sheep skeletal muscle

The objective of this study was to investigate the effects of ubiquitin‐proteasome pathway on meat tenderisation. The sheep muscle longissimus lumborum was injected with or without PYR‐41 (inhibitor of ubiquitination) or MG‐132 (inhibitor of proteasome). Muscle samples were collected at 6, 15, 24 and 48 h after injection. Myofibrillar protein degradation, muscle ultrastructure and sarcomere length were determined. Results showed that inhibition of proteasome or ubiquitination affected sarcomere length at 48 h after treatments. Destruction of muscle ultrastructure in both treatments was reduced when compared to control. Inhibition of proteasome produced different fragments of myofibrillar proteins in comparison with control at 48 h. In conclusion, ubiquitin‐proteasome plays a role in postmortem proteolysis and might contribute to meat tenderisation.     

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.     

Tenderization and fragmentation of myofibrillar proteins in bovine longissimus dorsi muscle using proteolytic extract from Sarcodon aspratus

The objective of this study was to examine the Sarcodon aspratus extract including protease how to affect tenderness of the bovine longissimus dorsi muscle. In addition, we investigated myofibrillar protein fragmentation, particularly in myosin, and its influence on meat tenderness. Beef loin chunks were marinated with 0.5 g/100 g, 1 g/100 g, and 2 g/100 g powdered S. aspratus extract, 0.2 g/100 mL papain, and distilled water (control), respectively. Although tenderness of meat is increased by adding S. aspratus extract, differences in meat quality traits, such as muscle pH and meat color, were small and not considered to have practical importance between the control and enzyme-treated samples. Furthermore, the S. aspratus extract influenced the myofibril fragmentation index (MFI) as well as protein solubility. The changes in MFI and protein solubility were due to the myofibrillar protein degradation. Through Western blotting, we found that the S. aspratus extract, as well as papain, caused fragmentation of the myosin heavy chain, but the mushroom extract induced more fragmentations of myofibrillar proteins, and caused more tender meat.     

Degradation of myofibrillar proteins by a myofibril-bound serine proteinase in the skeletal muscle of crucian carp (Carasius auratus)

A myofibril-bound serine proteinase (MBSP) in the skeletal muscle of crucian carp (Carasius auratus) was identified. Hydrolysis of myofibrillar proteins by the endogenous MBSP was studied. Myosin heavy chain (MHC) was degraded markedly when crucian carp myofibril was incubated at 55 °C, as shown by SDS-PAGE. Prolonged incubation of myofibril at 55 °C also caused the obvious degradation of tropomyosin, while the decomposition of other myofibrillar proteins, such as α-actinin and actin, was slight, as detected by Western blotting. The results suggest the existence of an endogenous myofibril-associated proteinase in crucian carp myofibril, which efficiently cleaves MHC and tropomyosin. Serine proteinase inhibitors (Lima bean trypsin inhibitor, PMSF and benzamidine) greatly suppressed the degradation of MHC, caused by the enzyme, while inhibitors for cysteine, metallo-, and aspartic proteinases showed only partial or incomplete inhibitory effects, indicating that the endogenous proteinase is a serine proteinase. Substrate specificity analysis, using partially purified crucian carp MBSP, suggested that the enzyme is a trypsin-like serine proteinase.