RELATIONSHIP OF pH AND TEMPERATURE TO DISRUPTION OF SPECIFIC MUSCLE PROTEINS AND ACTIVITY OF LYSOSOMAL PROTEASES

From a review of the literature, and from specific data presented in this paper, it was concluded that both postmortem temperature and pH have effects on meat tenderness and on disruption of specific myofibrillar proteins. Increased postmortem temperature porduces more tender muscles and increases the disruption of troponin-T, myosin, Z-lines, connectin and gap filaments. Elevated postmortem temperature also increases the activity of enzymes which cause the disruption of myofibrillar proteins. Higher ultimate postmortem pH (above 6.0) produces more tender muscle, but also produces dark-cutting meat Except for one experiment, lower pH in the first few hours postmortem (in muscle with normal ultimate pH; i.e., 5.8 or below) improves meat tenderness. High pH increases the activity of CAF and low pH increases the activity of lsosomal cathepsins. Both high and low pH increase the degradation of troponin-T, Z-lines, gap filaments and connectin, but the degradation of these proteins (except for Z-lines) is greater at a low pH. Low pH increases the degradation of myosin; conversely, high pH retards it degradation.     

DEGRADATION OF PROTEOGLYCANS IN THE SKELETAL MUSCLE OF PACIFIC ROCKFISH (SEBASTES SP.) DURING ICE STORAGE

Soluble collagen, proteoglycans and two lysosomal enzymes known to degrade proteoglycans were studied during ice storage of skeletal muscle from Pacific rockfish. The solubility of muscle collagen progressively increased during storage in ice for 7 days. Proteoglycans isolated from prerigor muscle by extraction with guanidine HCl and density gradient ultracentrifugation contained only 43 μg hexuronic acid/100 g of wet tissue. The sulfated glycosaminoglycans portion of proteoglycans were isolated by trypsin digestion of an extract from muscle acetone powder and contained 58 μg hexuronic acid/20 g of acetone-dried muscle tissue. Sulfated glycosaminoglycans had several components which separated by sodium dodecylsulfate polyacrylamide gel electrophoresis. Electrophoresis of sulfated glycosaminoglycans from stored rockfish revealed disappearance/decrease of specific zones after 5 days at 0C. The degradation of proteoglycans occurs in postmortem fish muscle and this may contribute to the destabilization of the extracellular matrix and texture softening during postharvest storage of fish.     

IDENTIFICATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE IN THE SKELETAL MUSCLE OF SILVER CARP

Myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was characterized. Myosin heavy chain (MHC) degraded markedly when silver carp myofibril was incubated at 55–60C as shown by SDS‐PAGE. Prolonged incubation of myofibrils also caused the degradation of other myofibrillar proteins such as α‐actinin, actin and tropomyosin to some degree. The results suggest the existence of an endogenous myofibril associated proteinase. Serine proteinase inhibitors (Pefabloc SC and Lima bean trypsin inhibitor) greatly suppressed the degradation of myosin heavy chain, while inhibitors for cysteine, metallo, and aspartic proteinases did not show any effect, indicating that the endogeneous proteinase is a myofibril‐bound serine proteinase.     

HYDROLYSIS OF Z-DISC ACTIN BY Ca2+-ACTTVATED PROTEASE1

A 42,000 dalton protein has been identified in chicken breast muscle which differs from actin in that it is soluble at low ionic strength and insoluble in 1 MKI solutions. This protein is readily hydrolysed by Ca⁺⁺-activated neutral protease from chicken breast muscle, and is not present in Ca⁺⁺-activated neutral protease treated myofibrils. This protein may be involved in the integrity of the Z—disc in skeletal muscle.     

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

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

NEUTRAL AND ALKALINE MUSCLE PROTEASES OF MARINE FISH AND INVERTEBRATES A REVIEW

Muscle proteases active at neutral and alkaline pH's include calcium-activated neutral proteases, heat-activated thiol proteases, serine proteases, and metallo-proteases. They participate to different extents in postmortem degradation of fish muscle myofibrillar and scaffold proteins. Their activity in fish, the presence of endogenous activators and inhibitors, pH, and temperature. Recent studies indicate that neutral and alkaline proteases have more impact on the postmortem deterioration in quality of fish muscles than the cathepsins active at acid pH. Significant quality losses are caused by enzymatic collagen degradation in raw tissues and by heat-activated enzymes in fish gels.     

PROPERTIES OF AN ALKALINE PROTEASE FROM THE SKELETAL MUSCLE OF ATLANTIC CROAKER1

An alkaline protease was partially purified from the skeletal muscle of Atlantic croaker. The protease is a cytoplasmic enzyme and heat stable. The enzyme preparation was shown to degrade fish actomyosin in vitro between 50–60°C. The enzyme is a sulfhydryl protease and does not require Ca⁺⁺ ions for its activity. Preparations of the enzyme do not hydrolyze TAME, BTEE or denatured hemoglobin. Column chromatographic analyses suggest an apparent molecular weight of 80,000 ± 4,000 and the isoelectric point is 6.0 ± 0.2.     

Effect of Calcium Activated Protease (CAF) on Bovine Myofibrils Under Different Conditions of pH and Temperature

This study examined the in vitro effects of calcium-activated protease (CAF) on bovine myofibrillar proteins and structure under postmortem-like conditions of pH and temperature. Effects usually associated with this enzyme under optimal conditions were reduced as temperature and/or pH were lowered. However, significant activity remained at 15°C and pH 6.5, and some activity was detectable at even lower pH's and temperatures. Effects observed included: solubilization of myofibrillar protein, degradation of the myofibrillar protein titin and others, and an increase in the degree of myofibrillar fragmentation. These results suggest that CAF is able to hydrolyze proteins that are important to structural integrity under conditions mimicking those present in postmortem muscle.     

MOLECULAR PROPERTIES OF POST-MORTEM MUSCLE: EFFECT OF SULFHYDRYL REAGENTS AND POST-MORTEM STORAGE ON CHANGES IN MYOSIN B

Studies to determine the relationship of SH groups to certain changes of the myofibrillar proteins of post-mortem muscle were carried out with myosin B from at-death and post-mortem stored rabbit skeletal muscle (2° C and 25° C for 3 days) and with SH reagent modified myosin B from at-death and post-mortem stored muscle. Quantitative SH analysis, ATPase activity, turbidity rate and analytical ultracentrifugation were employed to determine the changes in myosin B associated with changes in SH groups. Post-mortem storage of muscle at 2°C for 3 days had no effect on SH content of myosin B; a decrease in SH groups, however, was observed in myosin B from muscle stored at 25°C for 3 days and for iodoacetamide (IAA) and N-ethylmaleimide (NEM) modified myosin B. ATPase activity was inhibited by reacting myosin B with enough NEM or IAA to block all SH groups. Dialysis of myosin B from at-death and post-mortem muscle against MCE to restore SH groups resulted in partial reversal of Mg++ and EGTA-activated ATPase of myosin B from post-mortem muscle and a less rapid rate of turbidity development. These results suggest that the state and nature of SH groups are partly involved in the actin-myosin interaction of post-mortem muscle; other constituents, however, in addition to SH groups are evidently modified and, in some instances, irreversibly modified, under certain post-mortem storage conditions.     

PURIFICATION AND CHARACTERIZATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE FROM THE SKELETAL MUSCLE OF SILVER CARP

Recently, a myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was identified. MBSP could be dissociated from myofibrils by treatment at pH 4.0. Following ultrafiltration concentration and chromatography on Sephacryl S‐200, High Q ion‐exchange and affinity column of Arginine Sepharose‐4B, MBSP was partially purified. The enzyme with an estimated molecular weight of 28 kDa cleaves synthetic fluorogenic substrates specifically at the carboxyl sites of arginine and lysine residues. MBSP activity is suppressed by serine proteinase inhibitors such as Pefabloc SC, lima bean trypsin inhibitor and benzamidine; it is insensitive to Pepstatin, l ‐3‐carboxy‐trans‐2, 3‐epoxypropionyl‐l ‐leucine‐4‐guanidinobutylamide and ethylenediaminetetraacetic acid, suggesting MBSP is a trypsin‐like serine proteinase. Optimal profiles of pH and temperature of the enzyme are 8.5 and 55C, respectively. Hydrolysis of myofibrillar proteins such as myosin heavy chain, actin and tropomyosin by purified MBSP occurred especially at around 55C, consistent with our proposal that MBSP plays a significant role in the Modori phenomenon.     

EFFECT OF THE REDOX POTENTIAL ON THE MUSCLE ENZYME SYSTEM

ABSTRACT This work presents the effect of redox potential in the range +100 to ?250 mV on the muscle cathepsins B, H and L, lysosomal acid lipase and acid esterase and on neutral and basic lipases and neutral esterase from adipose tissue. Cathepsins B, H and L need reducing conditions (below ?50 mV) for maximal activity. Lysosomal acid lipase remains unaffected while activity of muscle acid esterase decreases sharply below ?175 mV. The activity of basic lipase from adipose tissue is also reduced below ?80 mV while neutral lipase remains unaffected. The activity of neutral esterase is drastically reduced to negligible values below ?100 mV.     

EFFECTS OF ANTI-LMM ANTIBODIES ON THE SOLUBILITY OF CHICKEN SKELETAL MUSCLE MYOSIN

Monoclonal antibody Fab fragments which bind epitopes in the rod domain of skeletal muscle myosin had specific effects on the solubility properties of chicken muscle myosin in vitro. Two antibodies (NA4 and 5C3), which bind at the C-terminal portion of the rod domain caused myosin to remain soluble and monomeric in 0.1 M KCl, pH 7.2, conditions in which myosin normally aggregates into filamentous structures. Other antibodies (EB165 and AB8), which bind in the middle of the rod did not alter either myosin solubility or the morphology of the myosin assemblies formed. These results demonstrate the importance of the C-terminus of the myosin rod as a domain involved in regulating myosin interactions and solubility.     

ROLE OF pH IN GEL FORMATION OF WASHED CHICKEN MUSCLE AT LOW IONIC STRENGTH

This work was designed to test the hypothesis that it is not solubilization of the myofibrillar proteins per se that is required to form good gels at low salt concentrations, but the protein‐containing structures must be disorganized. Gels were made from washed minced chicken breast muscle at 0.15, 0.88, and 2.5% sodium chloride. The gels made with varying salt concentrations were evaluated either at pH 6.0–6.5 or pH 7.0–7.4. Strain values, an indicator of protein quality, were high only at neutral pH in the gels containing 0.15 or 0.88% salt. At 2.5% salt, strain values of gels made at acid pH were superior to those at the low salt concentrations at acid pH, but inferior to gels with 2.5% salt at neutral pH. Poor gels were obtained at 0.15% salt and low pH whether or not there was an intermittent adjustment to neutral pH. A neutral salt wash markedly increased the water content of the mince, suggesting that solubility‐inhibiting proteins were removed. Good quality gels were obtained in the absence of any detectable solubilization of myosin and only minimal solubilization of actin.     

CONTINUATION OF SYMPOSIUM: FUNDAMENTAL PROPERTIES OF MUSCLE PROTEINS IMPORTANT IN MEAT SCIENCE: ROLE OF NEW CYTOSKELETAL ELEMENTS IN MAINTENANCE OF MUSCLE INTEGRITY

Evidence suggests that desmin, titin and nebulin, three recently discovered proteins, have cytoskeletal roles in muscle cells. The three proteins have been purified from mature skeletal muscle and partially characterized. Properties of the three proteins are described, with special regard to their probable roles and importance in maintaining muscle cell integrity. Results will be shown that demonstrate ability of purified desmin to self-assemble into synthetic 10-nm (intermediate) diameter filaments. Taken together with immunoelectron microscope results (Richardson et al. 1981), it is evident that desmin is the major component of 10-nm filaments of mature skeletal muscle cells and that the desmin filaments link adjacent myofibrils at their Z-line levels and seemingly tie the myofibrils into the cell cyto-skeleton. Desmin is degraded at about the same rate as is the highly susceptible troponin-T in bovine semitendinosus muscle postmortem. Alterations in desmin and other recently discovered cytoskeletal proteins would be expected to disrupt muscle cell integrity and to have marked effects on properties of muscle important to its use as food.     

太平洋牡蛎冷藏过程中闭壳肌品质的变化

为了探究太平洋牡蛎（Crassostrea gigas）在0℃和4℃冷藏过程中的品质变化,首先对牡蛎不同组织（闭壳肌、外套膜、鳃和内脏团）在冷藏过程中的表观变化进行观察,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulphate-polyacrylamide gel electrophoresis,SDS-PAGE）分析冷藏过程中各组织中全蛋白的变化,进一步通过Western blotting鉴定闭壳肌中肌球蛋白重链（myosin heavy chain,MHC）、肌动蛋白、副肌球蛋白（paramyosin,PM）和原肌球蛋白（tropomyosin,TM）的降解情况,采用荧光光谱分析闭壳肌冷藏过程中内源性丝氨酸蛋白酶和组织蛋白酶B的活力变化,以闭壳肌的pH、K值和质构特性等为评价指标,分析其在冷藏过程中品质的变化。结果显示,随着冷藏时间延长,闭壳肌由紧实变得绵软,表观变化最明显。Western blotting结果显示,在0℃和4℃冷藏过程中,闭壳肌中MHC分别在第3天和第1天开始降解,表明低温有利于降低蛋白酶解速率;但是,肌动蛋白、PM和TM无明显变化。...     

CALMODULIN IN SKELETAL MUSCLE

The central role of Ca²⁺ in the regulation of muscular activity is well documented. At the level of the contractile proteins the dominant effect of Ca²⁺ is attributable to the Ca²⁺-bindingprotein, troponin C. This protein in the presence of Ca²⁺ elicits conformational changes which allow cross-bridge cycling and hence contraction. Another Ca²⁺-binding protein, similar in its physical properties to troponin C, has also been discovered and this is calmodulin. The latter differs from troponin C in that it is involved in the regulation of a wide range of enzymatic reactions and it appears to be a ubiquitous regulatory protein. In skeletal muscle, as in all other eukaryotic cells, the number of calmodulin-dependent systems is too large to be considered and only two enzymes have been selected. These are myosin light chain kinase and phosphory lose kinase. The activities of both are intimately related to the contractile mechanism and for this reason their actions could be important to meat quality.     

SOME PROPERTIES OF MYOFIBRILLAR PROTEINS OBTAINED FROM LOW IONIC STRENGTH EXTRACTS OF WASHED MYOFIBRILS FROM PRE- AND POST-RIGOR CHICKEN PECTORAL MUSCLE

SUMMARY— Changes in extractability of the proteins associated with the fragmentation phenomenon of myofibrils in chicken pectoral muscle were studied. The results indicate that the protein fractions extracted by neutralized water from muscle residue. from which water-soluble proteins have been washed out, increase in post-rigor muscle. The extracts from pre- and post-rigor muscle were fractionated with ammonium sulfate into two fractions: the fraction precipitated by 1.7 M ammonium sulfate (Fr.1) and the supernatant (Fr. 2). Depressing effect on the onset of ATP-induced superprecipitation of trypsin-treated myosin 6 which was initially present in Fr. 2 from pre-rigor muscle decreased to a great extent in that from post-rigor muscle, whereas promotive effect on gelation of F-actin and superprecipitation of the myosin 6 which was little in Fr. 1 from pre-rigor muscle appeared in that from post-rigor muscle. It is proposed that an increasing amount of protein which indicates α-actinin activity is released along with the destruction and final dissolution of the Z-line structure during postmortem storage of chicken pectoral muscle.     

HYDROXYL RADICAL MODIFICATION OF FISH MUSCLE PROTEINS

A xanthine oxidase-based system was used to generate hydroxyl free radicals in washed, minced cod muscle. Oxidation of protein was measured by increase in protein carbonyl content; the system used produced approximately 0.1 mol of carbonyl groups per 10⁵ g of protein. This degree of oxidation had only minor effects on the SDS-PAGE patterns of the muscle proteins. The solubility of the proteins was not affected by this amount of oxidation unless they were also subjected to a freeze/thaw cycle. With a freeze/thaw cycle, a highly significant decrease in protein solubility occurred compared to that which took place in a sample not exposed to the free radical system. Lowering the pH from 6.8 or 6.5 to 6.0 or 5.5 had a strong negative impact on protein solubility. Protein oxidation appeared in two phases in washed cod mince, an initial rapid increase followed by a second phase that may have been linked to oxidation of the small amount of lipid in the sample. Comparison of protein carbonyl formation in stored mackerel fillets or mince indicated that the range of oxidation studied in the cod model system was similar to what occurs in stored mackerel muscle postmortem.     

Characterisation of proteolytic enzymes from muscle and hepatopancreas of fresh water prawn (Macrobrachium rosenbergii)

BACKGROUND: Fresh water prawn in Thailand is widely consumed due to its delicacy. During postmortem handling and storage, prawn meat becomes soft and mushy, probably as a result of indigenous proteases. Therefore, an understanding of prawn proteases associated with the degradation of muscle proteins from fresh water prawn could pave the way for prevention of such a phenomenon during extended storage. RESULTS: Proteolytic enzymes in the crude extract (CE) from muscle and hepatopancreas of fresh water prawn (Macrobrachium rosenbergii) were characterised. CE from muscle exhibited the highest hydrolytic activities towards haemoglobin at pH 5 and 50 °C, while that from hepatopancreas had the highest activity on casein at pH 7 and 60 °C. Based on inhibitor study, cysteine protease and serine protease were dominant in CE from muscle and hepatopancreas, respectively. CE from muscle rarely hydrolysed natural actomyosin (NAM), but could not degrade pepsin‐soluble collagen (PSC). Conversely, NAM and PSC were susceptible to hydrolysis by CE from hepatopancreas as evidenced by the marked decreases in band intensity. Activity staining using haemoglobin, casein and gelatin as substrates revealed that no proteolytic or gelatinolytic activity was observed in CE from prawn muscle, while CE from hepatopancreas exhibited pronounced hydrolytic activities towards all substrates. CE from muscle showed calpain and cathepsin L activities but CE from hepatopancreas mainly exhibited tryptic and chymotryptic activities. CONCLUSION: Serine proteases, mainly trypsin‐like or chymotrypsin‐like, from hepatopancreas were probably responsible for the softening of prawn meat during postmortem storage via the degradation of both muscle and connective tissues. Copyright © 2010 Society of Chemical Industry     

宰后成熟期间结构蛋白对秦川牛肉嫩度的影响

为探究宰后成熟期间结构蛋白对秦川牛背最长肌嫩度的影响,测定不同贮藏期（0、2、4、6、8 d）内秦川牛背最长肌剪切力、肌原纤维小片化指数（myofibrillar fragmentation index,MFI）和蛋白含量等,并利用4D-非标记定量（4D-label free quantification,4D-LFQ）蛋白质组学技术分析蛋白质组学变化。结果表明：在贮藏期0～8 d内,剪切力总体呈先升后降的趋势（P＜0.01）,上升的幅度小于下降的幅度;秦川牛背最长肌MFI呈极显著上升趋势（P＜0.01）,总体增长了250.81%;总可溶性蛋白含量呈显著下降趋势（P＜0.05）,总体下降了34.60%;通过宰后肌肉组织代谢变化,肌肉组织结构蛋白发生降解,可能会影响到嫩度的形成,肌原纤维蛋白含量呈显著下降趋势（P＜0.05）,前期下降速度较快,后期下降速度减缓,总体下降了50.56%。在贮藏期0～4 d内,通过骨骼肌组织发育过程调控钙离子结合和细胞骨架蛋白结合途径,4 种蛋白（α-肌动蛋白-1、牛肌球蛋白重链9、牛肌球蛋白轻链2、肌球蛋白调节轻链12B）丰富度发生变化;在贮藏期0～8 d内,通过肌肉器官发育和横纹肌组织发育过程调控钙离子结合途径,8 种蛋白（肌球蛋白调节轻链2、肌球蛋白重链6、α-肌动蛋白-1、心肌肌动蛋白α1、牛肌球蛋白轻链2、肌钙蛋白I 1型、肌钙蛋白I 2型、肌球蛋白重链15）丰富度发生变化,通过肌球蛋白结合、钙离子结合、细胞骨架蛋白结合的肌原纤维组装、骨骼肌组织发育、肌肉器官发育、横纹肌组织发育过程等途径调控细胞的生理状态,结构蛋白降解造成肌原纤维小片化升高,进而促使嫩度提升。