POSTMORTEM CHANGES IN CYTOSKELETAL ELEMENTS OF FISH MUSCLE1

Transmission electron microscopic examination of samples from fresh and 7 days-cold-stored white croaker skeletal muscle did not reveal apparent changes in overall structure of contractile elements of muscle. However, when purified myofibrils obtained from fresh and stored muscles were extracted with 0.6 M KI and the residues were examined in the scanning electron microscope, a progressive disappearance of exosarcomeric longitudinal filaments connecting successive Z structures was observed. Electrophoretic analysis of myofibrillar proteins obtained from fresh and stored muscles showed a marked degradation of nebulin, a major component of the endosarcomeric cytoskeletal network, whereas the other protein constituents of myofibrils remained practically unchanged. These alterations in elements of both cytoskeletal lattices of muscle may have important effects on physical properties of fish meat.     

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.     

Mechanisms of water-holding capacity of meat: The role of postmortem biochemical and structural changes

Unacceptable water-holding capacity costs the meat industry millions of dollars annually. However, limited progress has been made toward understanding the mechanisms that underlie the development of drip or purge. It is clear that early postmortem events including rate and extent of pH decline, proteolysis and even protein oxidation are key in influencing the ability of meat to retain moisture. Much of the water in the muscle is entrapped in structures of the cell, including the intra- and extramyofibrillar spaces; therefore, key changes in the intracellular architecture of the cell influence the ability of muscle cells to retain water. As rigor progresses, the space for water to be held in the myofibrils is reduced and fluid can be forced into the extramyofibrillar spaces where it is more easily lost as drip. Lateral shrinkage of the myofibrils occurring during rigor can be transmitted to the entire cell if proteins that link myofibrils together and myofibrils to the cell membrane (such as desmin) are not degraded. Limited degradation of cytoskeletal proteins may result in increased shrinking of the overall muscle cell, which is ultimately translated into drip loss. Recent evidence suggests that degradation of key cytoskeletal proteins by calpain proteinases has a role to play in determining water-holding capacity. This review will focus on key events in muscle that influence structural changes that are associated with water-holding capacity.     

Biochemistry of postmortem muscle — Lessons on mechanisms of meat tenderization

It is certain that meat tenderness is a highly valued consumer trait and thus definition of the multiple processes that influence meat tenderness will provide clues toward improving meat quality and value. The natural process by which meat becomes tender is complex. Tenderness development is dependent on the architecture and the integrity of the skeletal muscle cell and on events that modify those proteins and their interaction. Specifically protein degradation and protein oxidation have been identified as processes that modify proteins as well as the tenderness of meat. The intracellular environment is a major factor that controls these events. Ultimately, the interplay between these events determines the rate and extent of tenderization. Given the intricacy of the structure of the muscle cell, coupled with the complexity of the regulation of protein modification and the ever-changing intracellular environment it is not surprising that this area of research is a very dynamic field. Just as the overall integrity and function of muscle cells does not depend on a single protein, but rather on the coordinated interaction of several proteins, the structural weakening of muscle cells during postmortem aging also must not depend on the degradation of a single myofibrillar or other cytoskeletal protein. The proteins mentioned in this review are located in different regions of the muscle cell, and most have been implicated in some manner as being important in maintaining the structure and function of the muscle cell. Oxidation of myosin heavy chain, a predominant protein in the myofibril, is known to promote aggregation and toughening of meat. Degradation of proteins such as desmin, filamin, dystrophin, and talin (all located at the periphery of the Z-line) may disrupt the lateral register and integrity of the myofibril themselves as well as the attachments of the peripheral layer of myofibril to the sarcolemma. Degradation of the proteins within the myofibril that are associated with the thick and thin filaments may allow lateral movement or breaks to occur within the sarcomeres of postmortem aged samples. Titin, nebulin, and troponin-T, by their ability to directly interact with, or modulate the interaction between, major proteins of the thick and thin filaments and (or) the Z-line, play key roles in muscle cell integrity. Disruption of these proteins, especially titin and nebulin, could initiate further physicochemical and structural changes that result in myofibril fragmentation and loss of muscle cell integrity, and ultimately in tenderization of the muscle. In order to make real progress in this area, the scientific community must have a global appreciation of how both the structural proteins and the key proteases are influenced by the vast changes that occur during the conversion of muscle to meat.     

SOLUBILITY OF THE PROTEINS OF MACKEREL LIGHT MUSCLE AT LOW IONIC STRENGTH

Over 90% of the proteins of mackerel light muscle were soluble in solutions of physiological ionic strength or less. To accomplish this solublization, it was necessary to extract certain proteins at moderate ionic strength and neutral pH before extracting the rest of the myofibrillar and cytoskeletal proteins in water. Six proteins were favorably solubilized by sodium chloride solutions of moderate ionic strength at neutral pH under conditions that allowed later dissolution of myofibrillar and cytoskeletal proteins in water. The possibility is suggested that three of these proteins were involved in preventing the solubilization in water of other myofibrillar and cytoskeletal proteins of mackerel light muscle. Based on molecular masses and relative abundance, these proteins could possibly be M-protein (166 kDa), α-actinin (95 kDa) and desmin (56 kDa).     

ROLE OF INITIAL MUSCLE pH ON THE SOLUBILITY OF FISH MUSCLE PROTEINS IN WATER

The solubility of the myofibrillar and cytoskeletal proteins in water was determined for the muscle tissue often species offish. The flesh of six white-muscled fish had pH's at the time of processing above pH 6.6 and greater than 80% of their myofibrillar/cytoskeletal proteins were soluble in water. The flesh of three pelagic species and a shark had pH values when processed below 6.6 and the water solubility of their myofibrillar and cytoskeletal proteins was less than 40%. When the washed minced muscle of one of the white-fleshed species, cod, was exposed to low pH, the solubility of its myofibrillar and cytoskeletal proteins decreased substantially. The water solubility of the cod myofibrillar and cytoskeletal proteins could be reestablished by washing the acid-treated cod flesh with neutral salt solutions. It is suggested that pH values below 6.6 modify certain proteins which prevent the water-extractability of the rest of the myofibrillar and cytoskeletal proteins from being expressed.     

Post-mortem changes in cytoskeletal proteins of muscle

Progressive changes have been identified in the solubility of muscle-cell proteins during post-mortem muscle ageing, particularly the cytoskeletal proteins, desmin and connectin. Ox sternomandibularis muscle was sampled immediately post mortem and up to six days later. It was homogenised and separated into three salt-soluble fractions: phosphate soluble, concentrated KI soluble and guanidine-HCl soluble. Proteins in each fraction were analysed on sodium dodecylsulphate polyacrylamide gels. Changes reported previously by other authors were confirmed. In addition desmin, which was restricted to the guanidine fraction, disappeared, apparently due to proteolysis during storage. Connectin was also partly lost from the guanidine fraction, possibly through increased solubility in the KI fraction. In this respect an unidentified polypeptide of 110 000 D appeared during storage. Desmin extracted from ox muscle was partially purified and identified by amino acid analysis. It apparently occurs in vivo as a network of linked collars around the Z-discs and its loss during post-mortem storage probably accounts for the ease with which stored muscle disintegrates into individual myofibrils on homogenisation. The disintegration of the cytoskeletal network can account for the post-mortem changes in the physical properties of muscle for the increased tenderness after cooking of stored meat. However, factors other than those related to changes in cytoskeletal proteins are responsible for the toughness of cooked, cold-shortened muscle since cold shortening prior to storage did not affect the distribution of proteins among the three salt fractions, nor the patterns obtained in gel analyses.     

POSSIBLE ROLE OF MUSCLE PROTEINS IN FLAVOR AND TENDERNESS OF MEAT

Evidence suggests that both the myofibrillar proteins and collagen play important roles in meat flavor and tenderness. The probable contributions of the purified proteins to flavor are reviewed in terms of their amino acid composition, especially the sulfur containing and certain other amino acids that have been implicated in meat flavor development. Myofibrils solubilized in sodium dodecylsulfate (SDS) undergoproteolysis on warming to room temperature overnight or on storing for several days at 0–4°C as demonstrated by extra protein bands. The extra proteins appear to be due to the presence of indigenous muscle proteases. The implications of some indigenous muscle proteases are reviewed in terms of their probable role in tenderization of postmortem meat.     

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.     

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.     

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.     

LOCALIZATION OF PARATROPOMYOSIN IN CHICKEN SKELETAL MUSCLE MYOFIBRILS AND ITS BEHAVIOR DURING POSTMORTEM STORAGE OF MUSCLES REVEALED BY IMMUNOELECTRON MICROSCOPY

Paratropomyosin is a minor myofibrillar protein which in freshly prepared myofibrils is exclusively localized at the A-I junction region of sarcomeres. We investigated the ultrastructural localization of paratropomyosin in intact and postrigor myofibrils by immunoelectron microscopy. Paratropomyosin was localized as two distinct stripes at the A-I junction in intact myofibrils. It also was localized at the position corresponding to the original A-I junction in thick filament-free myofibrils (I-Z-I brushes). However, following postmortem storage, paratropomyosin was found broadly distributed in thin filaments of myofibrils.     

Trends in postmortem aging in fish: understanding of proteolysis and disorganization of the myofibrillar structure

Postmortem tenderization is caused by enzymatic degradation of key structural proteins in myofibrils as well as in extracellular matrix, and of proteins involved in intermyofibrillar linkages and linkages between myofibrils and the sarcolemma. The function of these proteins is to maintain the structural integrity of myofibrils. Current data indicate that calpains and cathepsins may be responsible for degradation of these proteins. Other phenomena occurring in cells postmortem (pH drop, sarcoplasmic Ca2+ increase, osmotic pressure rise, oxidative processes) may act in synergy with proteases. Our understanding of the underlying mechanisms of muscle degradation should be improved for an accurate evaluation of the postmortem muscle changes and consequently of the fish quality.     

PSE-LIKE SYNDROME IN BREAST MUSCLE OF DOMESTIC TURKEYS: A REVIEW

There are several similarities between the development of pale, soft, exudative (PSE) meat in breast muscle (Pectoralis supeficialis) of domestic turkeys, swine longissimus and ham muscles. Although the ultimate cause of the syndrome is not known, it appears that the combination of antemortem stress sensitivity of the domestic turkey and predominantly glycolytic metabolism of the breast muscle, results in accelerated rigor mortis processes. The consequent low pH (<5.8) combined with high breast muscle temperature (>35C), typically causes protein denaturation leading to soft and discolored, PSE meat with reduced protein functionality. Our studies revealed that phosphorylase, a sarcoplasmic enzyme, becomes denatured and tightly associated with the myofibrils in PSE turkey breast     

M-line protein: Presence of two non-equivalent high molecular weight components

M-line enriched protein preparations were prepared from glycerated rabbit muscle myofibrils by means of three alternate procedures and analysed with SDS-polyacrylamide gel electrophoresis. A doublet corresponding to polypeptides with molecular weights of 193,000 and 182,000 was obtained in a ratio of2:3, respectively. The two bands are evident in SDS-gels of purified myofibrils, indicating that the proteins represent intrinsic M-line structural peptides. Transmission electron micrographs of the extracted myofibrils confirmed the effectiveness of the extraction procedures for removing M-line proteins. The possible significance of the M-line proteins to meat science is discussed.     

In vitro proteolysis of myofibrillar and sarcoplasmic proteins of European sea bass (Dicentrarchus Labrax L) by an endogenous m‐calpain

The effects of m‐calpain isolated from the skeletal muscle of sea bass on sarcoplasmic and myofibrillar proteins isolated from the same tissue were examined in vitro. Incubation of sarcoplasmic proteins with m‐calpain resulted in only a slight decrease (0.7 kDa) in the molecular weight (MW) of a 26.5 kDa protein. Degradation of myofibrils, monitored by quantification of TCA‐soluble peptides generated, resulted in the maximum amount of peptides being generated after 1 h of incubation at 25 °C. Noticeable modifications in the SDS‐PAGE profile of digested myofibrils were observed, including partial denaturation of myosin heavy chain and the release of tropomyosin, ～69 and ～27 kDa doublet bands and a few polypeptides of MW lower than 20 kDa in the soluble fraction. Examination of the degradation patterns of myofibrillar proteins using Western blotting showed that α‐actinin was partially degraded, with release of native α‐actinin and its fragments from myofibrils, whereas desmin was highly degraded after 2 h of digestion. © 2002 Society of Chemical Industry     

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.     

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.     

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.     

The effect of ageing on the water-holding capacity of pork: role of cytoskeletal proteins

The water-holding capacity (WHC) of pork decreases post-mortem but has been shown to increase during subsequent ageing. In order to test a hypothesis that water-holding capacity increases during ageing due to degradation of the cytoskeleton, WHC was followed 10 days post-mortem and related to the extent of proteolysis of cytoskeletal proteins. A fast method for measuring WHC in small meat samples was developed by the use of centrifugation. The WHC of fresh pork decreases in the first part of post-mortem storage after which it increases to the level of 1 day PM. No changes in total water content of the meat were observed which could explain changes in WHC during ageing. Vinculin and desmin degrade gradually during ageing while talin degrades rapidly. These observations are consistent with the hypothesis that degradation of the cytoskeleton slowly removes the linkage between lateral shrinkage of myofibrils and shrinkage of entire muscle fibres, so removing the force that causes flow into the extracellular space. Inflow of previously expelled water is then possible, so increasing WHC as observed in later periods of storage.