Molecular Properties of Post-Mortem Muscle.

SUMMARY— Post-mortem changes in nucleoside triphosphatase activity of bovine myosin B have been studied by using several different modifiers with either 5 mM ATP or 5 mM ITP as substrate at ionic strengths (r/2) of 0.09, 0.19, or 0.52. Enzymic activity was determined by measuring the release of inorganic phosphate. There was very little difference in enzymic activity between myosin B isolated from prerigor, rigor (24 hr post-mortem) or post-rigor (312 hr post-mortem) muscle stored at either 2° or 16°C except that the specific activity of myosin B prepared from muscle stored for 12–24, hr post-mortem was higher than activity of myosin B prepared immediately after death. This increase cannot be explained in terms of rigor shortening, but suggests that a change in myosin conformation or in the nature of the actin-myosin interaction occurs in post-mortem muscle. If an actin-myosin interaction occurs during rigor mortis and if this association remains unchanged during extraction of myosin B, then the very low Mg⁺⁺-modified myosin B enzymic activities obtained at Γ/2 = 0.19 and 0.52 indicate that this interaction is not irreversible. Extraction in the absence of ATP produced a myosin B whose ATPase activity was markedly inhibited by trace amounts of Mg⁺⁺. This may be due to the absence of a-actinin in these myosin B preparations. No consistent differences in activation energies were found either at Γ/2 = 0.19 or 0.52 among the NTPase reactions of myosin B samples prepared from muscle after various times of post-mortem storage.     

Molecular Properties of Post-Mortem Muscle. 6. Effect of Temperature on Protein Solubility of Rabbit and Bovine Muscle

SUMMARY– Studies were conducted to investigate the effect of temperature on the actin-myosin interaction of rabbit and bovine muscle during rigor and post-rigor shortening. Muscle was stored at four different temperatures (2°, 16°, 25° and 37°), corresponding to three types of post-mortem muscle shortening: cold, minimal and high temperature. These three types of shortening are presumably related to different states of the actin-myosin interaction in post-mortem muscle. Post-mortem tenderization may be the result of either actin-myosin dissociation or F-actin depolymerization.
To detect the occurrence of either of these possible changes, two salt solutions, differing widely in their myofibrillar protein extracting abilities, were used to compare post-mortem myofibrillar protein solubility after different times of post-mortem storage and to provide information about the actin-myosin complex. Myofibrillar protein solubility of both rabbit and beef muscle in 0.5M KCl, 0.1M phosphate, pH 7.4, increased markedly with increasing post-mortem storage at temperatures up to 25deg;. Similar solubility changes were obtained with 1.1M Kl, 0.1M K phosphate, pH 7.4, but these changes were much smaller in magnitude. Solubility in both salt solutions, in general, decreased for muscle stored at 37°.
Although time and temperature of post-mortem storage caused appreciable alterations in protein solubility, these alterations could not be directly related to changes in tenderness or sarcomere length or to species differences in the effects of temperature on post-mortem shortening. Viscosity, analytical ultracentrifugation, and ATPase assays all indicated the absence of "normal" actomyosin in all myofibrillar protein extracts in this study. It was suggested that the 1.1 M KI extracts contained G-actomyosin, but the available evidence indicated the presence of only myosin in 3-hr, 0.5 M KCI extracts.     

Molecular Properties of Post-Mortem Muscle. 7. Changes in Nonprotein Nitrogen and Free Amino Acids of Bovine Muscle

SUMMARY– Proteolysis and its relationship to tenderness were studied by measuring nonprotein nitrogen (NPN), free amino groups, and shear resistance during post-mortem aging of bovine muscle. Both NPN and free amino groups increased during post-mortem aging, indicating some degradation of proteins and/or peptides. However, neither the increase in NPN nor free amino groups was related to post-mortem tenderization since these quantities increased only after most of the improvement in tenderness had occurred. Much of the increase in NPN or free amino groups may originate from degradation of sarcoplasmic proteins or peptides. It is suggested that weakening or breaks at crucial points in the sarcomere, such as at the junction of the Z-line with the thin filaments, occur within the first 48-72 hr post-mortem and that this weakening or cleavage is responsible for tenderization. Cathepsin D may be responsible for this weakening but most of the available evidence is against proteolysis as the primary cause of post-mortem tenderization.     

Treatment and Post-Mortem Aging Effects on the Z-line of Myofibrils from Chicken Pectoral Muscle

SUMMARY: Changes in the morphology of myofibrils prepared from chicken pectoral muscle during post-mortem storage at 5°C were examined by light and electron microscopy. When the 24-hr stored samples were blendorized, electron micrographs showed two types of destruction in the Z-lines of sarcomeres and myofibrillar fragments: (1) The degradation and/or disappearance of Z-lines. (2) The breakdown of the junction of Z-line and I-filaments. A change in the state of the Z-line and the junction of the Z-line and I-filaments appeared to be indispensable for the fragmentation of the myofibrils. It was also shown through phase contrast microscopic observations that sarcoplasmic proteins, participating in the glycolytic cycle, may play a role in the fragmentation of the myofibrils. Evidence has not been obtained, to date, on the participation of proteolytic enzymes in the fragmentation phenomenon.     

Post-Mortem Changes in Subcelllular Fractions from Normal and Pale, Soft, Exudative Porcine Muscle. 2. Electron Microscopy.

SUMMARY— Myofibrillar, mitochondrial, heavy sarcoplasmic reticulum, and light sarcoplasmic reticulum fractions were isolated from homogenates of normal and pale, soft, exudative (PSE) porcine muscle at 0 and 24 hr post-mortem and examined by electron microscopy. No differences were observed between normal and PSE myofibrils obtained at death. PSE myofibrils prepared at 24 hr post-mortem had more granular appearing filaments and wider Z lines than normal myofibrils at 24 hr. The PSE heavy sarcoplasmic reticulum fraction obtained at death had a higher proportion of granular material than the same fraction from normal muscle. Several structural differences between the other PSE and normal fractions were also observed, especially at 24 hr postmortem. This study indicated that the composition of the subcellular fractions changed with time post-mortem and that this change should be considered when analyzing biochemical data from these fractions. However, the differences observed could not explain the large changes in calcium accumulating ability that have been shown to occur post-mortem.     

Storage Stability of Antioxidant-Washed Myofibrils From Chicken White and Red Muscle

Oxidation and gel-forming ability of chicken white (breast) and red (leg) muscle myofibrillar proteins during storage at 0°C were examined. Breast myofibril gels exhibited greater shear moduli than leg myofibril gels throughout 8 days storage. Shear moduli of both breast and leg gels in the intermediate temperature zone (45–55°C) decreased during storage, but at >55°C, they either increased or remained unchanged. Lipid oxidation was inhibited by washing myofibrils with antioxidants propyl gal-late, ascorbate, and tripolyphosphate. However, these antioxidants did not affect the content of protein carbonyls, and only slightly decreased the amine content during storage. Storage affected the kinetic process of myofibril gelation independently of antioxidant treatments.     

Thermal Gelation of Stretched and Cold-Shortened Bovine Sternomandibularis Muscle and Myofibrils

Muscle gels (10% protein) and myofibril gels (8% protein) were prepared at pH 6.0 with 2% NaCl and a heating rate of 0.7°C/min. No difference in gel strength occurred between stretched and cold-shortened muscles, but cooking loss was lower for stretched muscle. Stretched muscle sarcomeres were longer than those of cold-shortened muscle. The myofibril fraction from stretched muscle had higher gel strength, viscosity index, elasticity, and lower cooking loss than that from cold-shortened muscle. These results suggest that the contractile state of the muscle affects protein binding and water binding of the myofibrillar fraction.     

Heat Gelation Properties and Protein Extractability of Beef Myofibrils

At a heating rate of 1^oC/min suspensions (pH 6.0) of isolated beef myofibrils were found to start forming gels at 43-56^oC, as detected by dynamic rheological measurements. The increase in gel storage modulus levelled off at temperatures > 65^oC. At medium to high (0.3-0.6M) concentrations of sodium chloride, addition of pyrophosphate (plus magnesium chloride) had the following effects: (1) both the protein extractability of non-heated myofibrils and the storage moduli of heat-induced gels were markedly increased; (2) the apparent activation energy for gel formation was decreased. Increasing concentrations of sodium chloride, up to 0.5-0.6M, increased the protein concentration of the liquid phase of the gels.     

Transaminases of Skeletal Muscle. 1. The Activity of Transaminases in Post-Mortem Bovine and Porcine Muscles

SUMMARY: The activity of glutamic-oxaloacetic transaminase (GOT) and glutamicpyruvic transaminase (GPT) of bovine and porcine muscle tissue and muscle press juice was determined. The total GPT activity of muscle tissue is about one tenth of the GOT activity. There are no remarkable differences in the activities of GOT and GPT between these slaughter animals and other species (rat, rabbit and man). The GOT activity of the longissimus dorsi muscle of pigs is significantly higher than that of the same bovine muscle. The mitochondrial (GOT_M) and sarcoplasmic isozymes (GOT_B) of GOT in skeletal muscles of cattle and pigs were determined after electrophoretic separation. The ratio GOT_M:GOT_S in skeletal muscle was found to be about 1:1. There is only a small decrease in GOT activty during storage of muscle tissue at 0 or +4°C for several weeks postmortem. The small activity of GOT_M in the muscle press juice does not substantially change during storage of muscle tissue under these conditions, indicating that there is no drastic change of the mitochondrial structure during aging of meat. Bacterial spoilage of meat, however, results in the release of GOT_M from the mitochondria.     

Post-Mortem Softening of Fish Muscle During Chilled Storage as Affected by Bleeding

Bleeding caused the delay of muscle softening in yellowtail, horse mackerel, and striped jack, which are pelagic fish. Conversely, bleeding had no influence on the muscle firmness of red sea bream, flatfish, and rudder-fish, which are demersal fish. Transmission electron microscopy showed delay of degradation of pericellular collagen fibrils in bled yellowtail and horse mackerel. Striped jack showed slower weakening of the pericellular connective tissue in a compression test. However, the demersal fish had no structural difference due to bleeding. These results indicate that removal of blood could delay collagen fibril degradation and muscle softening of pelagic fish.