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.     

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: 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.     

POST-MORTEM ISOMETRIC TENSION CHANGES AND SHORTENING IN TURKEY MUSCLE STRIPS HELD AT VARIOUS TEMPERATURES

SUMMARY: A study of the physical changes associated with rigor mortis in breast muscle was undertaken to assess the factors that may influence ultimate tenderness. Isometric tension changes and shortening were measured at temperatures 2–37°C. These changes were measured while holding the muscle strips in a phosphate buffer, pH 7.2. Isometric tension was measured by transducers and recorded on a physiograph. A pattern of tension development and gradual relaxation has been demonstrated to occur post-mortem in strips of turkey breast muscle held isometrically. The time to maximum tension development occurs in 3.85 ± 0.19 hr and is not linearly related ( P <.05) to temperature. The amount of maximum tension developed averaged 25 g/cm² and was significantly ( P < .05) related to temperature. Relaxation to about 50% of maximum occurs in 18 hr. The amount of shortening that occurs post-mortem is linearly related ( P < .01) to temperature. No "cold shortening" of turkey breast muscle was evident.     

Pacific Cod Muscle 5'-Nucleotidase

SUMMARY: A 5'-nucleotidase, widely distributed in teleost fish muscles, was purified about 20-fold from Pacific cod (Gadus macrocephalus) by chromatography of a dialyzed aqueous extract of the muscle on DEAE-cellulose. The enzyme was unstable and lost 85% of its activity in 1 hr at 37°C 53% in 10 min at 42°C and 40% in 1 hr at 30°C. It was stable for 6 days at 0°C, could be dialyzed for up to 3 days at 0°C against 1 mM tris buffer pH 7.5 and quickly frozen and thawed without loss of activity. However, it was inactivated rapidly when held at −30°C. Brief exposure to pH 4.0 or 5.0 effected marked destruction. Attempts at further purification by means of chromatography on hydroxylapatite, adsorption using alumina Cγ and starch gel electrophoresis failed due to instability.
The enzyme was strongly inhibited by EDTA, pyrophosphate, KF and ZnCl₂ (1-10 mM); less markedly inhibited by GSH, 2-mercaptoethanol, carbonate and CaCl₂ (10 to 100 mM). It was strongly activated by Mn⁺⁺ and weakly activated by Mg⁺⁺. The optimum pH was 7.6, and the Km was 5 × 10⁻⁴M with UMP and 8 ⁻⁴M with IMP. It hydrolyzed, in order of effectiveness, LJMP, IMP, CMP, d-AMP, GMP, d-IMP, d-GMP, d-UMP and AMP, but not p-nitro phenylphosphate, sugar phosphates or a number of other compounds including 2',3'-nucleotides.     

Influence of Gonadal Stage of Hake (Merluccius Hubbsi Marini) on Biochemical Properties of Myofibrils Stored at 2 to 4 °C

ABSTRACT: The influence of the gonadal stage of hake on the biochemical properties of myofibrils stored at 2 to 4 °C was studied. At 0 time and during storage, both Mg²⁺-Ca²⁺-ATPase activity and Ca²⁺ sensitivity of myofibrils from post-spawned hake were significantly higher (p < 0.01) than those of pre-spawned fish. The profiles of SDS-PAGE gels of unstored and stored myofibrils from pre-spawned hake showed a partially denatured myosin heavy chain. The actin-myosin ratio in myofibrils from pre-spawned hake was significantly lower (p < 0.01) than the ratio in post-spawned hake. Irrespective of the gonadal condition of fish, no changes in the myosin-actin ratio of stored myofibrils were observed.     

Molecular Properties of Post-Mortem Muscle. 3. Electron Microscopy of Myofibrils

SUMMARY— A study was made of the fine structure of myofibril suspensions prepared from seven heifers immediately after death and after various times post-mortem. Studies on myofibrils sampled immediately after death showed that sucrose isolation gave the best structural preservation as indicated by maintenance of Z-line structure. Although the appearance of resting muscle was maintained in both sucrose and KCI preparations, several myofibrils from the KCI-treated preparations showed stretched sarcomeres. Glycerol-treated myofibrils usually had shorter sarcomere lengths than myofibrils prepared with the other two solvents. Although fibrillar preservation seemed adequate when glycerol was used, Z-line structure was seldom well-preserved with glycerol.
Myofibrils from muscle sampled 24 hr post-mortem at 2°C were supercontracted with thick filaments pushed against or through the Z-line, and no trace of l-bands remained. Myofibrils from muscle sampled 24 hr post-mortem at 16°C were contracted, but to a much lesser extent than 2°C-24 hr myofibrils. Storage at 2°C for 312 hr after death resulted in myofibrils that were contracted and that were structurally in a much poorer state of preservation than their 16°C counterparts. The 16°C-312 hr myofibrils were slightly contracted as indicated by the absence of H-zones and the presence of prominent, although narrowed, I-bands. All observations showed that shortening accompanying rigor mortis caused changes in banding patterns similar, and probably identical, to those predicted by Huxley's sliding filament model for contracting muscle.     

Studies on Bovine Natural Actomyosin 2. Physico-Chemical Properties and Tenderness of Muscle

SUMMARY: Studies were made of physicochemical characteristics of natural actomyosin from bovine longissimus of different post-mortem ages and tenderness classifications. Reduced viscosity, ATP sensitivity, and "actin" content (polyethylene sulfonate treatment) were higher for natural actomyosin prepared from muscle 12-24 hr post-mortem than from pre-rigor muscle, which confirms previous reports for rabbit natural actomyosin. A higher actin to myosin ratio in actomyosin from muscle 12-24 hr was therefore postulated. A stronger interaction of actin and myosin in actomyosin from muscle 12-24 hr post-mortem than from pre-rigor or aged muscle was also suggested by reduced viscosity and ultracentrifugation data. Reduced viscosity differences between actomyosins from tough and tender muscle suggested a higher gel character in actomyosin from tough muscle. This possibly indicated a higher content of α-actinin. No consistent differences in ATP sensitivity, myosin and actin content of natural actomyosin of tough and tender muscle were found. Natural actomyosin from muscle aged post-mortem showed the appearance during analytical ultracentrifugation of an additional component which sedimented at about 11S to 12S. This component appeared in the actomyosin prepared from tender muscle after 24 hr but did not appear until 10 days in the actomyosin from tough muscle.     

Post-Mortem Changes in Subcellular Fractions from Normal and Pale, Soft, Exudative Porcine Muscle. 1. Calcium Accumulation and Adenosine Triphosphatase Activities

SUMMARY– Myofibrillar, mitochondrial, heavy sarcoplasmic reticulum and light sarcoplasmic reticulum fractions were isolated by differential centrifugation of homogenates from normal and pale, soft, exudative (PSE) porcine muscle at various times post-mortem. Calcium uptake was measured using a solution containing⁴⁵Ca⁺⁺. The oxalate-stimulated calcium accumulating ability of the subcellular fractions declined 5-10 fold between 0 and 24 hr post-mortem. The major portion of this decline occurred in the first hour after death in fractions from PSE muscle but was more gradual in the normal fractions. The ATPase activities of normal and PSE fractions obtained at death did not differ significantly. These activities increased with time post-mortem in most normal fractions but decreased in those from PSE muscle. The subcellular site of ATP hydrolysis post-mortem was discussed. The results obtained point to the potential importance of the relaxing, factor in muscle post-mortem.     

ATPASE ACTIVITY, SURFACE HYDROPHOBICITY, SULFHYDRYL CONTENT AND PROTEIN DEGRADATION IN REFRIGERATED SEABASS MUSCLE IN MODIFIED ATMOSPHERE PACKAGING

The effect of modified atmosphere packaging (80% CO₂, 10% O₂, 10% N₂) on ATPase activity, surface hydrophobicity, sulfhydryl content and degradation of proteins in seabass muscle during storage at 4C was investigated. No changes in Ca²⁺-, Mg²⁺-, Mg²⁺-Ca²⁺-ATPase activities of natural actomyosin (NAM) in seabass slices kept under MAP were observed throughout the storage for up to 21 days (P > 0.05). However, a slightly increased Mg²⁺-EGTA-ATPase was found. For seabass slices stored under air atmosphere, Ca²⁺-ATPase activity decreased, whereas Mg²⁺-EGTA-ATPase activity increased (P < 0.05) with a concomitant loss in Ca²⁺-sensitivity. Lower decreases in total sulfhydryl content but higher increases in surface hydrophobicity were observed in samples stored under MAP, compared to those kept under air atmosphere. No marked autolytic degradation in samples kept under MAP was observed throughout the storage as monitored by no changes in myosin heavy chain, free α-amino acid and trichloroacetic acid soluble peptide. Conversely, a considerable degradation was found in samples kept under air atmosphere, especially after 9 days of storage. Therefore, MAP is a promising means to retard the changes in muscle proteins, especially degradation.     

Influence of the Physical State of Tissue during Rigor Mortis upon Protein Solubility and Associated Properties of Bovine Muscle

SUMMARY— Protein solubility and associated properties were studied in bovine sternomandibularis muscle allowed to pass into rigor in three physical states. Thirty min post-mortem, samples were incubated at 7°C for 48 hr in one of the following conditions: minced through 1/8-in. plate, free to shorten in a vertical position, stretched to 150% of equilibrium length. Stretched muscle exhibited greater protein solubility, higher pH values and longer sarcomeres than the remaining samples. For post-rigor muscle, protein solubility may be related to sarcomere length and moisture press ratio. Variations in sarcomere length may be related to post-mortem changes in pH. Possible relationships between the contractile state of proteins and the chemical, physical and quality characteristics of muscle are discussed.     

Sarcolemmae from Chicken Skeletal Muscle. 2. Properties

SUMMARY– Sarcolemmae are usually identified solely by morphological characteristics. We have determined several chemical and enzymic properties of sarcolemmae from chicken breast muscle prepared by homogenization of aged muscle in dilute CaCl₂ solution, washing 4 times in NaCl-histidine at pH 7.4, extraction with water buffered to pH 7 and isolation by differential centrifugation on a discontinuous sucrose gradient. The phospholipid content of the sarcolemmae was low, representing only 2 to 3% by weight compared with the 20 to 35% usually found in membraneous systems. This discrepancy may be due to the relatively small proportion of plasma membrane in the sarcolemma.
Analyses indicate little contamination by nuclei or mitochondria. The sarcolemmae have, like the microsomal fraction, high contents of RNA and glucose-6-phosphatase activity. The sarcolemma is either rich in these elements or is contaminated by other subcellular elements, such as the transverse tubules (T system), which are. The sarcolemmae display a Mg⁺²- activated ATPase activity which is typical for membraneous systems. Lactate dehydrogenase was shown to be associated with the sarcolemmae. Whether this represents the situation in vivo or is an artifact of preparation is not clear. The sarcolemmae are capable of binding soluble LDH.     

MOLECULAR PROPERTIES OF POST-MORTEM MUSCLE. 9. Effect of Temperature and pH on Tropomyosin-Troponin and Purified α-Actinin from Rabbit Muscle

SUMMARY– A study was done on the effects of in vitro storage of purified α-actinin, troponin, tropomyosin, and the tropomyosin-troponin complex on the activity of these protein fractions in the ATPase and superprecipitation assays. Storage was done at various combinations of temperatures between 0 and 40°C and pH values between 5.7 and 7.0. Even after 40 hr of storage, activities of purified tropomyosin and the tropomyosin-troponin complex were not affected by any combination of temperature and pH included in this study, but activities of purified α-actinin and troponin were almost completely lost after 16 hr at 40°C and pH 5.7. Storage for 40 hr at low pH (5.7) and low temperatures (0°C) did not affect the activity of either α-actinin or troponin, but 40 hr of storage at high temperatures (40°C) and neutral pH caused some loss in activity for both these proteins. This loss of activity caused by 40°C, pH 7.0 storage was much more noticeable in the case of troponin than in the case of α-actinin. Storage periods of 40 hr or longer were required before any loss of α-actinin activity could be detected at pH 7.0 and 40°C. Since most meat animal carcasses are chilled soon after exsanguination and attain muscle temperatures of 25°C or lower before the pH falls below 6.2, it is probable that α-actinin and tropomyosin-troponin activity remain almost unchanged in meat handled through normal market channels. However, myofibrillar tissue in those porcine animals whose musculature undergoes a very rapid post-mortem decline in pH so that values of 5.7 or less are reached while muscle temperatures are still 37°C or higher may lose much of its α-actinin and tropomyosin-troponin activity during the first 24 hr post-mortem.     

Calcium Chelators Influence Some Physical and Chemical Properties of Rabbit and Pig Muscle

SUMMARY: Intravenous antemortem injections of EDTA, EGTA and CDTA significantly inhibited muscle shortening during development of rigor mortis. Post-mortem micro-injections of CaCl₂ increased shortening, but MGCl₂ had no measurable effect. Even though average shear values for muscle from EDTA-treated rabbits were lower than those of untreated controls, the differences were not statistically significant. However, muscle from EDTA-treated pigs was significantly more tender than that from untreated controls. The interrelationships between ATP levels and pH values at 0 and 24 hr post-mortem were investigated and their effects upon muscle shortening and tenderness are considered and explained. A highly significant negative relationship was shown to exist between cooking losses and both initial pH and initial ATP values for both rabbit and pig muscle.     

A "Cold Shortening" Effect in Avian Muscle

SUMMARY— Experiments were conducted to determine the effect of post-mortem temperatures between 0 and 20°C on the degree of shortening in isolated pectoralis major muscles of chickens and turkeys. A "cold shortening" effect in these muscles is described and compared to post-mortem pH, average sarcomere length of isolated myofibrils, and relative solubility of myofibrillar and sarcoplasmic proteins.
The degree of muscle shortening at each temperature after various periods post-mortem indicated that shortening was essentially complete after 3 hr in chickens and 5 hr in turkeys. Shortening in muscles stored at 0°C was significantly greater (P < .01) than in the 12–18°C temperature range. Shortening was greatest in muscles stored at 20°C. The degree of gross shortening observed was directly related to the average sarcomere length of isolated myofibrils. Post-mortem decline in pH was not significantly correlated (P > .05) with shortening. Extractability of myofibrillar and sarcoplasmic proteins after 5 hr at either 0 or 16°C was determined and found to be unrelated to the degree of post-mortem shortening.     

EFFECT OF POSTMORTEM CONDITIONS ON CERTAIN CHEMICAL, MORPHOLOGICAL AND ORGANOLEPTIC PROPERTIES OF BOVINE MUSCLE

Paired sides from U.S. Choice grade beef were aged immediately after slaughter at 2 and 16°C. Samples were removed from longissimus and semitendinosus at slaughter and at 1, 3 and 7 days postmortem for ATPase assay, phase microscopy, shear and organoleptic evaluation. Rib steaks from sides aged at 16°C for 1-day postmortem were as tender as steaks from sides aged at 2°C for 7 days postmortem. Flavor development of rib steaks also was more rapid at 16°C than at 2°C. Tenderness of semitendinosus steaks was improved by aging sides at 16°C; the difference in improvement of tenderness of semitendinosus, however, was not as great between 2°nd 16° as it was for rib steaks. Ca⁺⁺, Mg⁺⁺ and EGTA-modified ATPase activity of myofibrils from both muscles increased with postmortem time, with myofibrils from muscles held at 16°C having slightly higher ATPase activity than myofibrils from muscles held at 2° Increased EGTA-modified ATPase activity was indicative of loss of calcium sensitivity of the myofibril. Sarcomeres of myofibrils from longissimus were longer at 1-day postmortem than those from at-death longissimus and they remained essentially unchanged during the remainder of postmortem aging; however, tenderness improved at 16°C for 1 day and at 2°C for 3 days. Also greater fragmentation of myofibrils from longissimus postmortem aged at 16°C for 1 day and at 2°C for 3 days was observed, suggesting that the rate of myofibril fragmentation is an important factor in tenderization.     

Effect of frozen storage on protein denaturation in bovine muscle 1. Myofibrillar ATPase activity and differential scanning calorimetric studies

The effect of frozen storage on myofibrillar ATPase activity and thermal transitions in bovine muscle was investigated. Myofibrillar ATPase activity and total enthalpy of denaturation (ΔH) decreased with time of storage. The rate of decrease was lower at −20°C than at −5°C or −10°C. Differences in behaviour during storage of muscle after fast or slow freezing could be attributed to differences in ice recrystallization. The observed decreases in area of the first peak seen in the thermograms and Ca²⁺-myo-fibrillar ATPase activity show that the myosin head denaturated progressively during storage. The myosin tail also denaturated during storage but the thin filament remained unaltered. Kinetic analysis suggested that the denaturation of the myofibrillar proteins took place through two consecutive first order reactions; an initial rapid reaction followed by a slower one.     

EFFECT OF POSTMORTEM AGING ON CHICKEN BREAST MUSCLE SARCOPLASMIC RETICULUM

FSR (fragmented sarcoplasmic reticulum) isolated from chicken breast muscle (Pectoralis major) at 0 hr, 48 hr and 7 day postmortem was purified using linear density gradient centrifugation. The Ca⁺⁺accumulating ability of the FSR was found to increase with postmortem aging. No loss in ATPase activity was noted nor was any significant change observed in the SDS-gel electrophoresis patterns of the proteins with postmortem aging. The FSR from the aged muscle contained a higher proportion of phospholipids These studies indicate that the Ca⁺⁺ sequestering properties of sarcoplasmic reticulum from chicken breast muscle are not impaired during postmortem aging.     

MYOSIN STABILITY IN INTACT CHICKEN MUSCLE AND A PROTEIN COMPONENT RELEASED AFTER AGING

SUMMARY– My osin from both red (myosin-R) and white (myosin-W) chicken muscle was extracted with Hasselbach-Schneider solution after the muscle had aged for 30 min and 24 hr post-mortem. Myosin-W from aged muscle contained a fast moving boundary in sedimentation velocity studies. When this fraction (component T) was separated on a DEAE-Sephadex column, the ATPase activity and sedimentation pattern of the resulting myosin were similar to those of chromatographed myosin from unaged muscle. The numbers of sulfhydryl groups in both myosin-R and myosin-W were not affected by aging. The extinction coefficient and absorption spectrum of component T were different from those of myosin, and component T had neither ATPase nor 5'-adenylic acid deaminase activity. Lowering the pH of myosin extracted from unaged muscle to the pH values found in muscle aged for 24 hr caused aggregation and loss of ATPase activity. These aggregates of myosin differed from component T in both sedimentation velocity and chromatographic pattern.     

Mechanisms of gelation of sardine proteins: influence of thermal processing and of various additives on the texture and protein solubility of kamaboko gels

Surimi prepared from freshly caught sardines was mixed with NaCl and other additives and used to prepare kamaboko gels. Protein-protein interactions involved in the setting (at 4 or 37°C) and/or the cooking (at 90°C) gelation steps were investigated (i) by assessment of kamaboko texture as a result of the type and concentration of additive added; (ii) by partial solubilization of kamaboko gels in buffers containing mercaptoethanol, sodium dodecyl sulphate (SDS) and/or urea, followed by determination of the soluble protein constituents by polyacrylamide gel electrophoresis. Cooked gels of high elasticity and of varying rigidity and gel strength were obtained in the 73–80% water range. Adequate gel texture required a NaCl content of 1.7–3.5% and a pH range of 6.4–8.4. Low concentrations of reducing agents (mercaptoethanol, dithiothreitol, cysteine) or of divalent cations (Ca²⁺, Mg²⁺) improved the texture of gels obtained by setting at 37°C with and without subsequent cooking at 90°C. On the other hand, the addition of N -ethyl maleimide or of ethylene diamine tetra-acetate led to texture deterioration after cooking. These data demonstrate the involvement of disulphide bonds and of electrostatic interactions in surimi gelation. Gel solubilization experiments indicate that the aggregation of myosin heavy chains through various types of protein-protein interactions may be responsible for the elastic gel network formed during setting at 37°C (30 min) or 4°C (24h). Strengthening of the gel network after cooking appears to be due to additional disulphide and hydrophobic interactions.