Myosin heavy chain isoform composition influences the susceptibility of actin-activated S1 ATPase and myofibrillar ATPase to pH inactivation

The objectives of this study were to determine the influence of pH and MyHC isoforms on myofibrillar and actin-activated myosin subfragment 1 (S1) ATPase activity and the protective effect of actin. Red (RST) semitendinosus and white (WST) semitendinosus myofibrils were incubated at pH 7, 6, or 5.5 with 0 or 2mM ATP. RST and WST S1 isolates were incubated at pH 7, 6, or 5.5 in the presence or absence of actin. Maximum calcium-activated myofibrillar and actin-activated S1-ATPase activity were then assayed at pH 7. Incubation of myofibrils with ATP caused ATPase activity of myofibrils to decrease (p<0.05) with the pH of the incubation. RST myofibrils maintained a higher (p<0.0001) relative activity than WST myofibrils after incubation at pH 6 with ATP. Myofibrils incubated without ATP exhibited higher (p<0.001) activities than those incubated with ATP following pH 5.5 treatments. WST myofibrils had a lower (p<0.05) relative activity than RST following incubation at pH 5.5 without ATP. S1 ATPase activities decreased (p<0.05) with incubation pH in WST samples, but not in RST samples. WST S1 activity was higher (p<0.01) in samples exposed to pH 6 and 5.5 with actin bound compared to those incubated without actin. RST S1 exhibited a higher (p<0.01) relative activity than WST samples following pH 5.5 treatment with bound actin. These data show that low pH inactivates myofibrils by altering actin-activated S1 ATPase. Furthermore, these results suggest that muscles with high proportions of fast fibers are more susceptible to pH inactivation of ATPase activity and that the protective effect of actin binding to myosin is less in fast fibers.     

Method of isolation, rate of postmortem metabolism, and myosin heavy chain isoform composition influence ATPase activity of isolated porcine myofibrils

The objective of this study was to determine the influence of myofibril isolation procedures and myosin heavy chain (MyHC) isoform composition on myofibrillar ATPase activity as related to postmortem muscle metabolism. Myofibrils from the red (RST) and white (WST) portions of semitendinosus muscles were isolated using two different methods (A and B) at 3 min and 24 h postmortem in control (NS) and electrically stimulated (ES) pork carcasses. Comparison of the relative MyHC isoform profiles between the two different myofibril isolation methods and myosin extracts from the RST and WST at 3 min showed that method B myofibrils were more similar to the myosin extract than method A. Myofibrillar ATPase activity remained constant or increased (P<0.01) from 3 min to 24 h postmortem in NS carcasses and decreased (P<0.0001) in ES carcasses. From the RST, method A myofibrils had higher (P<0.0001) ATPase activity compared to method B across sampling time and carcass treatment. In the WST, method A myofibrils had lower (P<0.01) activity at 3 min, were not different at 24 h in NS carcasses, but had higher (P<0.05) activity at 24 h in ES carcasses versus method B myofibrils. Compared to method B, isolation method A biased the isoform profile of myofibril samples more towards faster MyHC (2A and 2X) in the RST and towards MyHC 2X in the WST. Results suggest that the ATPase activity and MyHC isoform profile of isolated myofibril samples are influenced by method of myofibril isolation, postmortem sampling time, and the rate of postmortem metabolism. Thus, differences in MyHC isoform profile and method of myofibril isolation must be taken into account to determine accurately the relationship between myofibrillar ATPase activity and rate of postmortem metabolism.     

Influence of myosin heavy chain isoform expression and postmortem metabolism on the ATPase activity of muscle fibers

The objective of this study was to determine the effects of postmortem muscle pH and temperature declines on the actomyosin ATPase activity of muscle fibers expressing different MyHC isoforms. Using a quantitative histochemical procedure to determine ATPase activity, the maximum actomyosin ATPase activity was determined on individual fibers classified by MyHC expression. Samples were collected from the red (RST) and white (WST) semitendinosus muscles at 3 min and 24 h postmortem from electrically stimulated (ES) and control (NS) pork carcasses. In samples taken at 3 min postmortem, type I fibers had the lowest ATPase activity staining and type 2X and 2B had the highest activity staining, with type 2A fibers intermediate. Postmortem time and carcass treatment did not influence the ATPase activity staining of type I muscle fibers. ATPase activity staining of 2A fibers was lower (p<0.001) in 24 h samples than in 3 min samples from ES carcasses. In 3 min and NS-24 h samples, RST type 2A fibers had lower (p<0.05) activities than type 2A fibers from the WST. In type 2X fibers, ATPase activity staining decreased (p<0.01) from 3 min to 24 h postmortem in ES carcasses. This decrease was more severe in WST 2X fibers compared to RST 2X fibers. ATPase activity staining in type 2B fibers did not decrease from 3 min to 24 h postmortem in NS carcasses. In ES carcasses, activity staining of 2B fibers decreased (p<0.0001) with time postmortem. The results of the experiment indicate that fibers expressing fast MyHC isoforms have a higher ATPase activity early postmortem than slow muscle fibers but are more prone to inactivation by a rapid pH decline.     

Emulsion properties of pork myofibrillar protein in combination with microbial transglutaminase and calcium alginate under various pH conditions

In this study, the effects of microbial transglutaminase (MTG) and calcium alginate (CA) systems in combination with soybean oil on the emulsion properties of porcine myofibrillar protein (MP) were evaluated under various pH conditions. MTG was shown to improve emulsifying capacity and creaming stability, which increased with increasing pH values up to 6.5. The CA did not influence emulsifying capacity, but it improved the creaming stability of the MP-stabilized emulsions. Both MTG and CA enhanced the rheological properties, but their effects on the physical characteristics of the protein evidenced an opposite trend in relation to pH, i.e., the MTG system improved both the emulsion and gelling properties with increasing pH, whereas the CA system was effective when the pH was lowered. By combining the two MP gelling systems, a stable and pH-insensible emulsion could be produced.     

Myosin light chain 1 release from myofibrillar fraction during postmortem aging is a potential indicator of proteolysis and tenderness of beef

The objective of this study was to identify proteins in bovine longissimus dorsi muscle that are related to tenderness. Two dimensional difference in gel electrophoresis (2D-DIGE) was used to compare the sarcoplasmic fractions from steaks that differed in star probe values at 14 days postmortem. The intensity of myosin light chain 1 (MLC1) was greater in the sarcoplasmic fraction prepared from steaks that had lower star probe values. It was hypothesized that μ-calpain catalyzes the release MLC1 into the sarcoplasmic fraction. Myofibrils from beef longissimus dorsi were purified and incubated with μ-calpain and the appropriate buffer controls. μ-Calpain was added at 1.23 μg (0.0875 U) of pure μ-calpain/mg myofibrillar protein. Incubations of one and 120 min had a greater abundance of MLC1 in the supernatants than the control incubations. As a consequence of μ-calpain proteolysis, MLC1 is rapidly released from the myofibril and is a potential indicator of proteolysis and improvement in beef tenderness.     

Proteolytic activity of isolated lamb calpains on myofibrils under the conditions of pH, Ca2+ concentration and temperature existing in postmortem muscle.

The two calcium-activated neutral proteinases (calpains I and II) and their specific inhibitor were isolated by ion exchange chromatography in DEAE-Sephacel from lamb skeletal muscle (longissimus dorsi). Their proteolytic activities were then determined using myofibrils as substrate. The Ca2+ requirements were different for each form of the enzyme: calpain I needed only 50 mumol Ca2+ for half-maximal activity, while the other isoenzyme, calpain II, needed 1,000 mumol Ca2+ for reaching 50% of its maximum activity. Both calpains showed a relevant activity in the pH range 5.5-6.5 (over 40% of maximum activity found at pH 7.5). With regard to the effect of temperature, both isoenzymes retained about 25% of their activity at 25 degrees C with a temperature reduction down to 4 degrees C. It is concluded that calpain I is an active protease under conditions similar to that prevalent in lamb meat during postmortem storage.     

Proteolytic activity of isolated lamb calpains on myofibrils under the conditions of pH, Ca2+ concentration and temperature existing in postmortem muscle

Summary The two calcium-activated neutral proteinases (calpains I and II) and their specific inhibitor were isolated by ion exchange chromatography in DEAE-Sephacel from lamb skeletal muscle (longissimus dorsi). Their proteolytic activities were then determined using myofibrils as substrate. The Ca²⁺ requirements were different for each form of the enzyme: calpain I needed only 50 mol Ca²⁺ for half-maximal activity, while the other isoenzyme, calpain II, needed 1000 mol Ca²⁺ for reaching 50% of its maximum activity. Both calpains showed a relevant activity in the pH range 5.5–6.5 (over 40% of maximum activity found at pH 7.5). With regard to the effect of temperature, both isoenzymes retained about 25% of their activity at 25° C with a temperature reduction down to 4° C. It is concluded that calpain I is an active protease under conditions similar to that prevalent in lamb meat during postmortem storage.

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Proteolytische Aktivität an Myofibrillen von aus Lammfleisch isolierten Calpainen bei nach dem Schlachten herrschenden Bedingungen wie pH, Ca-Konzentrationen und Temperatur

Zusammenfassung Die zwei von Calcium abhängigen Neutralproteinasen Calpain I und II sowie ihre spezifischen Inhibitoren wurden mittels Ionaustauschchromatographie mit DEAE-Sephacel aus Lammskelettalmuskel (Longissimus dorsi) isoliert. Ihre proteolytische Aktivität wurde danach mit Myofibrillen als Substrat bestimmt. Die Calciumerfordernis war für jede Enzymart sehr unterschiedlich; Calpain I brauchte nur etwa 50 mol Ca²⁺ für halbmaximale Aktivität, während Calpain II 1000 mol Ca²⁺ benötigte, um 50% der Maximalaktivität zu erreichen. Beide Calpaine zeigten hervorragende Aktivität innerhalb des pH-Intervalls 5,5–6,5 (über 40% der Höchstaktivität bei pH 7,5). Bezüglich des Einflusses der Temperatur hatten beide Calpaine rund 25% ihrer Aktivität bei 25 °C nach einer Temperaturverminderung bis zu 4 °C. Danach ist Calpain I eine aktive Protease während der postmortalen Reifung unter gleichartigen Zuständen im Lammfleisch.

     

Performance of a photochromic time–temperature indicator under simulated fresh fish supply chain conditions

The objective of this study was to investigate the performance of a photochromic time–temperature indicator (TTI) under dynamic temperature conditions simulating real fresh fish distribution chain scenarios. The work aimed at testing the possibility of extending the application of the TTI kinetic model, developed for specific temperature range of isothermal conditions, at low temperatures. The results showed that the TTI presented reproducible responses after being charged and during the discolouration process under different conditions, which revealed the reliability of the indicator. The TTI reflected well the temperature conditions of the studied scenarios, which indicates its potential application to continuously monitor the temperature history of the fresh fish supply chain. The kinetic model gave good fits in non‐abused scenarios at temperatures below 2 °C, presenting the potential for application of the model in determining the right charging level to suit a product’s shelf life at low temperatures.     

Effect of muscle lysosomal enzymes and calcium activated neutral proteinase on myofibrillar ATPase activity: Relationship with ageing changes

Myofibrillar ATPase activities have been used to study the in vitro effect of Ca-ANP (Calcium Activated Neutral Proteinase) and lysosomal proteolytic enzymes on the myofibrils at their respective optimal conditions activity. It appeared that the effect of Post-mortem ageing of meat on the MgCa and Mg-EGTA modified ATPase activities can be reproduced by incubating myofibrils with these hydrolytic systems. However, the effect of ageing on the Ca(++)-enhanced ATPase activity of myofibrils was not explained. It was concluded that Ca-ANP and at least one lysosomal thiol protease could be involved in the ageing process.     

Correlation between antiradical activity and stability of betanine from Beta vulgaris L roots under different pH,temperature and light conditions

When the antiradical activity and stability of betanine were studied at pH values of 3.5 and 8.5 and temperatures of 25, 50 and 75 °C, the results showed that the antiradical activity was greater at acidic pH and lower at higher temperatures. At basic pH the activity of betanine correlated well with its stability at the three temperatures assayed, suggesting that the degradation products, betalamic acid (BA) and cyclo DOPA 5‐O‐β‐D ‐glucoside (CDG), did not contribute to this activity under the experimental conditions used. However, at acidic pH the degradation product, CDG, did seem to contribute to the antiradical activity. Furthermore, at pH 3.5, betanine stability was so great that light conditions had no effect on the antiradical activity. At basic pH, too, light had no effect on betanine activity owing to the high instability of the pigment. © 2001 Society of Chemical Industry     

Effect of electrical stimulation, high temperature conditions and ageing on muscle myofibrillar proteins

Electrical stimulation, when accompanied by high temperature incubation, was found to lower the extractability of myofibrillar proteins and increase their degradation, particularly that of the myosin component. Troponin T and myosin light chain 2 were similarly affected. Troponin T and troponins I and C were degraded earlier during ageing in electrically stimulated muscles incubated at high temperature than in conventionally chilled muscles.     

Behaviour of polydiacetylene vesicles under different conditions of temperature, pH and chemical components of milk

Blue polydiacetylene vesicles were studied with regard to their behaviour under variations in storage temperature, heating, potentiometric titration and in the presence of chemical components of milk, to evaluate their application as a sensor in the food industry. Vesicles were prepared using 10,12-pentacosadienoic acid (PCDA)/1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC). Their changes were monitored using UV-Vis absorption. Temperatures not exceeding 25°C did not cause colour change in PCDA/DMPC vesicles for a period of up to 60days of storage. Heating for 10min at 60 and 90°C, exposure to pH higher than 9.0 and the simulant solutions of the whey proteins, β-lactoglobulin and α-lactalbumin, promoted colour change from blue to red for the vesicles studied. The effects of routine factors on the characteristics and stability of polydiacetylene vesicles is important in defining the parameters related to their application as a sensor for the food industry.     

Short communication: Urea hydrolysis in dairy cattle manure under different temperature,urea, and pH conditions

The objective of the study was to quantify the rate of urea hydrolysis in dairy cattle manure under different initial urea concentration, temperature, and pH conditions. In particular, by varying all 3 factors simultaneously, the interactions between them could also be determined. Fresh feces and artificial urine solutions were combined into a slurry to characterize the rate of urea hydrolysis under 2 temperatures (15°C and 35°C), 3 urea concentrations in urine solutions (500, 1,000, and 1,500 mg of urea-N/dL), and 3 pH levels (6, 7, and 8). Urea N concentration in slurry was analyzed at 0.0167, 1, 2, 4, 6, 8, 12, 16, 20, and 24 h after initial mixing. A nonlinear mixed effects model was used to determine the effects of urea concentration, pH, and temperature treatments on the exponential rate of urea hydrolysis and to predict the hydrolysis rate for each treatment combination. We detected a significant interaction between pH and initial urea level. Increasing urea concentration from 1,000 to 1,500 mg of urea-N/dL decreased the rate of urea hydrolysis across all pH levels. Across all pH and initial urea levels, the rate of urea hydrolysis increased with temperature, but the effect of pH was only observed for pH 6 versus pH 8 at the intermediate initial urea concentration. The fast rates of urea hydrolysis indicate that urea was almost completely hydrolyzed within a few hours of urine mixing with feces. The estimated urea hydrolysis rates from this study are likely maximum rates because of the thorough mixing before each sampling. Although considerable mixing of feces and urine occurs on the barn floor of commercial dairy operations from cattle walking through the manure, such mixing may be not as quick and thorough as in this study. Consequently, the urea hydrolysis rates from this study indicate the maximum loss of urea and should be accounted for in management aimed at mitigating ammonia emissions from dairy cattle manure under similar urea concentration, pH, and temperature conditions reported in this experiment.     

A 1H-NMR study of bovine casein micelles; influence of pH, temperature and calcium ions on micellar structure

The influence of pH, temperature and Ca depletion on bovine casein micelle suspensions in D2O containing simulated milk ultrafiltrate was studied by 1H-NMR spectroscopy. In the pH range of 5.8-7.5 the spectrum of the micelles showed very little pH dependence, indicating that no changes occurred in the dynamic behaviour of the proteins constituting the micelle. The NMR spectrum of casein micelles was strongly temperature dependent, particularly in the temperature range of 60-98 degrees C. Increase in temperature resulted in a strong increase in spectral intensity concomitant with changes in the spectral characteristics. In micelle suspensions these changes were reversible, and indicated that at elevated temperatures the rigid structure of the casein micelle started to melt, leading to an increased mobility of appreciable parts of the proteins in the micelle. Ca depletion of the casein micelles by addition of EDTA resulted in an increase in spectral intensity, which arose from the presence of casein components in the serum phase. The spectrum of these serum phase particles resembled closely the spectrum of a solution of total casein in simulated milk ultrafiltrate and was quite different from the spectrum of casein micelles. The implications of these results with respect to models of the structure of bovine casein micelles are discussed.     

Effects of camptothecin, etoposide and Ca2+ on caspase-3 activity and myofibrillar disruption of chicken during postmortem ageing

Recently, a novel consideration has focused on the potential relationship of apoptosis and the protease caspases and the underlying mechanism for meat postmortem tenderization. In this study, apoptosis inducers, camptothecin and etoposide as well as Ca(2+) were used to treat chicken muscle immediately after slaughter and follow the changes in caspase-3 activities and changes in the myofibrillar structures during 7 days of ageing. All three treatments resulted in significantly higher caspase-3 activities during storage (p<0.05), with the natural substrates, whereas Western blotting analysis of the α-spectrin cleavage product, 120 kDa peptide (SBDP 120), showed that Ca(2+) was more effective than either camptothecin or etopside, and all were most active up to day 3 (p<0.01). According to SDS-PAGE, each treatment enhanced the accumulation of the 30 kD Troponin-T degradation product, especially during the first 3 days (p<0.05), and this was supported by the degradation of myofibrils observed by electron microscopy (TEM). TEM images showed the treatments resulted in enlargement of the I-bands and shrinkage of A-bands; however Z-lines were only slightly affected, even at day 7. The findings revealed that the three apoptosis inducers could increase myofibrillar dissociation and proteolysis during the first 3 days of chicken meat ageing. Because of the high activity of caspase-3 during the early postmortem period, it is possible that caspase-3 contributes to the conversion of muscle into meat.     

Effect of calcium lactate on m-calpain activity and protein degradation under oxidising conditions

In two experiments, the effects of calcium lactate (CAL) on calpain activity were determined. In the model system, purified porcine skeletal muscle m-calpain was pre-incubated with various combinations of hydrogen peroxide (H₂O₂), calcium chloride, and/or different CAL concentrations. The m-calpain was activated by CAL, and the extent of m-calpain oxidation by H₂O₂ was significantly decreased with increasing CAL concentrations. In the muscle system, a beef longissimus lumborum (1 day postmortem) from each carcass (n = 6) was cut in half, randomly assigned to either 0.2 M CAL or water injection (WAT), and then packaged in a high-oxygen modified atmosphere. The CAL injected steaks resulted in less intact desmin and greater production of a 30-kDa troponin-T compared to the steaks in the WAT group. The CAL injection did not affect colour and lipid oxidation of steaks during display. These results suggest that CAL addition may improve tenderness of meat by enhancing activation of endogenous calpain and by protecting against calpain oxidation under oxidative conditions.     

酸的种类、pH和温度对酸性纤维素酶活力的影响

以8种酸(甲酸、冰醋酸、羟基乙酸、乳酸、柠檬酸、酒石酸、氨基乙酸和氨基磺酸)配制成pH=5.0的缓冲液,测定酸性纤维素酶在各缓冲液中的活力.同时以冰醋酸作为pH调节剂,采用中心合成设计法,分析和优化pH和温度对酶活力的影响,得出线性回归方程和优化值.结果表明,在pH=5.0的条件下,乳酸和酒石酸为酸剂的酶活力高于冰醋酸,羟基乙酸、甲酸和柠檬酸比冰醋酸略低,而氨基乙酸和氨基磺酸明显低于冰醋酸;采用冰醋酸调节pH,酶活力随着温度的升高和pH的降低而增加,pH影响的显著性要大于温度.酸性纤维素酶在49.8℃,pH=4.8可以实现最佳的活力.     

Growth kinetics of Vibrio parahaemolyticus O3:K6 under varying conditions of pH, NaCl concentration and temperature

The growth responses of Vibrio parahamolyticus to pH, NaCl concentration and temperature changes were studied using serotype O3:K6 and other strains. Growth curves were obtained for 27 different sets of conditions, comprised of three levels of NaCl concentration, pH and temperature. The temperature, pH and NaCl concentrations most favorable for growth were in the order of 25 degrees C, 20 degrees C and 15 degrees C, pH 8, 7 and 5.8, and 1%, 3% and 7%, respectively. The bacteria grew most rapidly at 25 degrees C, at a pH of 7 or 8 in the presence of 1% or 3% NaCl, with the population (initial, ca. 2.5 log CFU/mL) reaching a level log 7 CFU/mL at 12 h. A growth predictive model using the Gompertz equation was generated from the experimental data for any combination of NaCl concentration, pH and temperature within the range used in this study.