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.     

Myosin heavy chain isoforms influence myofibrillar ATPase activity under simulated postmortem pH, calcium, and temperature conditions

The pH and Ca(2+) sensitivity of myofibrillar ATPase activity plays an integral role in regulating postmortem muscle ATP utilization and likely paces postmortem glycolysis. The objective of this study was to determine the influence of pH and Ca(2+) concentration on the ATPase activity of myofibrils from red semitendinosus (RST) and white semitendinosus (WST) porcine muscles. Myofibrillar ATPase was measured at 39 °C over a pH range 5-7.5 and a [Ca(2+)] range pCa 4-9 (10(-4)-10(-9)M). At maximum Ca(2+)-dependent activation (pCa 4), RST myofibrils had lower (p<0.0001) ATPase activity than WST myofibrils. This maximum activity of myofibrils from both muscle regions was not influenced from pH 7.5 to 6.5, declined between pH 6.5 and 5.75 (Hill coefficient, n(H)=2.7-3.4; pH at half maximum activity, pH(50)=5.97) and was near zero at pH 5.5. At pH 7, pCa-activity relationships showed that RST required less Ca(2+) for half-maximum activation (higher pCa(50); 6.50) than WST myofibrils (pCa(50)=6.35) but had no difference in n(H). At pH 7, both RST and WST myofibrils had maximum Ca(2+)-dependent, actin-activated ATPase activity at pCa ?6 and Ca(2+)-independent myosin ATPase activity at pCa ?6.75. pCa-activity relationships at different pH levels indicated that pCa(50) decreased with pH from pH 6.5 to 6.125 in both RST and WST myofibrils. At pH <5.75, [Ca(2+)] did not influence ATPase activity in RST or WST myofibrils. These data show that myofibrils with predominantly fast MyHC (WST) have a higher actin-activated myosin ATPase activity than myofibrils with primarily slow MyHC isoforms (RST) at Ca(2+) concentrations and pH values characteristic of postmortem muscle.     

Quantification of myosin heavy chain isoform in porcine muscle using an enzyme-linked immunosorbent assay

The aim of the present study (part of an EU AIR programme on boar taint) was to make objective the perception of boar taint in entire male pork, and to relate the perception to skatole and androstenone levels. Trained analytical sensory panels in seven European countries assessed pig meat with known levels of androstenone and skatole. The panels performed a sensory profiling using the attributes pig, urine, manure/stable, naphthalene/mothballs, rancid, sweet, sweat and abnormal, both for odour and flavour in separate sessions. It turned out to be difficult to harmonise sensory methodology for seven sensory panels throughout the EU, especially with respect to the exact level of training the panellists received. Sensory panels in general were able to differentiate between the two compounds and between different levels of the compounds, though substantial differences between the panels in the different countries existed. Androstenone was found to relate mostly to the urine attribute, while skatole related mostly to manure and, to a lesser extent, to naphthalene.     

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.     

Influence of myosin heavy- and light chain isoforms on early postmortem glycolytic rate and pork quality

This study addressed the influence of the content of myosin heavy- (MHC) and light chain (MLC) isoforms on early postmortem glycolytic rate and meat quality traits in the porcine longissimus muscle. The fast-glycolysing group showed lower contents of MHC slow and MLC 1s isoforms (P<0.05), and higher MHC fast isoform contents than the normal-glycolysing group (P<0.05). The MHC fast/slow ratio was correlated with lactate content (r=0.41) and early postmortem muscle pH (r=-0.51), and the content of the MLC 1s isoform was negatively correlated with lactate content and glycolytic potential (r=-0.38 and -0.36, respectively). Hence, both the MHC and MLC isoforms did influence metabolite contents, thus also affecting glycolytic rate, and suggested that the myosin isoforms, in particular the MHC isoforms, might also have some bearing on the extent of protein denaturation and pork quality during the early postmortem period.     

Muscle fiber type characterization and myosin heavy chain (MyHC) isoform expression in Mediterranean buffaloes

This study aimed to evaluate myosin heavy chain (MyHC) isoform expression and muscle fiber types of Longissimus dorsi (LD) and Semitendinosus (ST) in Mediterranean buffaloes and possible fibers muscles modulation according to different slaughter weights. The presence of MyHC IIb isoforms was not found. Only three isoforms of MyHC (IIa, IIx/d and I) were observed and their percentages did not vary significantly among slaughter weights. The confirmation of the presence of hybrid muscles fibers (IIA/X) in LD and ST muscles necessitated classifying the fiber types into fast and slow according to their contractile activity, by m-ATPase assay. For both muscles, the muscle fiber frequency was higher for fast than for slow fibers in all weight groups. There was a difference (P<0.05) in the frequency of LD and ST muscle fiber types according to slaughter weights, which demonstrate that the slaughter weight influences the profile of muscle fibers from buffaloes.     

Effects of myosin heavy chain isoforms on meat quality, fatty acid composition, and sensory evaluation in Berkshire pigs

The main objective of this study was to investigate the effects of myosin heavy chain (MHC) isoforms on meat and sensory quality in Berkshire pigs. A total of 85 pigs were evaluated, and muscle samples were taken for the analyses of MHC isoform, meat quality, fatty acid composition, and sensory evaluation. Content of the MHC slow isoform was significantly correlated with pH(24h) (r=0.26, P<0.05) and drip loss (r=-0.32, P<0.01), although the content of MHC isoforms showed limited relationships with individual fatty acids. In the case of sensory evaluation of meat by a trained panel test, the MHC fast/slow ratio was correlated with the juiciness (r=-0.33, P<0.01), off-flavor (r=0.34, P<0.01), tenderness attributes (r=-0.43 to -0.47). These results imply that the content of MHC isoforms can influence various aspects of quality including pork and sensory quality in Berkshire pigs.     

Evidence for expression of IIb myosin heavy chain isoform in some skeletal muscles of Blonde d’Aquitaine bulls

In cattle, expression of the IIb myosin heavy chain (MyHC) isoform has been demonstrated only in extraocular muscles. In this study, we demonstrated for the first time its expression in the Semitendinosus and Longissimus thoracis muscles of a French beef breed, Blonde d’Aquitaine. Several techniques were used: RT-PCR, electrophoresis, western blotting, histochemistry with ATPase staining and immunohistochemistry using a combination of anti MyHC antibodies on serial sections. We found that MyHC IIb was expressed at the mRNA level in the two muscles of the cattle studied. However, the protein was observed at the tissue and cellular levels in only five of the 22 young bulls analysed, suggesting complex regulation of its expression. Using immunohistochemistry we demonstrated the presence of this MyHC isoform in pure fibres and also in hybrid fibres in co-expression with other MyHC.     

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.     

The relation between glycogen, lactate content and muscle fiber type composition, and their influence on postmortem glycolytic rate and pork quality

This study examined the relation between glycogen, lactate content and muscle fiber type composition, and evaluated their influence on postmortem glycolytic rate and meat quality. Muscle samples were classified based on their glycogen and lactate content at 45 min postmortem. Muscles with low glycogen and high lactate levels showed low muscle pH_45 min and high R-values. However, muscles with low glycogen and lactate levels showed normal rates of postmortem glycolysis and normal meat quality. On the other hand, muscles with high glycogen and lactate content showed rapid postmortem glycolysis, paler surface color, higher drip loss, and higher extents of protein denaturation than muscles with high glycogen and low lactate content. These results may be partially explained by muscle fiber type composition. Muscles with low glycogen and lactate content at early postmortem are composed of significantly higher fiber type I and lower fiber type IIB as compared to muscles with high glycogen and lactate content.     

Myofibrillar 1-D fingerprints and myosin heavy chain MS analyses of beef loin at 36 h postmortem correlate with tenderness at 7 days

This study investigated the potential for relating changes in electrophoretic protein patterns derived from the longissimus dorsi of beef cattle 36 h postmortem with tenderness at 7 days. We report finding a significant correlation (R(2)=0.82) between electrophoretic l. dorsi myofibrillar fingerprints at 36 h postmortem and tenderness at 7 days, as determined by Warner-Bratzler shear analysis. In addition, we have used mass spectrometric analyses to identify fragments of bovine myosin heavy chain that are significantly correlated with tenderness. Furthermore, this method offers the potential to increase our understanding of the fundamental cellular mechanisms underlying the proteolytic breakdown of muscle proteins during the aging process.     

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.

     

Purification of transglutaminase and its effects on myosin heavy chain and actin of spent hens

The purpose of this study was to purify pig plasma transglutaminase (TGase) and examine its effects on the myosin heavy chain and actin of the breast muscles from spent hens at different temperatures. TGase (0.3 units/mg) was added to myofibrillar proteins solution (0.5 ml) at 4°C for 0, 4, 8, 12, 24 and 48 h; at 25°C for 0, 1, 2, 4, 8, 12, 24 and 48 h; at 37°C for 0, 5, 10, 30 and 60 min. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that plasma TGase was composed of units of molecular weights approximately 75,000 and 80,000. TGase added to the myofibrillar proteins solution indicated that the concentration of the myosin heavy chain and actin decreased when incubated at 4°C for 48 h and when incubated at 25°C for 2 h. Moreover, the relative intensity determined by scanning densitometry of the SDS-PAGE gel indicated that the myosin heavy chain and actin concentration decreased to 45 and 64%, respectively. In addition, the relative intensity of the myosin heavy chain and actin declined to 7 and 63%, respectively, when incubated at 37°C for 5 min. The relative intensity of both the myosin heavy chain and actin decreased with time when incubated at 25 and 37°C.     

The relationship between pig genetics, myosin heavy chain I, biochemical traits and quality of M. longissimus thoracis

The relationship between muscle biochemical traits, myosin heavy chain I and meat quality of longissimus thoracis was studied using gilts from five divergent porcine lines (A to E) (carcass weight: 83.7±8.7 kg). Intramuscular fat (IMF) and haem pigments content as well as myosin heavy chain 1 (MyHC I) percentage and the activity of lactate dehydrogenase (LDH) and isocitrate dehydrogenase (ICDH) were determined. Only group E, a well conformed line of pigs, included the halothane positive genotype. The presence of the Hal gene in this line resulted in meat of poorer quality in terms of meat of higher exudate compared to line D, also a well conformed line of predominantly Pietrain origin but being halothane gene free. Line C presented the highest IMF content (2.02%) as well as high oxidative characteristics (MyHC I, 10.0%; LDH/ICDH, 1.92 μmol nmol(-1); ICDH activity, 1.78 nmol min(-1) g(-1)) and the lowest drip losses (5.3%). According to a principal component analysis including MyHC I, biochemical traits and meat quality parameters, line C was characterized by a high IMF content and oxidative traits, and line B by a high glycolytic metabolism. Line E was distinguished by high drip losses and by low pH(45) and pH(u). In conclusion, several observed differences in muscle metabolism between lines free of the halothane gene (A, B, C and D) must be caused by other "genetic background" factors.     

Differences in molecular structure among the porcine myosin heavy chain-2a, -2x, and -2b isoforms

Full coding regions for fast type myosin heavy chain (MyHC) isoforms were sequenced from a porcine skeletal muscle to analyze sequence diversity relating to the contractile properties of muscle fibers. An approximately 6-kb fragment for each MyHC was amplified through RT-PCR using isoform type-specific primers, which were designed in the 5' and 3' non-coding regions of the porcine MyHCs. The lengths of deduced amino acid sequences were 1939, 1939, and 1937 for the porcine MyHC-2a,-2x, and-2b, respectively. The entire amino acid sequences were highly conserved among the three MyHCs, except for the 50/20 k junction region (loop 2) which would weakly bind actin molecules. The porcine MyHC-2b possessed different amino acids from MyHC-2a and-2x, in loop1 and ELC binding region. The sequence data suggested the diversity of contractile properties among the porcine MyHC isoforms.     

Relationships of myosin heavy chain fibre types to meat quality traits in traditional and modern pigs

Porcine skeletal muscle fibres were molecularly classified, using in situ hybridisation and immunocytochemistry, into four types, according to the isoform of myosin heavy chain (MyHC) that was present in each fibre (MyHC slow/I, MyHC 2a, MyHC 2x and MyHC 2b). The relationship between MyHC fibre types and meat quality traits between two phenotypically divergent muscles [longissimus dorsi (LD) and psoas], and between the same muscles of different breeds (traditional Berkshire and Tamworth, and modern Duroc-based and Large White-based) were examined. We found that the greater abundance of fast oxidative-glycolytic MyHC 2a and 2x fibres in the psoas was associated with superior meat quality traits, and that the greater presence of fast glycolytic MyHC 2b fibres in the LD could account for less favourable quality traits, both in terms of pH, drip loss, grain, colour, yield force and work done. Although significant correlations were found between specific fibre types and quality traits, within either the psoas or LD muscle of some breeds, no consistent correlation was found across both muscles and all breeds. This finding was in line with the view that a given fibre type could have considerable differences in phenotype between breeds, and between muscles. The observed inverse compositional and functional-meat quality relationship between MyHC 2b and 2x fibres, and MyHC 2b and 2a fibres could form a basis of fibre type manipulation to improve meat quality.     

The relationship between slow and fast myosin heavy chain content, calpastatin and meat tenderness in different ovine skeletal muscles

The present study investigated the relationship between fibre type distribution and slow (MHC-s) and fast (MHC-f) myosin heavy chain content on calpastatin and meat tenderness in longissimus dorsi (LD), tensor fasciae latae (TFL), semitendinosus (ST), trapezius (TZ) and supraspinatus (SS) muscles from six Mule×Charolais rams. Samples taken at slaughter were frozen either in liquid N(2) for analysis of MHC-s and MHC-f by immunoblotting, or in cooled isopentane for histochemical fibre typing. Calpastatin activity and an immunoreactive 135 kDa calpastatin band were analysed in samples taken 24 h postmortem. Shear force was determined on muscle chops taken at 24 h postmortem and conditioned until day 14. The intensity of MHC-s and MHC-f immunopositive bands correlated with %Type I and %Type II fibres identified histochemically (r(2)=0.612 and 0.366, respectively, p<0.001). Muscle specific differences were observed in MHC-s and MHC-f immunoreactivity, fibre type distribution, calpastatin activity, calpastatin 135 kDa immunoreactivity and shear force. MHC-s correlated positively with calpastatin activity (r(2)=0.725, p<0.001) and 135 kDa calpastatin (r(2)=0.228, p<0.01) across all muscle types. The data show that detection of MHC-s can be used to identify fibre type differences between ovine muscles and that this correlates with differences in calpastatin content and inhibitory activity, but not tenderness.     

猪胰脏淀粉酶的提取、部分纯化与性质研究

以猪胰脏为原料,采用异丙醇沉淀、硫酸铵盐溶盐析、透析和反渗透系列方法对淀粉酶提取纯化,并对其性质进行了研究。结果表明,制备的反渗透酶比活力达856U/mg,约为粗酶(146U/mg)的6倍,可替代市购粗淀粉酶用于实验,其最适温度和pH分别为37℃和6.9。     

重金属离子对鲫蛋白ATPase活性和SH基总量的影响

研究了体外添加Pb2+、Cd2+、Cu2+等不同浓度的重金属离子对肌动球蛋白和肌球蛋白ATPase活性和SH基总量的影响。随Pb2+和Cd2+浓度的升高,肌动球蛋白的ATPase活性和SH基总量先升高,后下降,肌球蛋白的ATPase活性逐渐降低,SH基量先略有升高,后下降。Cu2+浓度的升高引起蛋白中ATPase活性和SH基含量明显下降,显示了较强的毒性。