Myofibrils isolation

In 1987 the gene responsible for muscular dystrophy was identified, leading to the isolation of a protein, known as dystrophin, which is either totally absent in Duchenne, or partially absent in the Becker type. The protein is located on the inside of the plasma membrane of all muscles (and some neurones). Although its precise function is not known, the mutant form results in structural abnormalities of the plasma member which results in degradation of myofibrils, but the hnk between the abnormalities of the membrane and degradation is not known. One theory is that it leads to an increase in the activity of a Ca " ion channel in the membrane and, therefore, a marked increase in the Ca ion concentration in the cytosol. This chronic elevation results in the activation of calpain, which leads to protein breakdown and the degeneration within the fibre (Chapter 13). 

That actin and myosin are jointly responsible for contraction was demonstrated long before the fine structure of the myofibril became known. In about 1929, ATP was recognized as the energy source for muscle contraction, but it was not until 10 years later that Engelhardt and Ljubimowa showed that isolated myosin preparations catalyzed the hydrolysis of ATP.138 Szent-Gyorgi139 140 showed that a combination of the two proteins actin (discovered by F. Straub141) and myosin was required for Mg2+-stimulated ATP hydrolysis (ATPase activity). He called this combination actomyosin. 

In summary, a,-adrenergic inotropic responses in the A KO and AB KO reveal negative contractile effects in RV trabeculae mediated by the A or the B, primarily because of decreases in Ca2+ sensitivity of the myofibrils, and positive contractile effects of the A or the B in the isolated heart, with unknown mechanism. The D in coronary arteries mediates flow reductions and secondary negative contractile effects. 

The conversion of a-connectin to )8-connectin was confirmed using chicken breast muscle. The change was complete for whole muscle stored for 1 day at 0°C, and for isolated myofibrils stored for 3 days at O C (Hu et al., 1984). In myofibrils, addition of 10 mM EGTA retarded the conversion but did not prevent it completely. It is very likely that )8-connectin is a proteolytic product of a-connectin (mother molecule of connectin). However, it is not clear whether j8-connectin is functional in situ or not. It should be mentioned that there is a minor band just below /3-connectin in the total SDS extract of adult chicken muscle (Yoshidomi et al., 1985). This was not likely to be a proteolytic product of a- or j8-connectin, because it was still present unchanged in isolated /3-connectin. 

MSG is also an effective cryoprotective agent for myosin, HMM, LMM, and actin that have been isolated from carp muscle (63,64). Collectively, these results suggest that the cryoprotective effect of MSG extends to each constituent protein and each subunit of the myofibrils. 

The myofibrillar proteins make up 50-60% of the total protein of muscle cells. Insoluble at low ionic strengths, these proteins dissolve when the ionic strength exceeds 0.3 and can be extracted with salt solutions. Analysis of isolated mammalian myofibrils shows that nine proteins account for 96% or more of the protein myosin, which constitutes the bulk of the thick filaments, accounts for 43% and actin, the principal component of the thin filaments, 22%. 

Incubation of rat aortic rings with palytoxin led to microvesiculation of the endothelial cell cytoplasm [87], Dilatation of the sarcoplasmic reticulum, densification of mitochondrial cristae (possibly reflecting calcium uptake by the tissue), and disruption of myofibrils were observed in isolated rat muscle [88], Addition of palytoxin to the perfusion medium of the perfused heart caused cardiac arrest within minutes [13], and the spontaneous beating of isolated rat auricles was rapidly inhibited [9],... 

Liu G, Xiong YL, Butterfield DA (2000) Chemical, physical, and gel-forming properties of oxidized myofibrils and whey- and soy-protein isolates. J Food Sci 65 811-818... 

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