Myosin storage

Contraction of muscle follows an increase of Ca " in the muscle cell as a result of nerve stimulation. This initiates processes which cause the proteins myosin and actin to be drawn together making the cell shorter and thicker. The return of the Ca " to its storage site, the sarcoplasmic reticulum, by an active pump mechanism allows the contracted muscle to relax (27). Calcium ion, also a factor in the release of acetylcholine on stimulation of nerve cells, influences the permeabiUty of cell membranes activates enzymes, such as adenosine triphosphatase (ATPase), Hpase, and some proteolytic enzymes and facihtates intestinal absorption of vitamin B 2 [68-19-9] (28). 

In nature, extended helical conformations appear to be utilized in two major ways to provide linear systems for the storage, duplication, and transmission of information (DNA, RNA), and to provide inelastic fibers for the generation and transmission of forces (F-actin, myosin, and collagen). 

Storage Structural Contractile Ferritin Collagen (tendons), keratin (hair) Actin, myosin in muscle tissue... 

Actomyosin. At high salt concentrations ( . . 0.6 M KC1), actin and myosin combine to form actomyosin filaments giving a highly viscous solution. Actomyosin retains the ATPase activity of myosin and demonstrates "super-precipitation" on the addition of ATP (24,34). As expected, there are differences between actomyosins of rabbit and fish with respect to solubility (10,22,35,36), viscosity (46) and ultracentrifugal behavior (477. Since actomyosin is the most readily available form of myofibrillar proteins from fish muscle, its behavior relative to deterioration during frozen storage has been most frequently studied. 

Figure 2. Hypothetical mechanisms of aggregation of fish actomyosin during frozen storage. (A) King, 69 (B) Connell, 61 (C) Matsumoto ( proposal in the present paper). AM, actomyosin M, myosin MD1 and MDt, denatured myosin A, actin.

Jarenback and Liljemark (75,76) found similar changes in cod actomyosin solution and cod muscle during frozen storage. The denatured myosin was not extracted with salt solution. 

The above mentioned dissociation of actomyosin into actin and myosin could be due to a shift in the equilibrium, actomyosin 5= actin + myosin, by the highly concentrated salt solution of the unfrozen liquid portion in the protein-water system (22, 77). However, if this is true, the dissociated actin and myosin must re-associate immediately after thawing. This may be difficult since the ability to associate is decreased during frozen storage. 

ATPase activity, another property of myosin related to its contractile function, as is the actin-binding property, is also decreased by frozen storage. The specific ATPase activity of fish actomyosin decreases with increased time of frozen storage (66,67,72,78-82). This decrease should be due to a decrease in the ATPase activity of myosin. The rate of decrease is slower than that of free myosin (80,82). Connell (78) and Kawashima et al. (83) have detected some ATPase activity in insoluble aggregated actomyosin. 

Connell has proposed that insolubilization of actomyosin during frozen storage of cod muscle is attributable to the denaturation of myosin rather than actin (89). During 40 weeks storage at -14°C, extractability of actomyosin and myosin decreased in parallel, while that of actin appeared to remain constant. The decrease in extractability of myosin was biphasic, while that of actomyosin followed an exponential curve. 

However, our work on in vitro frozen storage of isolated carp actomyosin showed that actin is denatured progressively with myosin as demonstrated by SDS-polyacrylamide disc gel electrophoresis (90). 

Changes in solubility, viscosity, ATPase activity, and ultracentrifugal and salting-out profiles were found during frozen storage at -20°C of carp myosin solutions (in 0.6 M KC1) and carp myosin suspensions (in 0.05 M KC1) (82,93). 

After frozen storage of myosin solutions and suspensions of the filaments, reconstitution of filaments was attempted. The filament suspensions were thawed, dissolved in 0.6 M KC1, and then examined for reconstitution at low ionic strength. Filaments formed from either frozen-stored samples (filaments or myosin solution) were not as perfect in shape as those prepared from unfrozen, intact myosin. The spindle-shaped myosin was more stable in frozen storage than the dumbbell-shaped myosin. Dissolved... 

When 0.1 M sodium glutamate was added to solutions of myosin prior to freezing, solubility, viscosity, ATPase activity and filament-forming capacity remained at the level observed before frozen storage (82). 

HMM and LMM were stored frozen at -20°C, and the changes in properties were followed for each protein (82). While there were no significant changes in the solubility curves for HMM and LMM, appreciable changes were found in other properties. ATPase activity of HMM decreased to 50% of the pre-freezing value after 1 day and was not detectable after 2 weeks. No initial increase in activity was found with HMM. The rate of decrease in ATPase activity was much faster than with myosin solutions, where about 55% of the initial activity was retained after 7 days frozen storage. The ability of HMM to bind with F-actin, as determined by electron microscopy, was lost after 2 weeks frozen storage. 

Figure 4. Electron micrographs of reconstituted spindle-shaped filaments of carp myosin before and after frozen storage in 0.05M KCl at —20°C. A and B, no additives C and D, 0.2M sodium glutamate added. A and C, reconstituted before freezing B and D, reconstituted after 2 and 6 weeks of frozen storage, respectively stained (82).

Tropomyosin and troponin. Tropomyosin is apparently the most stable of the fish fibrillar proteins during frozen storage. It can be extracted long after actin and myosin become inextract-able however, it does denature gradually (90). 

The results with carp actomyosin after various times of frozen storage are shown in Figure 8. Different solvents gave different results. Myosin gave similar patterns. 

These results led to the conclusion that denaturation and/or insolubilization of actomyosin and myosin during frozen storage is a result of aggregation caused by the progressive increase in intermolecular crosslinkages due to formation of hydrogen bonds, ionic bonds, hydrophobic bonds and disulfide bonds. 

Among the above hypotheses, effects of lipids (4-17,59-62, 69-71,155-159), formaldehyde (160-166), and gas-solid interface TMJ appear to be very important in Gadoid fishes. Denaturation of myofibrillar proteins caused by free fatty acids and/or lipid peroxides must occur during frozen storage. To prove this, Jarenback and Liljemark have shown by electron microscopy that, in muscle stored frozen with added linoleic and linolenic hydroperoxides, myosin became resistant to extraction with salt solution (168). 

Data on myosin (50,51,82,91) and LMM (82) support side-to-side aggregation of molecules without appreciable change in conformation during frozen storage, as proposed by Connell (91). 

Unfolding of globular proteins and subunits. Data on frozen storage of HMM, actin and sarcoplasmic enzymes have led us to propose that denaturation involves unfolding of the protein chain based on a decrease in enzymatic activity (myosin, HMM, and sarcoplasmic enzymes), polymerizing ability (actin) and filament forming properties (myosin) (82,99,113-116,122). 

Oscillatory shear data on dilute solutions of the stiff biopolymer myosin, from R. W. Rosser et al., Macromolecules 11, 1239 (1978). Open circles are the dimensionless storage... 

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