Actomyosin denaturation

Electron microscopic analyses of isolated preparations of fish actomyosin denatured by frozen storage (68,72-74) showed that actomyosin filaments with arrowhead structures aggregated side-to-side and crosswise when thawed immediately after freezing. As time of frozen storage increased further aggregations formed network structures (Figure 3). 

When 0.1 M sodium glutamate was added to carp actomyosin, denaturation during frozen storage was almost eliminated, as measured by changes in solubility, viscosity, ultracentrifugal behavior, ATPase activity and electron microscopic profiles (66,72) (Figure 3). This protective effect of sodium glutamate will be discussed below. 

Actomyosin denatures also in situ under the influence of hypertonic salt solutions. When cod muscle is immersed in various concentrations of sodium chloride, there is a critical salt content in the fillet (8% to 10% NaCl) at which denaturation occurs together with a rapid loss of water and uptake of salt (Duerr and Dyer, 1952 Fougere, 1952). In herring, however, this critical salt concentration is much lower (3 % NaCl) (Nikkila and Linko, 1954b). The stability of the native configuration appears very variable, but we are unable to explain such differences. The need for a better knowledge of the protein itself is clearly stressed by these researches. Let us now consider the recent progress made in this direction. 

Actomyosin. Frequently, the change in amount of soluble actomyo-sin is regarded as the primary criterion of freeze denaturation. It must be remembered that solubility data do not tell precisely how much protein is denatured and how much is native rather, it provides a relative measure of denaturation. Solubility decreases have been found in frozen storage experiments with either intact muscle, protein solutions or with suspensions of isolated actomyosin. 

On the other hand, King (69) and Anderson and coworkers (70,71), based on detailed analyses of ultracentrifugal patterns of extracts of frozen stored cod muscle and experiments on the effect of lipids on protein denaturation, have proposed that denaturation of F-actomyosin occurs by two parallel pathways which lead to insolubilization (Figure 2). As indicated by Connell (61), the occurrence of G-actomyosin at an intermediary stage needs experimental verification. Possibility of an alternate pathway involving lipids will be discussed later. 

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. 

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). 

Analysis of the rates of insolubilization of isolated carp actomyosin indicated that denaturation proceeds by two or more first order processes with different rate constants (73). 

Some enzymes and enzyme systems are still active at the temperature of frozen storage (123-132). Such enzymatic activity, especially of proteases, may cause loss of biological activity of actomyosin and other muscle proteins. Products of such enzymatic activity, e.g. free fatty acids and formaldehyde, may effect a secondary denaturation of muscle proteins. 

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. 

Denaturation of myosin and actomyosin has so far been ascribed to intermolecular aggregation, but recent investigations have shown that intramolecular transconformation, the unfolding of the polypeptide chains, occurs in globular proteins and in subunits with globular structures. 

It was proven over 20 years ago that both native and denatured proteins bind volatile flavouring substances [7,9,12,32-34], Studies at the beginning of the 1980 s revealed that aldehydes (such as nonanal) and ketones (such as 2-heptanone, 2-octanone, 2-nonanone) formed exceptionally strong bonds to native proteins (soy protein, bovine serum albumin, fish actomyosin) in aqueous systems [35-39],... 

The investigations relative to fish actomyosin have first been oriented towards the study of its denaturation. Fish actomyosin becomes easily insoluble in salt solutions and its instability is increased by successive precipitations (Bailey, 1944 Atlantic Fisheries Experimental Station, 1953). This change of solubility has been used to study its denaturation. 

Dyers outstanding work stimulated many studies of a similar nature leading to the firm conclusion that denaturation of actomyosin occurs at a significant rate during frozen storage of fish muscle and that the rate of denaturation can be used to estimate the rate of quality change of the fish. 

Most of the early work on this subject was done by extracting actomyosin from fish fillets following frozen storage and thawing. On the other hand, many studies designed to determine the mechanisms of denaturation have involved frozen solutions or suspensions of isolated protein preparations. 

Effect of Mineral Salts. As cellular water is frozen, mineral salts and soluble-organic substances become concentrated in the remaining unfrozen phase. This increase in solute concentration, with corresponding changes in ionic strength and pH, is believed to affect dissociation and/or denaturation of proteins (1,2,62,96-98). Experiments by Fukumi et al. (99) support this theory. They found that freeze denaturation of washed actomyosin from Alaska pollack muscle was accelerated by the presence of Ca2+, Mg2+, K+, and Na+ ions and reduced by their removal. 

Pig. 23. Sedimentation curves of L-myosin and actomyosin. Points O,pure, homogeneous L-myosin V, denatured, homogeneous L-myosin A, components from mixtures of pure and denatured L-myosin 4 homogeneous natural actomyosins -(-, natural actomyosins with two components a, actomyosin from actin and L-myosin. Curve 1 L-myosin curve la, denatured L-myosin of Sjo = 15 curves 2, 3, and 4 actomyosin. The broken curve 2 is extrapolated by means of the... 

When actin and myosin have once combined to give actomyosin, it is not possible by any known method to separate them completely on a preparative scale. There is no doubt, however, that natural actomyosin is really a complex of actin and mj osin, (a) because Straub (1942) obtained in small yield from actomyosin the same actin as obtained from the dry acetone powder of muscle, and (b) natural and artificial actomyosins react with ATP in the same typical manner (Section III, 5d). It can therefore be concluded that complex formation is thermodynamically irreversible, for by repeated fractional precipitation a preparation can be obtained from muscle extracts in which no free L-myosin can be detected by methods at present available. The ultracentrifugal peak of L-myosin reappears, however, when the actomyosin in solution by its history and its properties, e.g., disappearance of ATP-sensitivity, may be regarded as denatured (Portzehl et al., 1950 see also Johnson and Landolt, 1950). 

The actomyosin fraction can be freed from L-myosin by further reprecipitation. It too tends to denature on repeated precipitation and on ageing, and although the actual viscosity does not change much, the ATP sensitivity decreases at first slowly, and then more rapidly. When... 

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