Actomyosin solubility

Actomyosin. Solubility. Studies have dealt with changes in the solubility of proteins during frozen storage of fish muscle or solutions of isolated actomyosin (33,51,52). Analysis by gel filtration of the salt extracts has shown that the actomyosin fraction decreases in solubility during frozen storage whereas the sarcoplasmic proteins remain essentially unchanged (53). 

Before selecting a method to measure a specific aspect of protein functionality, one must decide on the complexity of the testing matrix. Researchers have used a single purified protein, a crude extract of proteins, a prototype food product, or an actual product to study protein functionality. For meat studies, formulated meat systems, ground muscle, myofibrillar proteins, salt-soluble proteins, actomyosin,... 

Ryania. The root and stem of the plant Ryania speciosa, family Flacourtiaceae, native to South America, contain from 0.16—0.2% of insecticidal components, the most important of which is the alkaloid ryanodine [15662-33-9], C25H3509N (8) (mp 219-220°C). This compound is effective as both a contact and a stomach poison. Ryanodine is soluble in water, methyl alcohol, and most organic solvents but not in petroleum oils. It is more stable to the action of air and light than pyrethrum or rotenone and has considerable residual action. Ryania has an oral LD5Q to the rat of 750 mg/kg. The material has shown considerable promise in the control of the European com borer and codling moth and is used as a wettable powder of ground stems or as a methanolic extract. Ryanodine uncouples the ATP—AD P actomyosin cycle of striated muscle. 

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. 

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. 

Viscosity of soluble actomyosin fractions decreased with increasing time of storage (64-68). This suggests that the actomyosin filaments have become less rod-like or less filamentous either by individual molecular folding or by aggregation of the filaments. 

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. 

In 1959 Bettex-Galland and Liischer succeeded in extracting from human blood platelets such a (iontractile protein, which was subsecpiently named thrombosthenin. Its solubility properties, as well as its dependence for activity on the presence of ATP and metal ions, soon led to its classification as a member of the actomyosin group. Work on thrombosthenin has since continued, both with respect to its properties as a complex protein with enzymatic activity and to its biological significance. 

Actomyosin is generally extracted from fresh rabbit muscles by the use of buffered KCl solutions of an ionic strength of 0.5-0.6 y. (Weber-Edsall solution). The solubility curve of the isolated actomyosin at pH 7 shows an inflection at 0.25 y, above a value of 0.3 u the protein is completely soluble (Hasselbach et al., 1953). At low ionic strengths, actomyosin upon addition of ATP and provided Mg++ ions are present shows superprecipitation. By glycerol extraction, muscle fibers may be prepared to contain essentially only the contractile system. Such fibers will contract normally under the conditions mentioned above for the isolated actomyosin (Weber and Portzehl, 1952). The muscle fibril contains the actomyosin in the insoluble state and in an optimal spatial arrangement (cf. Section IV, A,2). 

Fresh uterus contains from 1.1 to 3.5 % contractile protein, which is extractable under the same conditions as the actomyosin from striated muscle (Table III). It is interesting to note that the solubility of this material changes in the course of gravidity, the actomyosin from nongravid uterus passing into solution at an ionic strength of 0.3-0.4 n, independent of the amount of ATP present. The material from gravid uterus is already soluble at 0.2-0.3 and its solubility is influenced by ATP (Ledermair, 1959). 

Most likely, proteins of the actomyosin type also exist in other smooth muscles. litiegg and Strassner (1963), for instance, have isolated from arterial walls a protein which is soluble at high ionic strengths and which exhibits ATPase activity. More detailed ijiformation about this material is not yet available, and therefore a comparison with the other contractile proteins is not possible at the present time. 

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. 

Solubility. The application to fish extracts of fractionation methods elaborated for the separation of rabbit actomyosin and myosin has allowed the comparison of the two actomyosins (Hamoir, 1955). The precipitation of actomyosin at neutral pH in presence of a phosphate-NaCl buffer is complete at M 0.25 in the case of the carp (see p. 249) and at p 0.30 in the case of the rabbit. A similar difference is observed with the phosphate-acetate method of fractionation (Hamoir, 1947). Carp actomyosin is therefore slightly, but definitely, more soluble than rabbit actomyosin. 

Viscosity. The reduced viscosity of the protein extracted from frozen-stored fish muscle (44,54) and of the soluble fraction of the frozen-stored solutions of isolated actomyosin decreases with increasing time of storage (51, 52). 

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. 

Findings in the authors laboratory (100) demonstrated that the number of cross bonds in carp actomyosin and myosin increases during frozen storage and that solubility of these proteins decreases. Based on the types of chemicals that resolubilized these proteins at various rates, it was concluded that ionic bonds, hydrogen bonds, covalent bonds (S-S), and hydrophobic associations all are involved in the aggregation process. 

Water Soluble Proteins. In studies with frozen minced Alaska pollack, the possibility has been raised that removal of water soluble proteins may render the residual actomyosin more stable to frozen storage (107). Whether the effect of the washing is because of the removal of the water-soluble proteins or of the organic substances of lower molecular weight is left to further study. 

Myosin phosphatases are prepared from both the soluble (cytosolic) and the myofibrillar fraction of smooth muscle. If the source of phosphatase is the soluble fraction, then chromatographic procedures are usually preceded by fractionation with ammonium sulfate. Myosin phosphatase from myofibrils, crude actomyosin, or myosin is solubilized at high ionic strength by 0.6 NaCl plus detergent (Alessi et al.,... 

There is no longer any doubt that actomyosin is identical with the S-myosins these actin-containing complexes will always be referred to as actomyosins. General data on these proteins e.g., solubility, viscosity, birefringence, will be entered in the Tables as L-myosin. and actomyosin, but footnotes will be added for data obtained on a and /3 myosins electrophoretic data will be discussed using Dubuisson s nomenclature, though for the sake of clarity actomyosin will be added in parentheses after a-myosin, and L-myosin after /3-myosin. 

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