Actin extraction from muscle

Figure 15 shows the temperature dependence of the piezoelectric ooostani d for Psoas muscle with a few percent water content 27]. Tbe decieaae of d above (fC should be due to the conductivity of adsorbed water. The oriented fUnis of myosin and of actin extracted from muscle showed similar behavior [28]. 

The muscle fibrils are embedded in sarcoplasm, each individual fibril showing the banding pattern of the whole fiber (Bowman, 1840). Myosin could be extracted from muscle with strong salt solutions. From the altered appearance of the bands after extraction it was suggested that this protein was a major component of the A bands (Kuhne,1864 Danilewsky, 1881). The localization of myosin in the A bands and of actin in the I bands was convincingly shown by Jean Hanson and Hugh Huxley (1954-1955) in electron micrographs of transected fibers and confirmed after selective extraction to remove myosin (Hasselbach, 1953 Hanson and H.E. Huxley, 1953-1955). 

The fermentation broth of an unidentified endophytic fungus isolated from plants growing in Hawaii has afforded microcarpalide I (59), an alkyl-substituted nonenolide that is weakly cytotoxic as a result of its ability to disrupt microfilaments. Extracts from the strain 112/13 collected from the bark oiFicus microcarpa L. (Moraceae) demonstrated a strong abrogation of microfilament activity. In A-10 rat smooth muscle cells, a 5 Xg/mL dose was able to induce a 50%—75% loss of actin filaments. Microcarpalide 1... 

Most of the machinery of living cells is made of enzymes. Thousands of them have been extracted from cells and have been purified and crystallized. Many others are recognized only by their catalytic action and have not yet been isolated in pure form. Most enzymes are soluble globular proteins but an increasing number of RNA molecules are also being recognized as enzymes. Many structural proteins of the cell also act as catalysts. For example, the muscle proteins actin and myosin together catalyze the hydrolysis of ATP and link the hydrolysis to movement (Chapter 19). Catalysis is one of the most fundamental characteristics of life. 

Actin. Rabbit muscle G-actin is globular with a molecular weight of 4.2 X 104. In the presence of salts it is polymerized into F-action (34). The principal properties of fish actin (35-37,40, 43,44), including amino acid composition (41), are similar to rabbit actin, but fish actin is more readily extracted from wet muscle by salt solutions as a viscous solution of actomyosin (22,35,36,45). 

Myosin is easily extractable from homogenized muscle at higher ionic strength. Since actin seems to be more solidly linked to the structural element, this offers a convenient means for the separation of the two components. Myosin has been studied in some detail, and a molecular weight of the order of 400,000 to 500,000 has been reported (cf. review by Perry, 1960). 

These experiments with reconstituted thin filaments agree with the stoichiometry and stability of CD present in native thin filaments extracted from smooth muscles (Marston, 1990). The stoichiometry is 1 CD 16 actin and the estimated affinity of CD for the thin filament is 2 x 10 M (/ = 0.09 M). Thus a complex of 1 CD 2 TM 14 actin, which resembles the native thin filament, may be reconstituted in the test tube. Actin activation of myosin MgATPase and motility is switched off in this complex and a Ca2+-regulated thin filament can be reconstituted by the further addition of calmodulin (Smith et al., 1987 Marston and Smith,... 

The location of CD and its cellular concentration are compatible with its proposed function. A CD actin ratio of 1 30 in the whole muscle cell corresponds to 1 18 if CD is confined to the contractile domain. This is close to the CD actin ratio obtained when thin filaments are extracted from actomyosin (1 16 Marston,... 

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). 

G-actin, when extracted from a muscle fiber into water, had bound ATP. During polymerization to F-actin, this ATP was hydrolyzed to ADP and inorganic phosphate [7]. The ADP was kept bound in F-Actin. In the process of depolymerization of F-actin to G-actin, rephosphorylation of ADP did not happen. After depolymerization, bound ADP was replaced with ATP in solution. Later it was found that G-actin having ADP instead of ATP also polymerizes to F-actin, although the rate of polymerization is much slower than G-actin having ATP [8]. Even G-actin without ATP or ADP can polymerize, if denaturation of this nucleotide-free G-actin is inhibited by a high concentration of sucrose [9]. The G-F transformation of actin was described by the scheme shown in Fig. 1. 

The principal molecular constituent of thin filaments is actin. Actin has been highly conserved during the course of evolution and is present in all eukaryotes, including single-celled organisms such as yeasts. Actin was first extracted and purified from skeletal muscle, where it forms the thin filaments of sarcomeres. It also is the main contractile protein of smooth muscle. Refined techniques for the detection of small amounts of actin (e.g., immunofluorescence microscopy, gel electrophoresis, and EM cytochemistry) subsequently confirmed the presence of actin in a great variety of nonmuscle cells. Muscle and nonmuscle actins are encoded by different genes and are isoforms. 

Actin is generally obtained from acetone-dried muscle powder by extraction with either water or, provided ATP is present, with KI solutions. Under these conditions the globular form of the protein is obtained, which polymerizes to the fibrillar form in 0.1 M KCl containing traces of Mg++ or Ca++ ions. Fibrillar actm combines with myosin to give actomyosin. Nevertheless, the exact structure and mode of action of the actomyosin complex are still far from being fully understood. 

This protein was recently prepared from cod muscles and investigated by Connell (1954). Acetone-dried muscle fiber was extracted with water by the method of Feuer, et al. (1948) omitting the final treatment with Na2COa (Tsao and Bailey, 1953). The G-actin obtained polymerizes on addition of salts to a viscous solution of F-actin showing pronounced double refrac-... 

Actin can be extracted by itself from fresh muscle brei provided the L-myosin has been exhaustively removed (cf. IV, 1). This is done by repeatedly extracting relatively coarsely-minced muscle with 0.6 M KCl at pH 6, but the process takes so long that an appreciable amount of actin becomes denatured (Hasselbach and Schneider, 1951). The significance of the acetone treatment is that it renders L-myosin insoluble, breaks its combination with actin, changes F-actin into G-actin, and at the same time arrests denaturation processes. 

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