Actomyosin preparation

The viscosity of all actomyosin preparations investigated varies with concentration according to the Arrhenius formula log ijrei = K.C. Their viscosity numbers Z, are therefore equal to 2.303 K. The results obtained with 1-hour actomyosins are reproduced in Fig. 20, but those which relate to 10-minute actomyosins are in no way different, in contradiction with... 

From this summary it is clear that several phosphatases may dephosphorylate myosin and there is no obvious way to identify the in vivo activity. One approach was to characterize the phosphatase that is associated with myosin (or actomyosin) on the assumption that binding to myosin is a property expected for MLCP. Using okadaic acid as a probe it was found that the major phosphatase in gizzard actomyosin preparations was PPl (Ishihara et al, 1989). Subsequently several groups isolated MLCP using myosin or actomyosin as a source. The preparation of Mitsui et al. 

There are a number of actin-associated proteins proposed to be involved in the regulation of SM. These are discussed in the following sections and elsewhere in this volume. Although it remains debatable whether these proteins contribute to the regulation of the SM cross-bridge in vivo, there is ample biochemical evidence in which the activity of actomyosin preparations and in vitro motility assays are affected by caldesmon (CD), calponin (CP), and tropomyosin (TM), as well as several other proteins. 

Dissymmetry measurements have been made on an actomyosin preparation by Jordan and Oster (1948). The results were interpreted as indicating that the actomyosin particle becomes more highly coiled in the presence of adenosine triphosphate. 

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. 

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). 

With Ztj values between 0.1 and 0.2, solutions of thrombosthenin appear less viscous than comparable preparations of actomyosin obtained from striated muscle. As shown in Fig. 8, the sudden decrease in viscosity which accompanies the addition of ATP is followed by a slow rise owing to the enzymatic breakdown of the nucleotide, until the original level is almost reached again. On repeated addition of ATP the same sequence of events is observed. 

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). 

The ATPase activity of the actomyosins from smooth muscle is in the order of 0.1 jumole P, per milligram of protein per minute (cf. Table IV). At an ionic strength of 0.6 n and in the presence of Mg++ ions the enzyme is less active than at 0.1 ix the opposite is found in the presence of calcium ions (Needham and Cawkell, 1956 Ledermair, 1959). Cretins and Jaisle (1960) attribute a considerable proportion of the observed ATPase activity to the presence of granular particles in their preparations this might then explain at least some of the observed descrepancies. 

The discussion of the similarities and dissimilarities of thrombosthenin and other contractile proteins will be restricted to the group of actomyosin-like proteins. As already mentioned, contractile substances of other types have not yet been isolated in a purified form and most of our knowledge about them comes from studies of glycerol-extracted cell models, which have not yet been prepared from blood platelets. Lastly, there can be little doubt that the mode of action of these other systems must be quite different from thrombosthenin. 

Electrophoretic behavior. The very constant mobility of carp actomyosin whatever may be its conditions of preparation and its identity with... 

Fid. 20. Viscosity of several 1-hour actomyosins of the carp. The triangles correspond to the same preparation without and with ATP (after hamoir, 1955). 

Fig. 22. Variation of 2o.w X 10 with concentration of the solution for carp acto-myosins prepared by (a) ten-minute or (6) 1-hour extraction at neutral pH of muscles kept frozen for different periods of time (a) 2 days , 21 days A (6) 5 days X, 24 days O, 38 days . The lower figures (O and ) correspond to 10-minute actomyosins extracted at acid pH (after hamoir, 1955).

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. 

The key step in LC isolation is the preparation of pure smooth muscle myosin (Hasegawa et al., 1988). This is usually executed by homogenizing the muscle mince with salt solutions (60-100 mM ionic strength) to remove the cytoplasmic proteins and to a certain extent the thin filament proteins. A crude actomyosin is then extracted form the well-washed muscle residue with salt solutions containing ATP and the actin component is removed by ultracentrifugation. The LCs are... 

The possible differences in on- and off-positions of tropomyosin in smooth and skeletal muscle thin filaments may relate to significant differences in enzymatic behavior of the two systems. The well-known observation that tropomyosin activates the smooth muscle actomyosin ATPase (Chacko et al., 1977) and also accelerates the motility of smooth muscle preparations in vitro (Shirinsky et al., 1992) should be taken into account when evaluating caldesmon function. Indeed, the degree of actomyosin ATPase potentiation by tropomyosin in the smooth muscle system is considerably greater than that in skeletal muscle preparations (Chacko et al., 1977 Sobieszek and Small, 1977 Chacko, 1981 Lehrer and Morris, 1984 Williams et al.,... 

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.,... 

Sobieszek A, Bremel RD (1975) Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin. Eur J Biochem 55 4960 Sobue K, Morimoto K, Inui M, Kanda K, Kakiuchi S (1982) Biomed Res 3 188196 Sobue K, Muramoto Y, Fujita M, Kakiuchi S (1981) Purification of a cahnodulin-binding protein from chicken gizzard that interacts with F-actin. Proc Natl Acad Sci U S A 78 56525655... 

An important problem with any latch theory is that the latch-bridge in many ways remains descriptive and has not been coupled to specific biochemical actomyosin states or mode of actomyosin turnover. Skinned fibres could provide a way to explore this problem but at present there is a striking lack of a latch state in skinned smooth muscle preparations with a few exceptions. The hyperbolic phosphorylation force relationship has been demonstrated in e.g. skinned smooth muscle strips from chicken... 

In this review, the contractile protein complex is called actomyosin, and its components actin and L-myosin. To avoid ambiguity, the word myosin is used only for preparations which may contain actomyosin and L-myosin, i.e., for all preparations made before the discovery of actin, and for the A and B myosins of Szent-Gyorgyi. 

Some of the contractile properties of the models, however, differ markedly from those of the particular muscle from which they have been prepared (Table II, columns 1, 5 and 7). One of the most important of these differences, perhaps, is the slowness with which the models redevelop their tension after a release the time in the case of living muscle is short and characteristic of the speed of contraction (A. V. Hill, 1926 Gasser and Hill, 1924). It is hardly surprising that an actomyosin thread from a striated muscle no longer has the same short recovery time as that of the muscle (Table II, column 5), for the actomyosin from both slow and fast muscles appears to be much the same (Hamoir 1949). Actin and I.-myosin from quite different animals combine to give acto-myosins with the characteristic properties of the natural ones (Cigada... 

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