Purification of Skeletal Muscle Actin
Skeletal muscle is the most important preparative source for actin since the discovery of the protein in the 1940s. The original purification procedure developed by Feuer et al. (1948) is still the basis of modern protocols. In a first step, fresh skeletal muscle tissue is ground and most of the myosin is extracted by 0.5M KCl. The tissue is then washed several times with distilled water and is dehydrated with acetone at -15°C to yield acetone powder, which can be stored conveniently. The second step, the actual actin preparation from this acetone powder, is based on cycles of actin extraction/depolymerization in low salt buffer, actin polymerization by the addition of salt, and sedimentation of the polymerized actin. Important methodical improvements were reached by the development of additional purification steps aimed at removing major protein contaminants. In particular, α-actinin (Ebashi and Maruyama, 1965) is eliminated by low-speed sedimentation of a thick gel formed with actin at 3 M KCl, and tropomyosin (Spudich and Watt, 1971) is dissociated from F-actin at high ionic strength. The procedure presented here is based on the widely used protocol of Spudich and Watt (1971; reviewed in Pardee and Spudich, 1982) followed by a gel filtration step at low ionic strength to isolate monomeric ATP-G-actin (MacLean-Fletcher and Pollard, 1980). It yields very high purity actin suitable for all common biochemical applications, such as actin binding and polymerization assays or chemical labelling. To store and handle actin efficiently, it is paramount to have an understanding of its biochemistry, especially of its interaction with Ca2+ and Mg2+ (for a review, see Carlier et al., 1994). At slightly alkaline pH, Ca-G-actin can be stored on ice at 2-3mg/ml (50-75µM) without detectable selfassembly for weeks. This is not the case with Mg-actin, the prevalent species in vivo. The following protocol includes a procedure to convert Ca-actin to Mg-actin (Gershman et al., 1984).
II. MATERIALS AND INSTRUMENTATION
If not stated otherwise, chemicals are p.a. grade and are purchased from Merck. NaaATP is from Roche Molecular Diagnostics (0519987). Water is MilliQ grade. Acetone should be pure (99.5%). Sephadex G- 200 is from Amersham Biosciences.
III. LABORATORY EQUIPMENT
Acetone powder preparation: sharp knife, large glassware (4-liter beaker, 4-liter Btichner flask), inox beaker, meat grinder, kitchen blender, gauze, and centrifuge. Actin purification: tissue grinder, thermometer, preparative ultracentrifuge, glass wool, dialysis tubes, and low-pressure chromatography system.
A. Preparation of Acetone Powder from Rabbit Skeletal Muscle
Note that acetone powder is also available commercially (Sigma M-0637). To prevent protease activity/contamination, the material should not be allowed to warm above 4°C and gloves should be worn throughout the preparation.
B. Preparation of Ca-G-Actin from Acetone Powder
Gloves should be worn throughout the purification to avoid protease contamination.
To prepare ATP-containing buffers, it is convenient to use a stock solution of ATP in H2O buffered to pH 7.0. Store this stock solution at -20°C.
C. Conversion of Ca-Actin to Mg-Actin
Actin can also be stored frozen or lyophilized, and lyophilized actin is available commercially (Sigma A- 2522). However, these techniques lead to partial denaturation of the protein. If actin is frozen or lyophilized, sucrose should be added (2mg/ml per mg/ml actin), and thawed or rehydrated actin should be dialysed against buffer G and recycled by a cycle of polymerization, high-speed sedimentation, and depolymerization. The protein concentration should be redetermined after this procedure.
Carlier, M.-E, Valentin-Ranc, C., Combeau, C., Fievez, S., and Pantaloni, D. (1994). Actin polymerization: Regulation by divalent metal ion and nucleotide binding, ATP hydrolysis and binding of myosin. Adv. Exp. Med. Biol. 358, 71-81.
Ebashi, S., and Maruyama, K. (1965). Preparation and some properties of alpha-actinin-free actin. J. Biochem. (Tokyo) 58, 20-26.
Feuer, G., Molnar, E, Pettko, E., and Straub, E (1948). Studies on the composition and polymerization of actin. Hung. Acta Physiol 1, 150-163.
Gershman, L., Newman, J., Selden, L., and Estes, J. (1984). Boundcation exchange affects the lag phase in actin polymerization. Biochemistry 23, 2199-2203.
MacLean-Fletcher, S., and Pollard, T. (1980). Identification of a factor in conventional muscle actin preparations which inhibits actin filament self-association. Biophys. Res. Commun. 96, 18-27.
Pardee, J., and Spudich, J. (1982). Purification of muscle actin. Methods Enzymol. 85, 164-181.
Spudich, J., and Watt, S. (1971). The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J. Biol. Chem. 246, 4866-4871.
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