Purification of Skeletal Muscle Actin

I. INTRODUCTION
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.

IV. PROCEDURES
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.

Solutions

1. Extraction solution: For 8 liters, add 298.2 g KCl and 40 g KHO₃ to 8 liters H₂O
2. Acetone: Chill to -20°C (acetone denatures proteins above 0°C)
3. H₂O: Chill to 4°C

Steps

1. Mince preparation. If possible, work in a group of two or three. Sacrifice two male rabbits (New Zealand white or equivalent). Skin and place in a large flat ice box. With a sharp knife, quickly excise the large muscles on both sides of the spine and from both hind legs. Chop muscles coarsely with scissors, removing as much connective tissue as possible. Grind. You should obtain about 600 g mince.
2. Myosin extractions. Place the mince in a large beaker and add 3 liters chilled extraction solution. Stir for 10 min and then centrifuge for 10 min at 2500g. Discard supernatant and repeat this step once.
3. Water extractions. Discard supernatant and add pellet to 3 liters distilled water. Stir for 10min and centrifuge as in step 3. Repeat this step three more times or until pellet begins to swell visibly; in this case, discontinue water extraction.
4. Acetone washes. Discard supernatant and mix washed tissue with cold acetone. Homogenize briefly with a kitchen blender. Centrifuge at 1200g for 5min. Repeat this step two more times.
5. Drying. After last acetone wash, pool all pellets in a large inox beaker and add 2 liters cold acetone. Stir vigorously for 20min. Lay out a large Bfichner funnel with several layers of gauze and place on a 4-liter Bfichner flask. Apply suction and filter the acetone suspension. Squeeze out residual acetone by gathering the ends of the gauze and wringing. Spread the dehydrated material on filter paper and let dry overnight. Store dry acetone powder at -20°C.

B. Preparation of Ca-G-Actin from Acetone Powder
Gloves should be worn throughout the purification to avoid protease contamination.

Solutions
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.

1. X buffer: 2 mM Tris-HCl, 0.5 mM ATP, 0.1 mM CaCl₂, 1 mM dithiothreitol (DTT), 0.01% (w/v) NAN₃, pH 7.8. Store at 4°C.
2. Xb buffer: 2 mM Tris-HCl, 1 mM MgCl₂, 1 mM DTT, pH 7.8. Store at 4°C.
3. G buffer: 5 mM Tris-HCl, 0.2 mM ATP, 0.1 mM CaCl₂, 1 mM DTT, 0.01% (w/v) NaN3, pH 7.8. Store at 4°C.
4. 4M KCl: Store at room temperature
5. 1M MgCl₂: Store at 4°C.

Steps

1. G-actin extraction. Add 270ml of cold X buffer to 9 g acetone powder in a beaker on ice. Stir gently with a glass rod to completely moisten the acetone powder; excessive stirring will lead to increased α- actinin extraction. Leave on ice for 30min, repeat stirring every 10 min.
2. Clarification. Centrifuge for 45min at 25,000g 4°C. Filter the supernatant through glass wool and determine the volume after filtration.
3. Polymerization/α-actinin elimination. Add solid KCl to a final concentration of 3.3M, taking into account a volume increase of about 10%. Stir at room temperature until the solution has reached a temperature of 15°C to allow complete solubilization of the salt. Subsequently, chill the solution on ice without stirring until its temperature has fallen back to 5°C. Centrifuge for 30min at 25,000g / 4°C. Discard the F-actin/α-actinin pellet and filter the supernatant through glass wool.
4. Dialysis. Dialyse at 4°C overnight against 32 volumes of buffer Xb to bring [K⁺] to 0.1M.
5. Tropomyosin elimination. Add 0.22 volumes of 4M KCl to bring [K⁺] to 0.8M. Stir for 90min at 4°C. Centrifuge for 3h 30min at 70,000g/4°C. Remove tropomyosin-containing supernatant. Collect the transparent F-actin pellets with a clean spatula and pool into a small glass tissue grinder. To collect residual actin, rinse centrifuge tubes with buffer X and add to pool. Homogenize the F-actin solution on ice, avoiding excessive foaming. Transfer to a 50-ml measuring cylinder. Add 75 µl 1M MgCl₂ and 375 µl 4M KCl and adjust volume to 38.6 ml with buffer X. (If necessary, the protocol can be interrupted at this point and the solution left to sit overnight at 4°C.) Repeat step 5 to remove tropomyosin completely, i.e., add 9ml 4M KCl, make up to 50ml with buffer X, stir for 90min at 4°C, centrifuge for 3h 30min at 85,000g/4°C, and remove tromomyosin-containing supernatant.
6. Depolymerization. Resuspend and homogenize the F-actin pellets as in step 5 in a total volume of 20-30ml buffer X. Dialyse overnight against 1 liter buffer G.
7. Sonication. Sonicate 2 × 10s in the dialysis bag. Change dialysis buffer and continue dialysis for another 24h.
8. Clarification and gel filtration. Centrifuge the actin for 2h at 250,000g / 4°C. Run the supernatant over a Sephadex G-200 gel filtration column equilibrated in buffer G at 15-20ml/h. Collect 5-ml fractions. You should observe a first small and a second large peak by following the A₂₉₀; the second one is the actin peak. Avoid collecting the peak front because of possible CapZ contamination.
9. Concentration determination. Pool actin fractions and determine the concentration by spectrophotometry (ε_290nm = 26,600M^-lcm^-1) using the G buffer as blank. Typically this will be 40-50 µM. Ca-G-actin can be stored on ice for several weeks.

C. Conversion of Ca-Actin to Mg-Actin
Solutions

1. 10 mM MgCl₂
2. 25 mM EGTA, pH 9.0

Steps

1. Dilute Ca-G-actin to a concentration not higher than 10 µM with G buffer.
2. Add MgCl₂ to a final concentration of (x + 10) µM, where x is the actin concentration in µM.
3. Add EGTA to a final concentration of 200µM. Mix gently by pipetting. The cation exchange will be complete in about 3 min.

V. COMMENTS
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.

References
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.