Characterization of cytoplasmic actin isolated from Acanthamoeba castellanii by a new method.

Cytoplasmic actin has been isolated from Acanthamoeba castellanii by a new method, employing chromatography on DEAE-cellulose, that improves the yield by more than 20-fold over the previously reported method. This procedure should be particularly useful for isolating actin from cells in which it is present in relatively low concentration because the method does not depend initially on the polymerization of actin or its interaction with myosin. Systematic comparison of the properties of purified Acanthamoeba actin and rabbit skeletal muscle actin shows them to be similar in many ways: viscosity of F-actin, stoichiometry of bound nucleotide, stoichiometry of binding of muscle heavy meromyosin and myosin subfragment 1 in the absence of ATP, and ability to inhibit the KATPase activity of heavy meromyosin. The amino acid compositions of Acanthamoeba and muscle actin are also quite similar, but significant differences, especially the presence of epsilon-N-methyllysines in Acanthamoeba actin, have been confirmed. In addition to this structural difference, we find that Acanthamoeba actin is only one-third as effective as muscle actin as an activator of the MgATPase of muscle heavy meromyosin and subfragment 1. For Acanthamoeba actin, as for muscle actin, this activation exhibits hyperbolic dependence on actin concentration; i.e. the double reciprocal plot of ATPase activation versus actin concentration is linear. From these plots we find that the two actins give the same extrapolated ATPase activity at infinite actin concentration (Vmax) but differ by a factor of three in the concentration of actin needed to produce half-maximal activation (Kapp).


Culture
of Acanthamoeba castellanii-Amebae were grown in 15liter aerated carboys at 28" as described by Pollard and Korn (13). One carboy grown to a density of 1.2 x lo6 cells/ml provided about 100 g wet weight of amebae.
Bound Nucleotide-F-actin was diluted to a concentration of 1.0 to 1.5 mg/ml and free ATP was removed by addition of Dowex l-X4-Cl in three batches of 0.15 g of resin/ml of actin solution. The actin was then divided into two portions.
Water (0.1 ml/ml) was added to one portion and its protein concentration determined by its A,,,. To the second portion, 70% HClO, (0.1 ml/ml) was added to precipitate the actin and release the bound nucleotide.
The concentration of the released ADP was determined from the AZ,, of the supernatant solution using a molar extinction coefficient of 15.0 x IO3 Mm ' cm ~.
Buffers-Buffer G contained 3 rn~ imidazole base/O.1 rn~ CaCl,/0.5 rn~ ATP/0.75 rn~ fi-mercaptoethanol, pH 7.5. In this buffer, actin assumes the monomeric globular or "G" form. Buffer F contained 5 rn~ imidazole chloride/Z mM MgCl,/0.5 mM ATP, pH 7.0. In this buffer, actin assumes the polymeric filamentous or "F" form. Buffer D, the buffer for DEAE-cellulose chromatography, was the same as Buffer G, except that the imidazole concentration was 10 rn~; pH was adjusted to 7.5 with HCI.
Amino Acid Analysis-Protein samples were incubated for 2 hours at 37" in 5 M guanidine-HCl/5 rn~ dithiothreitoU0.2 M NaHCO,, pH 8.0, and then cooled to 22", made 0.2 M in iodoacetic acid (recrystallized and preadjusted to pH 8.0) and incubated for 15 min. P-Mercaptoethanol was added to 2.0 M. The reduced, alkylated protein was then dialyzed against several changes of distilled water and lyophilized. Two milligrams of the lyophilized sample was dissolved in 1 ml of 6 N HCl and hydrolyzed for 18 hours at 105". The hydrolysate was lyophilized, redissolved in water, adjusted to pH 9 with NaOH, lyophilized again, and finally dissolved in 2 ml of 0.01 M HCI and adjusted to pH 2. Muscle Proteins-Actin, HMM, and S-l were prepared from rabbit back and leg muscle. Actin was extracted at low ionic strength from an Neutral and acidic amino acids were determined by the method of  Kielley and Harrington (16). HMM was prepared by tryptic digestion which resolves lysine from its mono-, di-, and trimethyl derivatives as of myosin (17). S-l was prepared by papain digestion of myosin (18).
well as histidine and 3.methylhistidine. All amino acid analyses were carried out on a Beckman 121 amino acid analyzer with automatic sample injector and equipped with an Infotronics integrator. Amino The pooled polymerized actin was centrifuged at 100,000 x g acid compositions were normalized to a total mOkCUkW weight of 42,000 for actin.
for 3.5 hours at 200. The clear gelatinous pellets were combined and homogenized in 20 to 30 ml of Buffer G containing 0.02% RESULTS NaN, to retard growth of bacteria. The suspension was then Purification of Acanthamoeba actin-Acanthamoeba actin dialyzed against two to three changes of this buffer for 60 to 80 was usually prepared from about 2 x 10 lo amebae obtained hours to allow complete depolymerization of the actin. Residfrom one 15-liter carboy. All purification steps were carried out ual material that had not depolymerized was removed by at 0-4O unless otherwise noted. Cells were harvested by low centrikgation at 100,000 x g for 90 min. The depolymerized speed centrifugation and washed three times in 600 to.800 mi of supematant solution contained about 5% of the protein of the 10 mM imidazole chloride, pH 7.5. The cells were then original extract (Table I); its actin content by gel electrophoresuspended in Buffer G, 2 ml/g of cells, and ruptured by release sis was about 70% (Fig. 1). from a Parr bomb equilibrated with nitrogen at 400 p.s.i. The The depolymerized actin was applied to a column containing homogenate was centrifuged at 100,000 x g for 90 min and the Sephadex G-150 and eluted with Buffer G containing 0.02% pellet was discarded. The low ionic strength extract thus NaN, (Fig. 4). Actin was eluted as an asymmetric peak with a obtained generally consisted of 200 to 300 ml of a variably slowly rising ascending limb and a sharply declining descendturbid (depending on the lipid content), amber-colored liquid ing limb similar to the pattern reported for muscle actin (26). containing 12 to 16 mg/ml of protein of which about 15% was Contaminating protein was removed in a voided fraction and in actin (45,000-dalton band) by dodecyl sulfate gel electrophore-material eluting between the voided and included (actin) sis ( Fig. 1).
fraction. The eluted actin was concentrated to 5 to 8 mg/mI by The ameba extract was fractionated on DEAE-cellulose as ultrafiltration on an Amicon PM-10 membrane, polymerized described in the legend to Fig. 2, which shows a typical elution by adjusting the solution to 0.1 M KC1 and 2 mM MgC12, and pattern. Eluted fractions were monitored for the presence of dialyzed for 40 hours against two to three changes of Buffer F to actin filaments by observing through crossed polarizers to remove KCI. The purified actin accounted for about 3% of the detect flow birefringence. As long as these fractions were kept protein of the original extract and was 95% pure according to cold, no flow birefringence developed. However, when they the scans of electrophoretic gels (Fig. 1). The gel scans indicate were warmed to 25' and MgCl, was added to 2 mM, fractions that 15 and 20% of the actin was recovered from the original eluting between 0.19 and 0.24 M KC1 rapidly developed flow extract. birefringence.
Dodecyl sulfate gel electrophoresis of selected Specific Actiuity of Act&-Each of the fractions obtained fractions throughout the gradient confirmed the presence of during the purification procedure was also assayed for its actin as the major component in these fractions, the absence of ability to activate the MgATPase of HMM. Because of the actin in fractions eluting before 0.17 M KCI, and the gradual high intrinsic ATPase activity of the crude extract it was decrease of actin content in fractions eluting beyond 0.24 M impossible to detect activation of HMM MgATPase by this KC1 (Fig. 3). The material eluting between 0.19 and 0.24 M fraction. An increase in the specific activity of actin at each of KCI, about 15% of the protein applied to the column, was the three following purification steps was consistently observed pooled as the DEAE-actin fraction (Table I). Its actin content (Table I). was 40% by gel electrophoresis (Fig. 1).
There are two apparent discrepancies between the specific  Table I were analyzed: the crude extract, fraction eluted from DEAE-cellulose between 0.19 to 0.24 M 0.6 -KCl, polymerized-depolymerized actin, and purified actin from the included 0.4peak on Sephadex G-150. These scans were used to obtain the data in Table I The extract was applied as soon as possible because after 4 to 6 hours it became increasingly turbid, eventually forming a precipitate. Fifty milliliters of Buffer G were applied immediately before and after the sample; this procedure insured that ameba actin would not be exposed to 0.1 M KC1 until after it was adsorbed to the DEAE-cellulose.
The column was then eluted with 200 to 300 ml of 0.1 M KC1 Buffer D and 2 liters of a linear gradient from 0.1 to 0.5 M KC1 Buffer D.
1593. Actin is the major band of the initial extract and is otherwise The gel on the extreme left is the original extract. The next 15 gels from present only in Gaels 12 to 15, which correspond to the action peak in left to right are aliquots from material eluting at the following volumes Fig. 2, and in Gel 16. Approximately equal amounts of total protein (milliliters): (2) 294, (3) 367, (4) 493, (5) 538, (6) 586, (7)