70

MYOSIN- AND ACTOMYOSIN-RELATED PROTEINS

[7]

[7] Isolation of A c t i n - B i n d i n g P r o t e i n s from

Dictyostelium discoideum By A. R. BRESNICKand J. CONDEELIS Dictyostelium discoideum has become an important model system for the study of cell motility during ameboid chemotaxis and morphogenesis. This popularity derives from a number of different properties. First, large volumes of homogeneous cell populations can be grown easily in suspension culture, simplifying biochemical analysis and pharmacological studies. Second, the motility exhibited by Dictyostelium amebas is very similar if not identical to that in higher organisms during morphogenesis and leukotaxis. Third, much is known about signal transduction, chemotaxis, and the cytoskeleton (Table 1)1-2° in this organism. Finally, Dictyostelium is haploid throughout most of its life cycle and can be genetically manipulated efficiently using allele replacement and antisense techniques, thus providing a means for assessing protein function in vivo. Analysis of the molecular mechanisms involved in the regulation of the actin cytoskeleton during motility and chemotaxis requires methods for 1 j. Condeelis, S. Geosits, and M. Vahey, Cell Motil. 2, 273 (1982). 2 j. Condeelis, M. Vahey, J. Carboni, J. DeMey, and S. Ogihara, J. CellBiol. 99, 119s (1984). 3 j. Condeelis, A. Hall, A. Bresnick, V. Warren, R. Hock, H. Bennett, and S. Ogihara, Cell Motil. Cytoskeleton 10, 77 (1988). 4 R. Hock and J. Condeelis, J. Biol. Chem. 262, 394 (1987). 5 j. Condeelis and M. Vahey, J. CellBiol. 94, 466 (1982). 6 M. Fechheimer, J. Brier, M. Rockwell, E. Luna, and D. Taylor, CellMotil. 2, 287 (1982). 7 H. Bennett and J. Condeelis, CellMotil. Cytoskeleton 11, 303 (1988). s M. Clarke and J. A. Spudich, J. Mol. Biol. 82, 209 (1974). 9 G. C6t6, J, Albanesi, T. Ueno, J. Hammer, and E. Korn, J. Biol. Chem. 260, 4543 (1985). 1oM. Demma, V. Warren, S. Dharmawardhane, and J. Condeelis, J. Biol. Chem. 265, 2286 (1989). i1 F. Yang, M. Demma, V. Warren, S. Dharmawardhane, and J. Condeelis, Nature (London) in press (1990). ~2M. Fechheimer and D. Taylor, J. Biol. Chem. 259, 4514 (1984). 13S. S. Brown, Cell Motil. 5, 529 (1985). 14S. S. Brown, K. Yamamoto, and J. A. Spudich, J. CellBiol. 93, 205 (1982). ~s K. Yamamoto, J. D. Pardee, J. Reidler, L. Stryer, and J. A. Spudich, J. CellBiol. 95, 711 (1982). 16M. Schleicher, G. Gedsch, and G. Isenberg, EMBO J. 3, 2095 (1984). t7 H. Hartmann, A. Noegel, C. Eckerskorn, S. Rapp, and M. Schleicher, J. Biol. Chem. 264, 12639 (1989). ,s C. Stratford and S. Brown, J. CellBiol. 100, 727 (1985). 19L. Wuesthube and E. Luna, J. CellBiol. 105, 1741 (1987). 20j. Scheel, K. Ziegelbauer, T. Kupke, B. Humble, A. Noegel, G. Gerisch, and M. Schleicher, J. Biol. Chem. 264, 2832 (1989).

METHODS IN ENZYMOLOGY, VOL. 196

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Dictyostelium ACTIN-BINDING PROTEINS

[7]

71

TABLE I

DictyosteliumACTIN-B1NDINOPROTEINS Protein

Homology/function

?/filament cross-linkerin pseudopods Filamin/filament cross-linkernear cell membrane pH and Ca2+-regulatedfilament cross-linker, actomyosin regulation Fodrin/actin- membrane binding ABP-220 Conventional myosin/mechanochemicalenzyme Myosin II Brush border 110K/mechanochemicalenzyme Myosin I ABP-50 EF-la/filament bundler, monomer binding ?/Ca2+-regulatedfilament bundler p30a ?/filament bundler p30b Severin Gelsolin/Ca2+-regulatedcapping, severing,nucleating Cap 32/34 Cap Z/capping p24 ?/actin- membrane binding Ponticulin ?/actin - membrane binding Histactophilin ?/pH-regulatedactin binding, induces actin polymerization ABP-120 ABP-240 a-Actinin

Refs. 1-3 3, 4 2, 5, 6 7 8 9 10, 11 12 13 14, 15 16, 17 18 19 20

purification of key cytoskeletal proteins. We have identified several actinbinding proteins in Dictyostelium which cross-link and bundle actin filaments in the cell cortex and whose association with the cytoskeleton is regulated during motility or chemotaxis. These include ABP-220, a fodrinlike protein, 7 ABP-240, a filamin-like protein, 3,4 a-actinin, 5 ABP-120, t and ABP-50) ° In this chapter we describe the purification protocols which are routinely used in our laboratory for the preparation ofABP-240, a-actinin, ABP-120, ABP-50, actin, and myosin II from Dictyostelium amebas. P r e p a r a t i o n o f Cells

Dictyostelium discoideum strain AX-3 is g o w n in HL5 medium. 2~ Amebas are harvested from 4.0 liters o f m e d i u m at a density of 8 - 1 0 × l0 s cells/ml by centrifugation at 1700g for 6 min at 4 ° in a 6.0-liter swinging bucket rotor. It is important that this cell density is not exceeded. Dictyostelium grows logarithmically and 8 - 10 × 106 cells/ml is the density at which cell growth begins to plateau. Attempts to grow cells to higher densities result in cell lysis and an unhealthy cell culture. Buffers for purification o f Dictyostelium actin and actin-binding protein are listed in Table II. The pelleted cells are gently resuspended in an excess volume o f buffer A by gentle scraping against the wall o f the centrifuge bottle with a 2LW. Loomis, Exp. CellRes. 64, 484 (1971).

72

MYOSIN- A N D ACTOMYOSIN-RELATED PROTEINS

[7]

TABLE II BUFFERS FOR PURIFICATION OF Diclyostelium ACTIN AND ACTIN=BIND1NGPROTEINS

Buffer~ Buffer A Buffer B Buffer C Buffer D Buffer E Buffer F Buffer G Buffer H Buffer I Buffer J Buffer Kc Buffer L Buffer M Buffer N

Composition b 5 mM PIPES, pH 7.0 5 mM EGTA, 1 mM DTT, 5 mM PIPES, pH 7.0 0. I mM CaCI:, 0.5 mM ATP, 0.75 mM 2-mercaptoethanol, 0.02% (w/v) NAN3, 10 mM PIPES, pH 7.5 0.1 mMCaC12, 0.5 mMATP, 0.75 mM 2-mercaptoethanoi, 0.02% NAN3, 3 mM PIPES, pH 7.5 0.2 mMATP, 0.5 mM DTT, 0.2 mMCaC12, 0.02% NAN3, 2 mM Tris-HC1, pH 8.0 50 mM KC1, 0.5 mM EDTA, 0.25 mM DTT, 0.02% NaN 3, 10 mM PIPES, pH 7.0 500 mM NaC1, 2 mM EGTA, 0.02%, NAN3, 5 mMPIPES, pH 6.8 500 mM NaC1, 2 mM EGTA, 0.1 mM DTT, 0.02% NaNa, 5 mM PIPES, pH 6.8 100 mM KCI, 0.1 mM DTT, 1.0 mM EDTA, l0 mM PIPES, pH 6.5 0.05 mM DTT, 0.02% NaN3, 10 mMPIPES, pH 7.0 0.5 M sucrose, 5 mM DTT, 0.5 INMATE 10 mM Tris-HCl pH 8.5, 5 mM PIPES pH 7.0, 2 mMEGTA, pH 7.3 100 mM KC1, I mM EDTA, 0.5 mMDTT, 20 mMPIPES, pH 7.0 0.5 mMDTT, 1 mM EDTA, 0.02% NAN3, 20 mM PIPES, pH 7.0 0.05 mM DTT, 0.02% NAN3, 20 mM Tris-HCl, pH 7.5

a The pH of all buffers is adjusted at 4*. b PIPES, Piperazine-N, N-bis (2-ethane-sulfonic acid); EGTA, glycol bis(fl-aminoethyl ether)-N,N'-tetraacetic acid; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid. c This buffer requires ultrapure sucrose from Schwarz/Mann Biotech, Cat. #821713, and should be brought to pH 7.3 with acetic acid.

10 ml-polystyrene pipette, and recollected by centrifugation at 1700 g. The cell pellet is resuspended in two pellet volumes of buffer B at 0 °. The average yield is 40 ml packed cells from 4.0 liters.

Purification of Actin, c~-Actinin,ABP-240, Myosin, and ABP-50 Cell Lysis. Two methods of lysis have been utilized for purification of these proteins. The cells can be homogenized at 0 ° with 30 passes of a motor-driven Potter-Elvehjem (Teflon-glass) homogenizer at setting 80 on an overhead stirrer (NSE-34, Bodine Electric Co., Chicago, IL). Prior to lysis the following protease inhibitors are added slowly to the cell slurry: 0.03 ml/ml Trasylol [10,000 kallikrein international units (IU)/ml, FBA Pharmaceuticals, New York, NY] and 10 gg/ml each of pepstatin, leupeptin, and chymostatin. Care must be taken with this method to avoid

DictyosteliumACTINoBINDINGPROTEINS

[7]

73

frothing and foaming of the cell slurry. The second method of lysis is by nitrogen cavitation in a Parr bomb (Parr Instruments, Moline, IL). This method of lysis is preferred since more efficient and more consistent cell breakage is achieved with nitrogen cavitation. The cells are equilibrated at 0 ° for 10 min at 100 psi and then lysed. Immediately following lysis, the inhibitors listed above are added to the cell lysate. The percentage cell breakage is monitored by light microscopy and quantitated by hemacytometer counting. Cell breakage usually ranges from 80 to 90%. Breakage greater than 90% generally results in lysis of nuclei and lysosomes with a subsequent decrease in protein yield presumably due to proteolysis. The homogenate is immediately ultracentrifuged at 100,000 g for 1 hr at 4 ° to remove nuclei, organelles, and membranes. Anion-Exchange Chromatography. The supernatant from the ultracentrifugation (S~) is applied to an ATP-saturated DE-52 (Whatman, Hillsboro, OR) anion-exchange column (2.5 × 30 cm) equilibrated in buffer C + 0.1 M NaC1. Immediately prior to sample addition, the column is pulsed with 50 ml of buffer D at 50 ml/hr. After loading the sample, the column is washed with an additional 50 ml of buffer D, 200 ml of buffer C + 0.1 M NaC1, and developed with a 2000-ml linear gradient of 0.1 0.4 M NaC1 in buffer C at 50 ml/hr. The colulmn fractions are monitored

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tant. The supematant was applied to a 2.5 X 30 cm ATP-saturatedDE-52 column equilibrated in bufferC + 0.1 M NaC1and pulsed with 50 ml of bufferD. Ten-milliliterfractions were collected. SDS-PAGEreveals that fractions 9-14 contain ABP-50, fractions 55-63 contain ABP-240and myosinII, and fractions73-85 containactin,a-actinin,and ABP-120.

74

MYOSIN- AND ACT•MYOSIN-RELATED PROTEINS

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Fraction Number FIG. 2. Sephadex G-150 gel-filtration chromatography of the actin pool. The actin-containing fractions pooled from the DE-52 column were polymerized, ultracentrifuged, depolymerized, and clarified prior to loading onto a 1.5 X 70 cm Sephadex G-150 column equilibrated in buffer E. Two-milliliter fractions were collected. Fractions 36-46 contained the pure actin.

by SDS-PAGE. From this initial column, actin, 22 a-actinin, 22 ABP-240, 4 myosin II,4 and ABP-50 ~° are fractionated (Fig. 1). ABP-50 elutes in the column flow through (fractions 9-14), ABP-240 and myosin elute between 0.17 and 0.19 M NaCl (fractions 55-63), and actin, a-actinin, and ABP-120 begin eluting at 0.22 M NaC1 and continue back through the last peak of the elution profile (fractions 73-85). Actin Purification. Traditionally, the initial column in the actin purification is an ATP-saturated DE-52 resin. ATP is thought to maintain actin in its native state by fulfilling its nucleotide requirement. We have performed actin purifications using both ATP-saturated DE-52 resin and resin without ATP. When we did not bind ATP to the DE-52 resin, not only did the actin remain native (DNase I binding, polymerizationdePolymerization cycling), but the final yield of actin was equivalent. Fractions 7 3 - 8 5 are pooled from the DE-52 column (Fig. 1). MgC12 is added to 2 m M and the solution is allowed to polymerize for 15 min at 22 °. The solution should become viscous. The sample is ultracentrifuged at 100,000 g for 3 hr at 4 °. The supernatant ($2) is saved for purification of a-actinin. The resulting pellet (P2) is resuspended in 2 - 3 ml of buffer E 22 j. Carboni and J. Condeelis, J. Cell Biol. 100, 1884 (1985).

Dictyostelium ACTIN-BINDINGPROTEINS

[7]

75

and dialyzed for 48 hr at 0 ° against buffer E with at least two changes. During this step the actin should be fully depolymerized. After dialysis the actin is clarified by ultracentrifugation at 100,000 g for 2 hr at 4 °. The supernatant ($3) is applied to a Sephadex G-150 (Pharmacia, Piscataway, NJ) column (1.5 × 70 cm) equilibrated in buffer E at a flow rate of 16 ml/hr. Actin elutes in fractions 3 6 - 4 6 (Fig. 2). The average yield is 25 mg of 980/0 pure material from 40 ml of packed cells. ot-Actinin Purification. a-Actinin coelutes with actin in fractions 7 3 - 8 5 on the DE-52 column. For purification of a-actinin, S 2 from the actin purification scheme is dialyzed overnight at 4* against buffer F with two buffer changes. The sample is directly applied to a hydroxylapatite (BioGel HTP, Bio-Rad, Richmond, CA) column (1 × 30 cm) equilibrated in buffer F. After loading, the column is washed with 20 ml buffer F and developed with a 400-ml linear gradient of 0 - 0.15 M KPO4 in buffer F at 22 ml/hr. a-Actinin elutes between 0.05 and 0.062 M KPO4, fractions 4 7 - 56 (Fig. 3). The pool is dialyzed overnight at 4 ° against buffer F. The protein is applied to a second hydroxylapatite column (1 X 2.5 cm) equilibrated in buffer F. The column is washed with 4 ml of buffer F and pulsed with 7 ml of 0.1 M KPO 4 in buffer F at 20 ml/hr, a-Actinin elutes in fractions 41 - 44 (Fig. 4) with an average yield of 2 mg of 95% pure material from 40 ml of

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76

[7]

MYOSIN- AND ACT•MYOSIN-RELATED PROTEINS 50 I

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packed cells. This last column step can be used to concentrate the a-actinin as well as remove contaminating proteins. ABP-120 also coelutes with a-actinin and actin from the DE-52 column, but is usually degraded at this step. A more efficient isolation procedure for ABP-120 is described below. ABP-240 Purification. The initial step in the purification of ABP-240 is also the DE-52 column described above. As with the other proteins purified from this column, we have tried purifying ABP-240 from a DE-52 column not saturated with ATP. ABP-240 was the only protein whose yield was markedly affected by the presence or absence of ATP in the first column step. In the absence of ATP the final yield of ABP-240 decreased by approximately twofold. ABP-240 shares antigenic cross-reactivity with filamin.4 SDS-PAGE also revealed massive proteolysis of the protein. This is not surprising since Chen and Stracher2a have demonstrated that filamin in platelets is phosphorylated in situ by cAMP-dependent kinase and that this phosphorylation stabilizes the protein against proteolysis by calpain. ABP-240 elutes in fractions 55-63 (0.17-0.19 M NaC1) from the DE-52 column. Trasylol (0.03 ml/ml) and chymostatin (10/zg/ml) are 23 M. Chen and A. Stracher, J. Biol. Chem. 264, 14282 (1989).

Dictyostelium ACTIN-BINDINGPROTEINS

[7]

77

added to the pool and it is immediately applied to a hydroxylapatite column (I X 26 cm) equilibrated in buffer G. Following sample loading, the column is washed with one column volume of buffer G and developed with a 190-ml linear gradient of 0-0.35 M Na_PO4 in buffer G at 22 ml/hr. ABP-240 elutes between 0.18 and 0.27 M NaPO4, fractions 106-126 (Fig. 5). After the addition of protease inhibitors (as above), the hydroxylapatite pool is vacuum concentrated 13-fold (ProDiCon, Bio-Molecular Dynamics, Beaverton, OR) against buffer H. The concentrated protein is clarified by ultracentrifugation at 100,000 g for 1 hr at 4 °. The supernatant is loaded onto a Sephacryl S-300 (Pharmacia) column (1.6 × 70 cm) equilibrated in buffer H at 12 ml/hr. ABP-240 elutes immediately after the void volume, fractions 26-31 (Fig. 6). The average protein yield is 1.5 mg of 99% pure material from 40 ml of packed cells. Myosin H Purification. Myosin II coelutes with ABP-240 in fractions 55-63 of the DE-52 column and is the major contaminant during the ABP-240 purification. Myosin can be separated from ABP-240 by cycling the myosin from monomer to polymer and back to monomer. The DE-52 pool is dialyzed overnight at 4 ° against buffer I. MgC12 is added to 2 m M and the sample is allowed to polymerize on ice for 2 hr. The solution should become cloudy due to the formation of myosin thick filaments. The

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78

[7]

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sample is ultracentrifuged at 100,000 g for 30 min at 4 °. The resulting pellet is resuspended in 2 - 3 ml of buffer I + 0.5 M KC1 at pH 6.8. Alternatively, the myosin can be isolated from the hydroxylapatite column in the ABP-240 purification. Myosin elutes at a phosphate concentration greater than 0.3 M. The hydroxylapatite pool is dialyzed overnight at 4 ° against buffer I, polymerized, and centrifuged as above. The pellet is resuspended in 10 ml buffer I + 0.5 M KC1 and dialyzed overnight at 4 ° against buffer I + 0.5 M KC1 with two buffer changes. After dialysis, the myosin is clarified by ultracentrifugation at 100,000 g for 30 min at 4 °. The supernatant is applied to an A-15m (BioGel, Bio-Rad, 100-200 mesh) column (2.5 × 100 cm) equilibrated in buffer I + 0.5 M KCI at 40 ml/hr. Myosin elutes at fractions 24-30 and represents the single peak on the elution profile. The average protein yield is approximately 6 mg of 98% pure material from 40 ml packed cells. ABP-50 Purification. ABP-50 elutes from the DE-52 column in the unbound fraction. In order to isolate larger quantities of this protein we have scaled up to using 6.0 liters of cells at a harvest density of 8 X 106 cells/ml, which yields an average of 60 ml of packed cells. To permit the ABP-50 fractions to be collected at a faster rate, the configuration of the DE-52 column was changed to 5 X 7 cm and the column is run at 280

Dictyostelium ACTINoBINDINGPROTEINS

[7]

79

ml/hr in the absence of ATP. The unbound fractions are pooled and applied at 4 ml/min to a Fast-S Sepharose (Pharmacia) column (1 × 10 cm) equilibrated in buffer J. The column is washed with one column volume of buffer J at 4 ml/min and developed with a 240-ml linear gradient of 0-0.4 M NaC1 in buffer J at 2 ml/min. ABP-50 elutes between 0.18 and 0.25 M NaC1, fractions 55-75 (Fig. 7). The rapidity of the first and second column steps in this purification are necessary to prevent proteolysis of ABP-50. The Fast-S pool is applied to a hydroxylapatite column (1 X 7 cm) equilibrated in buffer F to remove minor contaminants at 90K, 40K, and 30K. The column is washed with one column volume of buffer F and developed with a 100-ml linear gradient of 0-0.3 MKPO4 in buffer F at 20 ml/hr. ABP-50 elutes between 0.1 and 0.15 MKPO4, fractions 43- 52 (Fig. 8). These fractions can be divided into two pools of ABP-50, the early fractions containing 1 - 1.5 mg of 95% pure material and the later fractions containing 1 - 2 mg of 60-80% pure material, all from 60 ml of packed cells.

Purification of ABP-120 ABP-120 Purification. ABP-120 coelutes with actin and t~-actinin at 0.22 M NaC1 from the DE-52 column. Further purification of ABP-120 30

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80

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from this column is not feasible due to the low recovery of ABP-120 from the column and its susceptibility to proteolysis. Therefore we have devised another method of purification for this protein. ~ Several modifications described here have been made to the originally published protocol. Amebas are harvested from 8.0 liters of medium at a density of 8 10 × 106 cells/ml by centrifugation for 6 min at 1700 g at 4 °. The ceils are washed in 0.2% NaC1 and resuspended in 2 vol of buffer K at 0 °. The following inhibitors are added with gentle mixing: 0.06 ml/ml Trasylol, 0.3 mg/ml soybean trypsin inhibitor, 0.5 #g/ml E64, which is a thiol protease inhibitor, phenylalanine to 2 mM, and 10 #g/ml each of pepstatin, chymostatin, and leupeptin. All subsequent steps are done on ice. The cell slurry is sonicated (W185D sonifier, 70% power; Heat Systems, Plainview, NY) in 100-ml batches in 250-ml beakers at 10-sec intervals until 80-90% cell breakage is achieved. Cell breakage is monitored by light microscopy. We have tried other methods of cell lysis for ABP-120, but other methods lead to massive degradation of the protein and consequently low protein yields. The homogenate is ultracentrifuged at 100,000 g for 1 hr at 4 °. The supernatant is recovered and dry powdered ammonium sulfate (ultrapure grade, Schwarz/Mann Biotech, Cleveland, OH) is added to 45% saturation. The sample is allowed to stir for 5 min, is equilibrated for 10 min, and is

Dictyostelium ACTIN-BINDINGPROTEINS

[7]

81

centrifuged at 45,000 g for 15 min at 4 ° to collect the precipitated material. Dry powdered ammonium sulfate is added to the supernatant to 60% saturation and treated as before. The 45-60% ammonium sulfate pellet is resuspended in 5 ml of buffer L containing 40/tg/ml each of pepstatin, chymostatin, and leupeptin, 20 gg/ml E64, 2 × 104 units of Trasylol, and 4 mg/ml soybean trypsin inhibitor. The resuspended pellets are diluted to 16 ml with buffer L and brought to 0.6 M KI by the slow addition of 3 M KI in order to prevent the polymerization of actin. The suspension is clarified by ultracentrifugation at 200,000 g for 20 min at 4 °. The supernatant is applied to an A-I 5m (BioGel, Bio-Rad, 100-200 mesh) gel filtration column (2.5 × 100 cm) equilibrated in buffer L. Immediately prior to sample addition, the column is pulsed with 20 ml 0.6 M K I at 40 ml/hr. ABP-120 elutes in fractions 35-42 (Fig. 9). Fortuitously, the end of the pool is marked by a yellow pigment which elutes offthe column immediately after ABP- 120. The pool is quickly desalted on a Sephadex G-25 (Pharmacia) column (4.8 × 16 cm) equilibrated in buffer M. ABP-120 elutes in the void volume of this column. The fractions are pooled and 10 gg/ml each of pepstatin, chymostatin, and leupeptin are added to the pool. Rapid desalting of the A-15m pool is necessary since removal of the salt results in proteolysis of

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82

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Froction N u m b e r Fro. 10. DE-52 anion-exchange c h r o m a t o g r a p h y o f the A - 1 5 m pool. T h e ABP-120-containing fractions were pooled from the A - 1 5 m c o l u m n a n d rapidly desalted on a Sephadex G-25 c o l u m n . T h e desalted protein was immediately pumped onto a 2.5 × 34 c m DE-52 column equilibrated in buffer M. Six-milliliter fractions were collected. ABP-120 elutes in fractions 145-159.

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Froction N u m b e r Fro. 1 l. Hydroxylapatite chromatography of the DE-52 pool. Fractions pooled from the DE-52 column were applied to a 1.5 X 10.5 cm hydroxylapatite column equilibrated in buffer F. Four-milliliter fractions were collected. ABP-120 elutes in fractions 47-55.

[7]

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Fraction Number FIG. 12. Mono Q chromatography of the hydroxylapatitc pool. Fractions pooled from the hydroxylapatite column were applied to a 0.5 × 5 cm Mono Q column equilibrated in buffer N. Fractions of 0.8 ml were collected. Fractions 7 2 - 7 7 contained the pure ABP-120.

the protein. The desalted protein is applied immediately to a DE-52 column (2.5 × 34 cm) equilibrated in buffer M at 80 ml/hr. The column is washed with one column volume of buffer M and developed with a 1440ml linear gradient of 0-0.45 M NaC1 in buffer M at 50 ml/hr. ABP-120 elutes between 0.21 and 0.24 MNaC1, fractions 145- 159 (Fig. 10). The fractions are pooled and protease inhibitors are added as above. The DE-52 pool is applied to a hydroxylapatite column (1.5 X 10.5 cm) equilibrated in buffer F at 24 ml/hr. After loading, the column is washed with one column volume of buffer F and developed with a 330-ml linear gradient of 0-75 m M KPO4 in buffer F at 12 ml/hr. ABP-120 elutes between 10 and 17 mMKPO4, fractions 47-55 (Fig. 11). The pooled fractions are pumped at 2 ml/min onto a Mono Q (Pharmacia) column (0.5 × 5 era) equilibrated in buffer N. The column is washed with four column volumes of buffer N at 2 ml/min and developed with a 20-ml linear gradient of 0.2-0.5 MNaC1 in buffer N at 0.5 ml/min. ABP-120 elutes at 0.3-0.35 M NaC1, fractions 72-77 (Fig. 12). The average protein yield is 1.5-2 nag of 98% pure material from 80 ml packed cells.

Isolation of actin-binding proteins from Dictyostelium discoideum.

70 MYOSIN- AND ACTOMYOSIN-RELATED PROTEINS [7] [7] Isolation of A c t i n - B i n d i n g P r o t e i n s from Dictyostelium discoideum By A. R. B...
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