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[ 10] A f f i n i t y C h r o m a t o g r a p h y - B a s e d Profilin : Actin
By M I C H A E L
[ 1 0]
Purification of
ROZYCKI, CLARENCE E. SCHUTT, a n d U N O LINDBERG
In nonmuscle cells, as much as 50% of the cytoplasmic actin appears to exist in the nonfilamentous state, 1,2 a significant portion of which is associated with profilin in a 1 : 1 complex called profilin:actin or profilactin. This pool of unpolymerized actin enables nonmuscle cells to respond to conditions requiring the rapid assembly and reorganization of actin filaments, such as the formation ofspikelike projections during platelet stimulation, with the dissociation of the profilin: actin complex apparently freeing actin for polymerization: Recent evidence indicates that this dissociation may arise from an interaction between phosphatidylinositol 4,5-bisphosphate and profilin: actin, and a model has been proposed which links transmembrane signaling, the phosphatidylinositol cycle, and actin polymerization.4-6 Originally, profilin: actin from mammalian nonmuscle tissue was purified using ammonium sulfate fractionation followed by successive chromatographic separations on Cy alumina gel, DEAE-Sephadex, and Sephadex G-100.1,7 Additional chromatography on hydroxylapatite could then be employed to separate the profilin:fl-actin and profilin: y-actin isoforms, s Recently, the affinity of profilin:actin to a poly(L-proline)Sepharose matrix9 has been used to develop an alternative single-step procedure for purifying profdin :actin from nonmuscle tissue extracts, l° Dimethyl sulfoxide (DMSO) is used to elute profilin: actin from the affinity matrix at a degree of purity comparable to the original method, but requiring less than l day to complete. Coupled to hydroxylapatite and gel-filtration chromatography, comparable yields of homogeneous profit L. Carlsson, L.-E. NystrOm, I. Sundkvist, F. Markey, and U. Lindberg, J. MoL BioL 115, 465 (1977). 2 I. Blikstad, F. Markey, L. Carlsson, T. Persson, and U. Lindberg, Cell (Cambridge, Mass.) 15, 935 (1978). 3 F. Markey, T. Persson, and U. Lindberg, Cell (Cambridge, Mass.) 23, 145 (1981). 4 I. Lassing and U. Lindberg, Nature (London) 314, 472 (1985). 5 p. A. Janmey and T. P. Stossel, Nature (London) 325, 362 (1987). I. Lassing and U. Lindberg, Exp. CellRes. 174, 1 0988). 7 I. Blikstad, I. Sundkvist, and S. Eriksson, Eur. J. Biochem. 105, 425 (1980). s M. Segura and U. Lindberg, J. Biol. Chem. 259, 3949 (1984). 9 M. Tanaka and H. Shibata, Fur. J. Biochem. 151,291 (1985). lo U. Lindberg, C. E. Schutt, E. Hellsten, A.-C. Tj~lder, and T. Hult, Biochim. Biophys. Acta 967, 391 0988).
METHODS IN ENZYMOLOGY,VOL 196
Copyright© 1991by AcademicPress,Inc. All fightsof reproductionin any formr'-~crved.
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lin: fl-actin and profilin: ~,-actin can be obtained in less time than with the original method, and the protein purified in this manner has been shown to be satisfactory for in vitro actin polymerization experiments and for the growth of profilin: actin crystals? ° This chapter will describe the purification of profilin: actin from mammalian nonmuscle tissue by poly(L-proline)-Sepharose and gel-filtration chromatography, and the separation of profilin :fl-actin from profilin:7actin using hydroxylapatite chromatography. Methods for the separation of profilin from actin and the crystallization of profilin: actin will also be discussed. Assay Methods The most useful assay during the early stages of the purification of profilin: actin is the measurement of the inhibition of DNase I activity by monomeric actin and actin complexed to profilin. DNase I nicks doublestranded DNA, leading to an unwinding to single-stranded DNA which can be followed spectrophotometrically by measuring the hyperchromic shift of DNA at 260 nm. Actin inhibits the hydrolysis of DNA by binding to DNase I in a 1 : 1 complex, H,12 thereby reducing the overall hyperchromic shift and permitting quantitation of actin, even in crude cell extracts. Procedure
The experimental procedure has been described previously2,13 and a detailed protocol will not be repeated here. Electrophoretically pure DNase I obtained from Sigma (St. Louis, MO) is used without further purification. One 5-mg vial of Sigma DNase I (DN-EP) is prepared as a 0.1 mg/ml solution in 10 m M Tris-HC1, pH 7.6 at 4 °, 150 m M KCI, 0.5 m M CaCI2, and 0.01 m M phenylmethylsulfonyl fluoride (PMSF), and is aliquoted in 50-/zl droplets by freezing in liquid nitrogen. This is done conveniently in a cold room using a peristaltic pump set to deliver a droplet every 4 - 5 sec; a faster rate leads to fusing of droplets. The frozen droplets are transferred to a convenient receptacle and can be stored in liquid nitrogen for at least 4 months without significant loss-of activity. Before use, one or two droplets are melted in a test tube in a 25 ° water bath and are then stored on ice. The thawed enzyme usually retains full activity for at least 3 hr; however, if loss of activity is a problem, the enzyme stability may be enhanced by further tl H. G. Mannherz,J. B. Leigh,R. Leberman,and H. Pfrang,FEBSLett. 60, 34 (1975). 12S. E. Hitchcock,L. Carlsson,and U. Lindberg,Cell (CambridgeMass.) 7, 531 (1976). 13j. A. Cooperand T. D. Pollard,this series,Vol. 85, p. 182.
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purification 14 to remove trace amounts of proteolytic enzymes. The DNA substrate solution is prepared by cutting DNA from salmon testes (Sigma D-1626) into fine pieces with scissors and dissolving it to a concentration of 40- 50/~g/ml by stirring gently at 21 ° in 0.12 M Tris-HC1, pH 7.1, 4.8 m M MgSO4 and 2.1 m M CaCI2 at 40-50/~g/ml for 24-48 hr. Small particles of undissolved DNA are removed on a 0.22-/zm pore size filter. The absorbance at 260 nm (A26o) of the solution should be 0.6-0.7. Each assay uses 3 ml of DNA substrate and 1 #g of DNase mixed with varying amounts of the solution containing actin. The change in A26o is plotted as a function of time for the first 3 min after initiating the reaction at 25 °, and the maximum rate of change in A26o is determined from the linear portion of the graph. A typical specific activity for 1 #g of DNase I is 0.12-0.15 rain -1 in the absence of actin. The percentage decrease in the specific activity is then calculated for samples containing actin, with a unit of inhibition activity being defined as the reduction of 1% in the specific activity of 1/tg of DNase. 2,1a For crude extracts, filamentous actin can be depolymerized in the presence of 10 m M Tris-HC1, pH 7.6 at 21 °, 0.75 M guanidinium hydrochloride, 0.5 M acetate, 0.5 m M CaC12, and 0.5 m M adenosine 5'-triphosphate (ATP). Comparison of the number of inhibition units before and after treatment of a sample with guanidinium hydrochloride then gives a measurement of the total amount of actin present in the extract as well as the distribution between the polymerized and unpolymerized states. 2 Actin in later stages of the profilin: actin purification can be assayed for polymerization activity by measuring the change in viscosity of profilin:actin solutions using a Cannon-Manning type Ostwald viscometer (Cannon Instrument Co., State College, PA)J 3 The presence of 2.5 m M MgCI2 is required to polymerize actin from profilin: actin solutions. ~5
Preparation of Poly(L-proline)- Sepharose Affinity Column
Buffers 1 m M HCI: Prepare 3 liters by dilution of concentrated HC1 into water Coupling buffer: Prepare 4 liters of 0.1 MKHCO3, 0.5 MKC1, pH 8.3 at21 ° Deactivation buffer: Prepare 500 ml of 0.1 M Tris-HC1, pH 8.0 at 21 °, by dissolving 4.44 g Trizma hydrochloride (Sigma) and 2.65 g Trizma base (Sigma) in 500 ml water 14F. Markey, FEBSLett. 167, 155 (1984). 15 B. Malm, H. Larsson, and U. Lindberg, J. MuscleRes. CellMotiL 4, 569 (1983).
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Acetate buffer: Prepare 2 liters 0.1 N acetic acid, 0.5 MKC1, pH 4.0 at 21 ° Buffer A: Prepare 1 liter 10 m M Tris-HC1, pH 7.25 at 21", 100 m M KC1, and 100 m M glycine. On cooling to 4 °, the pH increases to 7.8. To minimize oxidation of proteins, deaerate under vacuum and purge by bubbling nitrogen or argon through the solution for several minutes.
Coupling Procedure This procedure prepares enough affinity matrix to pack a column of 130 ml. Dissolve 1 g of poly(L-proline), of M r range 10,000 to 30,000 (Sigma P-2129), in 100 ml coupling buffer and stir overnight (at least 16 hr) at 4" The poly(L-proline) dissolves more readily at 4" than at room temperature. The following day, centrifuge the solution for 25 min at 60,000 g and 4 °, and save the pellet of undissolved poly(L-proline) for further use. As the supernatant is allowed to warm to 21 *, weigh 40 g CNBr-activated Sepharose CN-4B (Pharmacia, Uppsala, Sweden) into a 4-liter capacity beaker and swell for 20 min in 2 liters 1 m M HC1, stirring very gently every 5 min to avoid damaging the Sepharose beads. Filter the swollen Sepharose in a BOchner funnel and wash five times, each with 200 ml 1 m M HC1. Remove the excess HC1 by suction, then wash twice in rapid succession, each time with 200 ml coupling buffer. After the second wash, immediately suspend the gel in 100 ml coupling buffer, combine with the solubilized poly(L-proline), and add more coupling buffer to a final volume of 300 ml to initiate the coupling reaction. Stir the mixture gently on a rotating shaker (not with a magnetic stir bar since this could damage the Sepharose) for 2 hr, at which time the coupling reaction is complete. Because poly(L-proline) has no absorbance at 280 nm, the absorbance at 230 nm (A23o)is used to measure the extent of coupling. The A23o decreased by about 70% during the course of the reaction. For convenience, the coupling mixture can be transferred to a cold room at this point and shaken further overnight. Otherwise, deactivate any remaining unreacted Sepharose by filtering and suspending it in 500 ml of deactivation buffer for 2 hr. The filtrate contains unreacted poly(L-proline) which can be added to the pelleted material obtained earlier and used in subsequent coupling reactions. To remove unreacted poly(L-proline) which may remain bound to the Sepharose, treat the matrix with five wash cycles consisting of two washes in 200 ml coupling buffer followed by two washes in 200 ml acetate buffer, changing the filter paper between each wash cycle to prevent clogging.
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Preparation of the Column Wash the poly(L-proline)-Sepharose gel on a B0chner funnel in 200 ml buffer A containing 0.5 m M dithiothreitol (DTT). Choose a column suitable for a bed volume of 130-140 ml; a short column with a diameter of 5 to 10 cm is recommended for maximum flow rates. Pour the affinity matrix into the column and pack at a flow rate of 500 ml/hr, washing with at least two bed volumes of buffer A containing 0.5 m M DTT.
Preparation of Hydroxylapatite Column
Buffers Potassium phosphate (2 M): Make up a 2 M stock solution to be used in preparing subsequent buffers. Dissolve 258 g anhydrous K2HPO 4 and 70.8 g KH2PO4 in 1 liter of high-purity water HEB: 4 liters of hydroxylapatite equilibration buffer, 5 m M potassium phosphate, pH 7.5 at 21 °. Dilute 2.5 ml of the 2 M phosphate stock solution into l liter of water. The solution should be at the correct pH without adjustment. Deaerate before use
Preparation of Matrix and Column Hydroxylapatite (Hypatite-C) is obtained from Clarkson Chemical Company (Williamsport, PA). The choice of material is critical; after screening hydroxylapatite from a number of suppliers, reproducible separations of the actin fl and 7 isoforms have been observed only with Hyparite-C, and then only when using even-numbered lots corresponding to a finer particle size. Add 75 g Hypatite-C to 1.5 liters of HEB, stir gently with a glass rod to make a homogeneous flurry, and adjust the pH to 7.5 with KOH. After the gel settles, allow it to stand 1 day at 21 *, preferably under vacuum or inert gas atmosphere to minimize CO2 contamination. Decant the supernatant, add fresh buffer, readjust the pH, and allow to stand another day. Continue this procedure until the pH has stabilized; usually the pH does not vary significantly after two buffer changes. To minimize the precipitation and accumulation of calcium carbonate on the matrix, equilibrate the hydroxylapatite and pour it into the column at room temperature before transferring the sealed column to the cold room. Pour the column by changing the buffer once more, stirring well to make a homogeneous slurry, and filling a 5 X 30 cm (diameter X height) column with the flurry. Open the outlet tube to begin draining the column by gravity. When the matrix has mostly settled (surface is well defined), siphon offthe excess
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buffer, which is still slightly cloudy from the presence of fine particles, and add more slurry. Continue pouring in this manner until all of the slurry is used, seal the column with a flow adaptor, and wash the matrix at approximately 400 ml/hr with two bed volumes of HEB to ensure complete packing. The final bed volume should be about 150 ml.
Purification of Profilin : Actin and Separation of Actin Isoforms
Buffers Buffer A: Prepare 1 liter as described above Buffer B: Prepare 500 ml of 5 m M potassium phosphate, pH 7.5 at 21 °, 0.5 m M D T T Buffer C: Prepare 500 ml of 40 m M potassium phosphate, pH 7.5 at 21 °, 0.5 m M DTT, 1.5 M glycine Buffer E: Prepare 1 liter of buffer A as described above, but containing 30% (v/v) DMSO Buffer G: Prepare 2 liters of 5 m M potassium phosphate, pH 7.5 at 21 °, 2 m M DTT, 0.5 m M A T P , 0.2 mM CaCh Buffer H: Prepare 1 liter of buffer A as described above, but containing 0.5 m M DTT and 0.5% Triton X-100. The Triton X-100 should be added several hours before use to ensure complete solubilization A m m o n i u m sulfate: Prepare 5 liters of saturated (NH4)2SO4, pH 7.5 at 4 ° (adjusted with NH4OH ) To minimize oxidation of proteins, all buffers should be deaerated and purged with nitrogen or argon before use.
Tissue Homogenization At present, this purification procedure has been tested most thoroughly with calf spleen and thymus tissue, although preliminary results show that profilin:actin can be purified equally well from other cell types such as human platelets and human placenta. The methods described below will be presented assuming that calf spleen or thymus is used. Spleens and thymus glands from newborn to 3-month-old calves are obtained from Pel-Freez Biologlcals (Rogers, AK), specially ordered so that the tissue is removed from the animal as soon as possible after slaughter and shipped on dry ice. Tissue obtained in this manner yields profilin:actin which is indistinguishable from that purified using fresh tissue obtained at a local abattoir, as judged by SDS gel electrophoresis, DNase inhibition assays, actin polymerization assays, and crystallizability. The tissue can be stored for many months at - 8 0 ° without loss of quality.
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All steps are carried out at 4 ° unless otherwise specified. To minimize proteolysis, the tissue is homogenized from the frozen state without thawing. We have tried employing PMSF in the homogenization buffer without any significant improvement in the quality ofprofilin: actin on SDS gels; it appears that more effective prevention of proteolysis is achieved by minimizing the cell autolysis which accompanies slow thawing. In a typical preparation, 600-700 g of frozen tissue is washed very briefly with highpurity water, cut into small pieces, and added with 800 ml of buffer H to a Cuisinart or similar type of food homogenizer. Using the grating attachment, mince the frozen tissue into the buffer, then change to the cutting blade and homogenize for a total of 45 see, resting periodically to avoid overheating the material. Centrifuge this crude homogenate for 20 min at 5000 g and 4 ° in a large-capacity rotor. Decant the supernatant, filter through cheesecloth, and centrifuge for 30 min at 40,000 rpm (125,000 g) and 4 ° in a 45 Ti rotor (Beckman, Palo Alto, CA). Filter the 45 Ti supernatant through cheesecloth, and apply the dark red filtrate to the poly(L-proline)-Sepharose column as described in the following section. Poly(L-proline) - Sepharose Chromatography
Load the column by adding one or more bed volumes of 45 Ti supernatant to the column, using a pipette or glass rod to stir the matrix and supernatant into a homogeneous slurry for about 1 min. After a pause of several minutes to allow the gel to settle, open the column to a flow rate of 500 ml/hr. When almost all of the effluent has passed through the column, make a second addition of supernatant in the same way, and repeat the process until all of the supernatant is used. Wash the gel by adding one column volume of buffer A to the column, gently stirring the gel to make a homogeneous slurry, and draining the buffer. This step is important because, in the presence of crude tissue extract, pockets of gel pack very tightly under high flow rates and do not wash as thoroughly as other parts of the column bed. After one or two such rinses, most of the extract is removed and the gel becomes uniformly permeable to buffer again, so that the column can be sealed and eluted with a buffer inlet tube. At this point, attach the column to a UV monitor and recorder and continue washing until the A280falls to zero (approximately three bed volumes). Begin elution with buffer E, which removes profilin and profilin:actin from the column. DTT is absent during the washes in buffers A and E because of its potential to react with DMSO to form dimethyl sulfide, a potential modifier of protein sulfhydryl groups. In addition, precautions should be taken to minimize the exposure of laboratory personnel to DMSO because of its generally acknowledged ability to solubilize toxins through the skin and its
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potential as a teratogen and carcinogen. Laboratory personnel often complain of irritation of the skin and respiratory system if DMSO is allowed to escape into the laboratory environment. The protein yield from the elution of profilin:actin from the poly(Lproline)-Sepharose column in buffer E is estimated from A2s0 measurements. The ratio A2so/A26ois usually 2.0, indicating that profilin:actin is eluted from the column without bound ATP. SDS gel electrophoresis and gel densitometry show that approximately 95% of the protein eluted with buffer E consists of profilin and actin (Fig. 1). Since poly(L-proline) interacts primarily with profilin, there is usually a molar excess of profllin over actin at this stage, l° One minor component is a protein of M, 38,000 which may represent a degradation product of actin. I The remaining minor components are concentrated mainly in the first 20% of the buffer E effluent, and can be reduced by discarding this portion of the protein peak. However, since these components are also removed in subsequent chromatography steps, it is not necessary to sacrifice this amount of profilin: actin. No differences in protein quality are observed in SDS gels of protein purified from calf spleen compared to that from thymus. Table I summarizes the purification of profilin:actin from calf spleen and thymus. Although the total protein yield, measured in A2so units, is higher in the spleen than in the thymus crude extract, the total DNase I inhibition activity in the thymus extract is significantly higher than in the spleen extract, and a larger proportion of the total activity recovered after the poly(L-proline)-Sepharose column is found in the flow through than in the buffer E eluate for thymus extract, compared to spleen extract. Increasing the quantity of thymus extract applied to the column results in a proportional increase in profilin:actin recovered during elution with buffer E, and reapplication of the flow through to a second poly(Lproline)-Sepharose column does not yield a significant amount of protein by buffer E elution. Therefore, the affinity matrix does not appear to be overloaded for this quantity of tissue extract, and the difference in the total inhibition activity found in crude extracts of calf thymus and spleen must be due to the presence of a significant proportion of unpolymerized actin which is not bound to profilin. Nevertheless, the protein yield from elution of the poly(L-proline)-Sepharose column with buffer E is similar in both tissues, as is the DNase I specific inhibition activity, indicating that profilin: actin is present at comparable levels in the spleen and thymus extracts. After pooling the desired fractions from buffer E elution, the protein can either be applied directly to the hydroxylapatite column for profilin: pactin and profilin:~,-actin subfractionation (next section), or it can be stored as an ammonium sulfate precipitate. In the latter case, dialyze the pooled buffer E peak fractions under a nitrogen or argon atmosphere
108
MYOSIN- A N D ACTOMYOSIN-RELATED PROTEINS
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A I
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FIO. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) performed according to the method of Laemmli [U. K. Laemmli, Nature (London) 227, 680 (1980)] on profilin: actin from calf spleen at successive stages of a single purification. Protein eluted with buffer E from the poly(L-proline)-Sepharose column is shown in (a), protein fron~ peaks I, II, and III of the hydroxylapatite column profile in Fig. 2 is shown in (b), (c), and (d), respectively, and profilin:~actin after Sephacryl S-200 HR chromatography (peak PA of the column profile shown in Fig. 3) is shown in (e). The positions of actin (A) and profilin (P) are marked, as are the positions of significant contaminating proteins with molecular weights of 38,000, 32,000, and 27,000 (top, middle, and bottom arrowheads, respectively). Gels were 13% polyacrylamide, stained with Coomassie Blue. Approximately 10 #tg of protein was loaded in each lane. Gel densitometry was carried out using a Bio-Rad model 620 video densitometer (Bio-Rad, Richmond, CA).
[ 10]
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against 4 vol of ammonium sulfate solution for 6 hr, at which point small amounts of precipitate begin to appear. Turn the bag upside down several times to mix the contents, change to fresh ammonium sulfate of the same volume containing 0.5 m M 2-mercaptoethanol, and continue dialyzing overnight. Precipitation is usually complete after 24 hr, and profilin: actin is stable in this condition for several months at 4°. 1°
Hydroxylapatite Chromatography of Poly(L-proline) - Sepharose Column Fractions If hydroxylapatite chromatography is to be performed immediately following poly(L-proline)-Sepharose chromatography, dilute the buffer E peak with 2 vol of HEB and load onto the hydroxylapatite column at a flow rate of 400 ml/hr. Undiluted extract does not bind completely to the column. After all of the protein has been applied, wash the column at the same flow rate with HEB until the DMSO is washed out, as detected by monitoring the A23o. Usually, five bed volumes is required. The direct application of the poly(L-proline)- Sepharose-purified profilin: actin to the hydroxylapatite column offers the advantages of completing the first two chromatography steps in 1 day, removing completely the protein from DMSO, and eliminating the exposure of the protein to ammonium sulfate, which is often of variable quality even for "enzyme-grade" material. Profilin:actin precipitated with ammonium sulfate must be desalted before application to the hydroxylapatite column. Recover profilin:actin by taking enough ammonium sulfate-precipitated material to contain 200 A2so units and centrifuging for 20 min at 50,000 g and 4*. Suspend the pellet in a volume of buffer G to give a protein concentration of 1015 mg/ml, and centrifuge again to remove undissolved protein. Resolubilizing profilin: actin in the presence of ATP and calcium ions seems to be important in stabilizing the polymerization activity of actin, although protein loaded directly from the poly(L-proline)-Sepharose column appears to be stable without ATP or calcium. However, ATP is not included in the subsequent wash and elution of the hydroxylapatite column because of its high affinity to hydroxylapatite. After desalting the profilin: actin into buffer B by dialysis or Sephadex G-25 chromatography, apply the protein to the hydroxylapatite column at a flow rate of 400 ml/hr. Wash the column in one bed volume of buffer B, and elute the bound profilin: actin with a linear gradient prepared from two bed volumes of buffer B as the starting buffer and two bed volumes of buffer C as the final buffer, using a flow rate of approximately 50 ml/hr. A hydroxylapatite column elution profile for calf spleen profilin: actin is shown in Fig. 2 and contains three peaks, labeled I, II, and III. Peak I
[ 10]
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111
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EFFLUENT (ml) Fro. 2. Elution profile of hydroxylapatite chromatography performed on the buffer E peak from a po]y(L-proi/ne)-Sepharose column. SDS-PAGE analysis of the three peaks, labeled I, II, and III, is shown in Fig. I. Peak II corresponds to profii/n: 7-aetin and peak III corresponds to proSlin :/~-actin. s,'° Elution was with a linear gradient of 800 ral starting with buffer B and ending with buffer C. The elution of profilin: actin is complete after approximately 50% of the gradient has passed through the column.
contains mostly actin, profilin, and a protein of M, 38,000 which may be a degradation product of actin.~ A number of minor components with molecular weights ranging between 27,000 and 38,000 comprise approximately 12% of the total protein, as determined by gel densitometry. Compared to the poly(L-proline)-Sepharose-purified material, the amount of profdin in this peak is significantly reduced, although in previous studies the protein eluting at the same gradient position contained almost pure profilin, s,~° The reason for this difference is not known, although it must involve the ability of excess profilin to bind to hydroxylapatite, since the molar excess of profilin over actin found in the poly(L-proline)Sepharose-purified material is eliminated in profilin :actin after hydroxylapatite chromatography. The remaining peaks II and III show greater homogeneity of profilin and actin, and occur at the same positions in the phosphate/glycine gradient as the peaks corresponding to profilin: 7-actin and profilin :fl-actin, r e s p e c t i v e l y . 7,9 A significant contaminant in each case is the protein of Mr 38,000, comprising approximately 12% of the total protein in peak II and 4% in peak III. If this protein is a degradation
112
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product of actin, the level of contamination of peaks II and III by nonprofilin: actin proteins is less than 1%. From Table I, the relative proportions of profilin: y-actin to profilin: flactin are 30:70 from spleen and 28:72 from thymus. These values are almost identical to those reported previously for hydroxylapatite-fractionated profilin: actin from calf spleen, although a slightly higher proportion of profilin: 7-actin was reported for protein from calf thymus.7 For both peaks, the specific DNase I inhibition activities and the percentage yields of total activity are very close for spleen compared to thymus protein. Hydroxylapatite-fractionated profilin: actin can be used for gel-filtration chromatography (next section), or it can be stored by ammonium sulfate precipitation as described previously.
Gel-Filtration Chromatography The profilin: actin isoforms obtained by hydroxylapatite chromatography can be subjected to gel-filtration chromatography to separate profilin:actin from higher molecular weight species which may result from polymerization or nonspecific aggregation processes, as well as to remove small amounts of contaminants which may remain after hydroxylapatite chromatography. Of particular interest is the presence of minute amounts of a factor which apparently stabilizes profilin: actin against dissociationt5 and which is removed more effectively at low (5 mM) than at high (500 mM) potassium phosphate concentrations by Sephadex G-100 chromatography. Previously, Sephadex G- 100 or Superose 6 resins (Pharmacia) have been used following hydroxylapatite chromatography.t° However, the introduction of the Sephacryl high-resolution matrices from Pharmacia have enabled us to obtain separations of equivalent quality in approximately 10-20% of the time required for other separation materials. To pour a Sephacryl S-200 high-resolution (S-200 HR) column, add 450 ml of a well-suspended slurry from the bottle to 150 ml of HEB and stir gently (swirling is best to avoid damaging the Sephacryl beads). Pour the slurry into a Pharmacia 2.6 × 60 cm column equipped with a packing reservoir, and pump buffer at a flow rate of 150 ml/hr using positive pressure from the top of the column rather than negative pressure from below, adding more HEB as required to prevent the column from going dry. The gel height should stabilize in 2 hr, at which time the flow rate should be increased to 300 ml/hr and pumping continued for another hour. Disconnect the column from the pump, attach the upper flow adaptor, turn the column upside down, and wash with two bed volumes of buffer G at 150 ml/hr. The final packed column bed volume should be approximately 300 ml. Protein can be used either after storage as an
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113
ammonium sulfate precipitate, or immediately after elution from the hydroxylapatite column. If ammonium sulfate-precipitated protein is used, centrifuge the homogeneous suspension for 20 min at 50,000 g and 4°, resuspend the pellet in buffer G to a volume of 10 ml containing 100-200 A280units, and centrifuge a second time. Protein to be applied directly from the hydroxylapatite column must be concentrated to 10 ml on an Amicon PMI0 or YM10 ultrafiltration membrane (Amicon, Danvers, MA). We have not observed any difference in yield between the two membranes, but the PM 10 gives a higher flow rate. Recovery is usually greater than 90%, and the protein is then centrifuged to remove any aggregated material. Profilin: actin is applied to the S-200 HR column at a flow rate of 100 ml/ hr and eluted at the same rate with buffer G. The elution profile of an S-200 HR run on profilin :fl-actin is shown in Fig. 3 and features a small peak (A), running at the void volume, and consisting of actin and several minor components. No profilin is present in this peak. The presence of actin at the void volume is probably a result of an aggregation or polymerization phenomenon. The larger, second peak (PA) contains profilin: actin of very high purity. SDS gel electrophoresis shows the protein composition in this peak to be nearly identical to the composition of the starting material, hydroxylapatite-purified profilin :fl-actin. Table I shows the protein yields and DNase I inhibition activities to be comparable for S-200 HR-purified profilin:actin from spleen and thymus. Relative to the specific inhibition activities of the poly(L-proline)-Sepharose and hydroxylapatite-purified profilin: actin, the activity after S-200 HR chromatography has increased to the theoretical maximum for profilin: actin, ~°even though no significant purification differences are evident by SDS gel electrophoresis. This suggests that relatively minute changes in the protein composition of profilin: actin can affect significantly the behavior of the system.
Column Regeneration Immediately after completion of the buffer E elution, the poly(Lproline)-Sepharose column is washed in 5 vol of buffer A to remove DMSO. If regenerated carefully, the poly(L-proline)-Sepharose matrix is reusable with no apparent loss of profilin: actin binding capacity or deterioration of the quality of protein recovered. The most obvious contaminant on the column appears to be an insoluble off-white material. Although we have not analyzed the material, it appears to be lipid-like in consistency and is more pronounced in spleen than in thymus preparations, and when spleens larger than 150 g are used. Aside from its potential as a contaminant of profilin:actin, the material has a tendency to clog the column, thereby reducing flow rates. To remove this material, the column contents
I 14
MYOSIN- A N D ACTOMYOSIN-RELATED
PROTEINS
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MYOSIN- AND ACTOMYOSIN-RELATED PROTEINS
[ 10] A f f i n i t y C h r o m a t o g r a p h y - B a s e d Profilin : Actin
By M I C H A E L
[ 1 0]
Purification of
ROZYCKI, CLARENCE E. SCHUTT, a n d U N O LINDBERG
In nonmuscle cells, as much as 50% of the cytoplasmic actin appears to exist in the nonfilamentous state, 1,2 a significant portion of which is associated with profilin in a 1 : 1 complex called profilin:actin or profilactin. This pool of unpolymerized actin enables nonmuscle cells to respond to conditions requiring the rapid assembly and reorganization of actin filaments, such as the formation ofspikelike projections during platelet stimulation, with the dissociation of the profilin: actin complex apparently freeing actin for polymerization: Recent evidence indicates that this dissociation may arise from an interaction between phosphatidylinositol 4,5-bisphosphate and profilin: actin, and a model has been proposed which links transmembrane signaling, the phosphatidylinositol cycle, and actin polymerization.4-6 Originally, profilin: actin from mammalian nonmuscle tissue was purified using ammonium sulfate fractionation followed by successive chromatographic separations on Cy alumina gel, DEAE-Sephadex, and Sephadex G-100.1,7 Additional chromatography on hydroxylapatite could then be employed to separate the profilin:fl-actin and profilin: y-actin isoforms, s Recently, the affinity of profilin:actin to a poly(L-proline)Sepharose matrix9 has been used to develop an alternative single-step procedure for purifying profdin :actin from nonmuscle tissue extracts, l° Dimethyl sulfoxide (DMSO) is used to elute profilin: actin from the affinity matrix at a degree of purity comparable to the original method, but requiring less than l day to complete. Coupled to hydroxylapatite and gel-filtration chromatography, comparable yields of homogeneous profit L. Carlsson, L.-E. NystrOm, I. Sundkvist, F. Markey, and U. Lindberg, J. MoL BioL 115, 465 (1977). 2 I. Blikstad, F. Markey, L. Carlsson, T. Persson, and U. Lindberg, Cell (Cambridge, Mass.) 15, 935 (1978). 3 F. Markey, T. Persson, and U. Lindberg, Cell (Cambridge, Mass.) 23, 145 (1981). 4 I. Lassing and U. Lindberg, Nature (London) 314, 472 (1985). 5 p. A. Janmey and T. P. Stossel, Nature (London) 325, 362 (1987). I. Lassing and U. Lindberg, Exp. CellRes. 174, 1 0988). 7 I. Blikstad, I. Sundkvist, and S. Eriksson, Eur. J. Biochem. 105, 425 (1980). s M. Segura and U. Lindberg, J. Biol. Chem. 259, 3949 (1984). 9 M. Tanaka and H. Shibata, Fur. J. Biochem. 151,291 (1985). lo U. Lindberg, C. E. Schutt, E. Hellsten, A.-C. Tj~lder, and T. Hult, Biochim. Biophys. Acta 967, 391 0988).
METHODS IN ENZYMOLOGY,VOL 196
Copyright© 1991by AcademicPress,Inc. All fightsof reproductionin any formr'-~crved.
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101
lin: fl-actin and profilin: ~,-actin can be obtained in less time than with the original method, and the protein purified in this manner has been shown to be satisfactory for in vitro actin polymerization experiments and for the growth of profilin: actin crystals? ° This chapter will describe the purification of profilin: actin from mammalian nonmuscle tissue by poly(L-proline)-Sepharose and gel-filtration chromatography, and the separation of profilin :fl-actin from profilin:7actin using hydroxylapatite chromatography. Methods for the separation of profilin from actin and the crystallization of profilin: actin will also be discussed. Assay Methods The most useful assay during the early stages of the purification of profilin: actin is the measurement of the inhibition of DNase I activity by monomeric actin and actin complexed to profilin. DNase I nicks doublestranded DNA, leading to an unwinding to single-stranded DNA which can be followed spectrophotometrically by measuring the hyperchromic shift of DNA at 260 nm. Actin inhibits the hydrolysis of DNA by binding to DNase I in a 1 : 1 complex, H,12 thereby reducing the overall hyperchromic shift and permitting quantitation of actin, even in crude cell extracts. Procedure
The experimental procedure has been described previously2,13 and a detailed protocol will not be repeated here. Electrophoretically pure DNase I obtained from Sigma (St. Louis, MO) is used without further purification. One 5-mg vial of Sigma DNase I (DN-EP) is prepared as a 0.1 mg/ml solution in 10 m M Tris-HC1, pH 7.6 at 4 °, 150 m M KCI, 0.5 m M CaCI2, and 0.01 m M phenylmethylsulfonyl fluoride (PMSF), and is aliquoted in 50-/zl droplets by freezing in liquid nitrogen. This is done conveniently in a cold room using a peristaltic pump set to deliver a droplet every 4 - 5 sec; a faster rate leads to fusing of droplets. The frozen droplets are transferred to a convenient receptacle and can be stored in liquid nitrogen for at least 4 months without significant loss-of activity. Before use, one or two droplets are melted in a test tube in a 25 ° water bath and are then stored on ice. The thawed enzyme usually retains full activity for at least 3 hr; however, if loss of activity is a problem, the enzyme stability may be enhanced by further tl H. G. Mannherz,J. B. Leigh,R. Leberman,and H. Pfrang,FEBSLett. 60, 34 (1975). 12S. E. Hitchcock,L. Carlsson,and U. Lindberg,Cell (CambridgeMass.) 7, 531 (1976). 13j. A. Cooperand T. D. Pollard,this series,Vol. 85, p. 182.
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MYOSIN- AND ACTOMYOSIN-RELATED PROTEINS
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purification 14 to remove trace amounts of proteolytic enzymes. The DNA substrate solution is prepared by cutting DNA from salmon testes (Sigma D-1626) into fine pieces with scissors and dissolving it to a concentration of 40- 50/~g/ml by stirring gently at 21 ° in 0.12 M Tris-HC1, pH 7.1, 4.8 m M MgSO4 and 2.1 m M CaCI2 at 40-50/~g/ml for 24-48 hr. Small particles of undissolved DNA are removed on a 0.22-/zm pore size filter. The absorbance at 260 nm (A26o) of the solution should be 0.6-0.7. Each assay uses 3 ml of DNA substrate and 1 #g of DNase mixed with varying amounts of the solution containing actin. The change in A26o is plotted as a function of time for the first 3 min after initiating the reaction at 25 °, and the maximum rate of change in A26o is determined from the linear portion of the graph. A typical specific activity for 1 #g of DNase I is 0.12-0.15 rain -1 in the absence of actin. The percentage decrease in the specific activity is then calculated for samples containing actin, with a unit of inhibition activity being defined as the reduction of 1% in the specific activity of 1/tg of DNase. 2,1a For crude extracts, filamentous actin can be depolymerized in the presence of 10 m M Tris-HC1, pH 7.6 at 21 °, 0.75 M guanidinium hydrochloride, 0.5 M acetate, 0.5 m M CaC12, and 0.5 m M adenosine 5'-triphosphate (ATP). Comparison of the number of inhibition units before and after treatment of a sample with guanidinium hydrochloride then gives a measurement of the total amount of actin present in the extract as well as the distribution between the polymerized and unpolymerized states. 2 Actin in later stages of the profilin: actin purification can be assayed for polymerization activity by measuring the change in viscosity of profilin:actin solutions using a Cannon-Manning type Ostwald viscometer (Cannon Instrument Co., State College, PA)J 3 The presence of 2.5 m M MgCI2 is required to polymerize actin from profilin: actin solutions. ~5
Preparation of Poly(L-proline)- Sepharose Affinity Column
Buffers 1 m M HCI: Prepare 3 liters by dilution of concentrated HC1 into water Coupling buffer: Prepare 4 liters of 0.1 MKHCO3, 0.5 MKC1, pH 8.3 at21 ° Deactivation buffer: Prepare 500 ml of 0.1 M Tris-HC1, pH 8.0 at 21 °, by dissolving 4.44 g Trizma hydrochloride (Sigma) and 2.65 g Trizma base (Sigma) in 500 ml water 14F. Markey, FEBSLett. 167, 155 (1984). 15 B. Malm, H. Larsson, and U. Lindberg, J. MuscleRes. CellMotiL 4, 569 (1983).
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Acetate buffer: Prepare 2 liters 0.1 N acetic acid, 0.5 MKC1, pH 4.0 at 21 ° Buffer A: Prepare 1 liter 10 m M Tris-HC1, pH 7.25 at 21", 100 m M KC1, and 100 m M glycine. On cooling to 4 °, the pH increases to 7.8. To minimize oxidation of proteins, deaerate under vacuum and purge by bubbling nitrogen or argon through the solution for several minutes.
Coupling Procedure This procedure prepares enough affinity matrix to pack a column of 130 ml. Dissolve 1 g of poly(L-proline), of M r range 10,000 to 30,000 (Sigma P-2129), in 100 ml coupling buffer and stir overnight (at least 16 hr) at 4" The poly(L-proline) dissolves more readily at 4" than at room temperature. The following day, centrifuge the solution for 25 min at 60,000 g and 4 °, and save the pellet of undissolved poly(L-proline) for further use. As the supernatant is allowed to warm to 21 *, weigh 40 g CNBr-activated Sepharose CN-4B (Pharmacia, Uppsala, Sweden) into a 4-liter capacity beaker and swell for 20 min in 2 liters 1 m M HC1, stirring very gently every 5 min to avoid damaging the Sepharose beads. Filter the swollen Sepharose in a BOchner funnel and wash five times, each with 200 ml 1 m M HC1. Remove the excess HC1 by suction, then wash twice in rapid succession, each time with 200 ml coupling buffer. After the second wash, immediately suspend the gel in 100 ml coupling buffer, combine with the solubilized poly(L-proline), and add more coupling buffer to a final volume of 300 ml to initiate the coupling reaction. Stir the mixture gently on a rotating shaker (not with a magnetic stir bar since this could damage the Sepharose) for 2 hr, at which time the coupling reaction is complete. Because poly(L-proline) has no absorbance at 280 nm, the absorbance at 230 nm (A23o)is used to measure the extent of coupling. The A23o decreased by about 70% during the course of the reaction. For convenience, the coupling mixture can be transferred to a cold room at this point and shaken further overnight. Otherwise, deactivate any remaining unreacted Sepharose by filtering and suspending it in 500 ml of deactivation buffer for 2 hr. The filtrate contains unreacted poly(L-proline) which can be added to the pelleted material obtained earlier and used in subsequent coupling reactions. To remove unreacted poly(L-proline) which may remain bound to the Sepharose, treat the matrix with five wash cycles consisting of two washes in 200 ml coupling buffer followed by two washes in 200 ml acetate buffer, changing the filter paper between each wash cycle to prevent clogging.
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Preparation of the Column Wash the poly(L-proline)-Sepharose gel on a B0chner funnel in 200 ml buffer A containing 0.5 m M dithiothreitol (DTT). Choose a column suitable for a bed volume of 130-140 ml; a short column with a diameter of 5 to 10 cm is recommended for maximum flow rates. Pour the affinity matrix into the column and pack at a flow rate of 500 ml/hr, washing with at least two bed volumes of buffer A containing 0.5 m M DTT.
Preparation of Hydroxylapatite Column
Buffers Potassium phosphate (2 M): Make up a 2 M stock solution to be used in preparing subsequent buffers. Dissolve 258 g anhydrous K2HPO 4 and 70.8 g KH2PO4 in 1 liter of high-purity water HEB: 4 liters of hydroxylapatite equilibration buffer, 5 m M potassium phosphate, pH 7.5 at 21 °. Dilute 2.5 ml of the 2 M phosphate stock solution into l liter of water. The solution should be at the correct pH without adjustment. Deaerate before use
Preparation of Matrix and Column Hydroxylapatite (Hypatite-C) is obtained from Clarkson Chemical Company (Williamsport, PA). The choice of material is critical; after screening hydroxylapatite from a number of suppliers, reproducible separations of the actin fl and 7 isoforms have been observed only with Hyparite-C, and then only when using even-numbered lots corresponding to a finer particle size. Add 75 g Hypatite-C to 1.5 liters of HEB, stir gently with a glass rod to make a homogeneous flurry, and adjust the pH to 7.5 with KOH. After the gel settles, allow it to stand 1 day at 21 *, preferably under vacuum or inert gas atmosphere to minimize CO2 contamination. Decant the supernatant, add fresh buffer, readjust the pH, and allow to stand another day. Continue this procedure until the pH has stabilized; usually the pH does not vary significantly after two buffer changes. To minimize the precipitation and accumulation of calcium carbonate on the matrix, equilibrate the hydroxylapatite and pour it into the column at room temperature before transferring the sealed column to the cold room. Pour the column by changing the buffer once more, stirring well to make a homogeneous slurry, and filling a 5 X 30 cm (diameter X height) column with the flurry. Open the outlet tube to begin draining the column by gravity. When the matrix has mostly settled (surface is well defined), siphon offthe excess
[ 10]
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105
buffer, which is still slightly cloudy from the presence of fine particles, and add more slurry. Continue pouring in this manner until all of the slurry is used, seal the column with a flow adaptor, and wash the matrix at approximately 400 ml/hr with two bed volumes of HEB to ensure complete packing. The final bed volume should be about 150 ml.
Purification of Profilin : Actin and Separation of Actin Isoforms
Buffers Buffer A: Prepare 1 liter as described above Buffer B: Prepare 500 ml of 5 m M potassium phosphate, pH 7.5 at 21 °, 0.5 m M D T T Buffer C: Prepare 500 ml of 40 m M potassium phosphate, pH 7.5 at 21 °, 0.5 m M DTT, 1.5 M glycine Buffer E: Prepare 1 liter of buffer A as described above, but containing 30% (v/v) DMSO Buffer G: Prepare 2 liters of 5 m M potassium phosphate, pH 7.5 at 21 °, 2 m M DTT, 0.5 m M A T P , 0.2 mM CaCh Buffer H: Prepare 1 liter of buffer A as described above, but containing 0.5 m M DTT and 0.5% Triton X-100. The Triton X-100 should be added several hours before use to ensure complete solubilization A m m o n i u m sulfate: Prepare 5 liters of saturated (NH4)2SO4, pH 7.5 at 4 ° (adjusted with NH4OH ) To minimize oxidation of proteins, all buffers should be deaerated and purged with nitrogen or argon before use.
Tissue Homogenization At present, this purification procedure has been tested most thoroughly with calf spleen and thymus tissue, although preliminary results show that profilin:actin can be purified equally well from other cell types such as human platelets and human placenta. The methods described below will be presented assuming that calf spleen or thymus is used. Spleens and thymus glands from newborn to 3-month-old calves are obtained from Pel-Freez Biologlcals (Rogers, AK), specially ordered so that the tissue is removed from the animal as soon as possible after slaughter and shipped on dry ice. Tissue obtained in this manner yields profilin:actin which is indistinguishable from that purified using fresh tissue obtained at a local abattoir, as judged by SDS gel electrophoresis, DNase inhibition assays, actin polymerization assays, and crystallizability. The tissue can be stored for many months at - 8 0 ° without loss of quality.
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All steps are carried out at 4 ° unless otherwise specified. To minimize proteolysis, the tissue is homogenized from the frozen state without thawing. We have tried employing PMSF in the homogenization buffer without any significant improvement in the quality ofprofilin: actin on SDS gels; it appears that more effective prevention of proteolysis is achieved by minimizing the cell autolysis which accompanies slow thawing. In a typical preparation, 600-700 g of frozen tissue is washed very briefly with highpurity water, cut into small pieces, and added with 800 ml of buffer H to a Cuisinart or similar type of food homogenizer. Using the grating attachment, mince the frozen tissue into the buffer, then change to the cutting blade and homogenize for a total of 45 see, resting periodically to avoid overheating the material. Centrifuge this crude homogenate for 20 min at 5000 g and 4 ° in a large-capacity rotor. Decant the supernatant, filter through cheesecloth, and centrifuge for 30 min at 40,000 rpm (125,000 g) and 4 ° in a 45 Ti rotor (Beckman, Palo Alto, CA). Filter the 45 Ti supernatant through cheesecloth, and apply the dark red filtrate to the poly(L-proline)-Sepharose column as described in the following section. Poly(L-proline) - Sepharose Chromatography
Load the column by adding one or more bed volumes of 45 Ti supernatant to the column, using a pipette or glass rod to stir the matrix and supernatant into a homogeneous slurry for about 1 min. After a pause of several minutes to allow the gel to settle, open the column to a flow rate of 500 ml/hr. When almost all of the effluent has passed through the column, make a second addition of supernatant in the same way, and repeat the process until all of the supernatant is used. Wash the gel by adding one column volume of buffer A to the column, gently stirring the gel to make a homogeneous slurry, and draining the buffer. This step is important because, in the presence of crude tissue extract, pockets of gel pack very tightly under high flow rates and do not wash as thoroughly as other parts of the column bed. After one or two such rinses, most of the extract is removed and the gel becomes uniformly permeable to buffer again, so that the column can be sealed and eluted with a buffer inlet tube. At this point, attach the column to a UV monitor and recorder and continue washing until the A280falls to zero (approximately three bed volumes). Begin elution with buffer E, which removes profilin and profilin:actin from the column. DTT is absent during the washes in buffers A and E because of its potential to react with DMSO to form dimethyl sulfide, a potential modifier of protein sulfhydryl groups. In addition, precautions should be taken to minimize the exposure of laboratory personnel to DMSO because of its generally acknowledged ability to solubilize toxins through the skin and its
[ 10]
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107
potential as a teratogen and carcinogen. Laboratory personnel often complain of irritation of the skin and respiratory system if DMSO is allowed to escape into the laboratory environment. The protein yield from the elution of profilin:actin from the poly(Lproline)-Sepharose column in buffer E is estimated from A2s0 measurements. The ratio A2so/A26ois usually 2.0, indicating that profilin:actin is eluted from the column without bound ATP. SDS gel electrophoresis and gel densitometry show that approximately 95% of the protein eluted with buffer E consists of profilin and actin (Fig. 1). Since poly(L-proline) interacts primarily with profilin, there is usually a molar excess of profllin over actin at this stage, l° One minor component is a protein of M, 38,000 which may represent a degradation product of actin. I The remaining minor components are concentrated mainly in the first 20% of the buffer E effluent, and can be reduced by discarding this portion of the protein peak. However, since these components are also removed in subsequent chromatography steps, it is not necessary to sacrifice this amount of profilin: actin. No differences in protein quality are observed in SDS gels of protein purified from calf spleen compared to that from thymus. Table I summarizes the purification of profilin:actin from calf spleen and thymus. Although the total protein yield, measured in A2so units, is higher in the spleen than in the thymus crude extract, the total DNase I inhibition activity in the thymus extract is significantly higher than in the spleen extract, and a larger proportion of the total activity recovered after the poly(L-proline)-Sepharose column is found in the flow through than in the buffer E eluate for thymus extract, compared to spleen extract. Increasing the quantity of thymus extract applied to the column results in a proportional increase in profilin:actin recovered during elution with buffer E, and reapplication of the flow through to a second poly(Lproline)-Sepharose column does not yield a significant amount of protein by buffer E elution. Therefore, the affinity matrix does not appear to be overloaded for this quantity of tissue extract, and the difference in the total inhibition activity found in crude extracts of calf thymus and spleen must be due to the presence of a significant proportion of unpolymerized actin which is not bound to profilin. Nevertheless, the protein yield from elution of the poly(L-proline)-Sepharose column with buffer E is similar in both tissues, as is the DNase I specific inhibition activity, indicating that profilin: actin is present at comparable levels in the spleen and thymus extracts. After pooling the desired fractions from buffer E elution, the protein can either be applied directly to the hydroxylapatite column for profilin: pactin and profilin:~,-actin subfractionation (next section), or it can be stored as an ammonium sulfate precipitate. In the latter case, dialyze the pooled buffer E peak fractions under a nitrogen or argon atmosphere
108
MYOSIN- A N D ACTOMYOSIN-RELATED PROTEINS
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FIO. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) performed according to the method of Laemmli [U. K. Laemmli, Nature (London) 227, 680 (1980)] on profilin: actin from calf spleen at successive stages of a single purification. Protein eluted with buffer E from the poly(L-proline)-Sepharose column is shown in (a), protein fron~ peaks I, II, and III of the hydroxylapatite column profile in Fig. 2 is shown in (b), (c), and (d), respectively, and profilin:~actin after Sephacryl S-200 HR chromatography (peak PA of the column profile shown in Fig. 3) is shown in (e). The positions of actin (A) and profilin (P) are marked, as are the positions of significant contaminating proteins with molecular weights of 38,000, 32,000, and 27,000 (top, middle, and bottom arrowheads, respectively). Gels were 13% polyacrylamide, stained with Coomassie Blue. Approximately 10 #tg of protein was loaded in each lane. Gel densitometry was carried out using a Bio-Rad model 620 video densitometer (Bio-Rad, Richmond, CA).
[ 10]
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MYOSIN- AND ACTOMYOSIN-RELATED PROTEINS
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against 4 vol of ammonium sulfate solution for 6 hr, at which point small amounts of precipitate begin to appear. Turn the bag upside down several times to mix the contents, change to fresh ammonium sulfate of the same volume containing 0.5 m M 2-mercaptoethanol, and continue dialyzing overnight. Precipitation is usually complete after 24 hr, and profilin: actin is stable in this condition for several months at 4°. 1°
Hydroxylapatite Chromatography of Poly(L-proline) - Sepharose Column Fractions If hydroxylapatite chromatography is to be performed immediately following poly(L-proline)-Sepharose chromatography, dilute the buffer E peak with 2 vol of HEB and load onto the hydroxylapatite column at a flow rate of 400 ml/hr. Undiluted extract does not bind completely to the column. After all of the protein has been applied, wash the column at the same flow rate with HEB until the DMSO is washed out, as detected by monitoring the A23o. Usually, five bed volumes is required. The direct application of the poly(L-proline)- Sepharose-purified profilin: actin to the hydroxylapatite column offers the advantages of completing the first two chromatography steps in 1 day, removing completely the protein from DMSO, and eliminating the exposure of the protein to ammonium sulfate, which is often of variable quality even for "enzyme-grade" material. Profilin:actin precipitated with ammonium sulfate must be desalted before application to the hydroxylapatite column. Recover profilin:actin by taking enough ammonium sulfate-precipitated material to contain 200 A2so units and centrifuging for 20 min at 50,000 g and 4*. Suspend the pellet in a volume of buffer G to give a protein concentration of 1015 mg/ml, and centrifuge again to remove undissolved protein. Resolubilizing profilin: actin in the presence of ATP and calcium ions seems to be important in stabilizing the polymerization activity of actin, although protein loaded directly from the poly(L-proline)-Sepharose column appears to be stable without ATP or calcium. However, ATP is not included in the subsequent wash and elution of the hydroxylapatite column because of its high affinity to hydroxylapatite. After desalting the profilin: actin into buffer B by dialysis or Sephadex G-25 chromatography, apply the protein to the hydroxylapatite column at a flow rate of 400 ml/hr. Wash the column in one bed volume of buffer B, and elute the bound profilin: actin with a linear gradient prepared from two bed volumes of buffer B as the starting buffer and two bed volumes of buffer C as the final buffer, using a flow rate of approximately 50 ml/hr. A hydroxylapatite column elution profile for calf spleen profilin: actin is shown in Fig. 2 and contains three peaks, labeled I, II, and III. Peak I
[ 10]
PURIFICATIONOF PROFILIN'ACTIN
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EFFLUENT (ml) Fro. 2. Elution profile of hydroxylapatite chromatography performed on the buffer E peak from a po]y(L-proi/ne)-Sepharose column. SDS-PAGE analysis of the three peaks, labeled I, II, and III, is shown in Fig. I. Peak II corresponds to profii/n: 7-aetin and peak III corresponds to proSlin :/~-actin. s,'° Elution was with a linear gradient of 800 ral starting with buffer B and ending with buffer C. The elution of profilin: actin is complete after approximately 50% of the gradient has passed through the column.
contains mostly actin, profilin, and a protein of M, 38,000 which may be a degradation product of actin.~ A number of minor components with molecular weights ranging between 27,000 and 38,000 comprise approximately 12% of the total protein, as determined by gel densitometry. Compared to the poly(L-proline)-Sepharose-purified material, the amount of profdin in this peak is significantly reduced, although in previous studies the protein eluting at the same gradient position contained almost pure profilin, s,~° The reason for this difference is not known, although it must involve the ability of excess profilin to bind to hydroxylapatite, since the molar excess of profilin over actin found in the poly(L-proline)Sepharose-purified material is eliminated in profilin :actin after hydroxylapatite chromatography. The remaining peaks II and III show greater homogeneity of profilin and actin, and occur at the same positions in the phosphate/glycine gradient as the peaks corresponding to profilin: 7-actin and profilin :fl-actin, r e s p e c t i v e l y . 7,9 A significant contaminant in each case is the protein of Mr 38,000, comprising approximately 12% of the total protein in peak II and 4% in peak III. If this protein is a degradation
112
MYOSIN- AND ACTOMYOSIN-RELATED PROTEINS
[ 10]
product of actin, the level of contamination of peaks II and III by nonprofilin: actin proteins is less than 1%. From Table I, the relative proportions of profilin: y-actin to profilin: flactin are 30:70 from spleen and 28:72 from thymus. These values are almost identical to those reported previously for hydroxylapatite-fractionated profilin: actin from calf spleen, although a slightly higher proportion of profilin: 7-actin was reported for protein from calf thymus.7 For both peaks, the specific DNase I inhibition activities and the percentage yields of total activity are very close for spleen compared to thymus protein. Hydroxylapatite-fractionated profilin: actin can be used for gel-filtration chromatography (next section), or it can be stored by ammonium sulfate precipitation as described previously.
Gel-Filtration Chromatography The profilin: actin isoforms obtained by hydroxylapatite chromatography can be subjected to gel-filtration chromatography to separate profilin:actin from higher molecular weight species which may result from polymerization or nonspecific aggregation processes, as well as to remove small amounts of contaminants which may remain after hydroxylapatite chromatography. Of particular interest is the presence of minute amounts of a factor which apparently stabilizes profilin: actin against dissociationt5 and which is removed more effectively at low (5 mM) than at high (500 mM) potassium phosphate concentrations by Sephadex G-100 chromatography. Previously, Sephadex G- 100 or Superose 6 resins (Pharmacia) have been used following hydroxylapatite chromatography.t° However, the introduction of the Sephacryl high-resolution matrices from Pharmacia have enabled us to obtain separations of equivalent quality in approximately 10-20% of the time required for other separation materials. To pour a Sephacryl S-200 high-resolution (S-200 HR) column, add 450 ml of a well-suspended slurry from the bottle to 150 ml of HEB and stir gently (swirling is best to avoid damaging the Sephacryl beads). Pour the slurry into a Pharmacia 2.6 × 60 cm column equipped with a packing reservoir, and pump buffer at a flow rate of 150 ml/hr using positive pressure from the top of the column rather than negative pressure from below, adding more HEB as required to prevent the column from going dry. The gel height should stabilize in 2 hr, at which time the flow rate should be increased to 300 ml/hr and pumping continued for another hour. Disconnect the column from the pump, attach the upper flow adaptor, turn the column upside down, and wash with two bed volumes of buffer G at 150 ml/hr. The final packed column bed volume should be approximately 300 ml. Protein can be used either after storage as an
[ 10]
PURIFICATIONOF PROFILIN'ACTIN
113
ammonium sulfate precipitate, or immediately after elution from the hydroxylapatite column. If ammonium sulfate-precipitated protein is used, centrifuge the homogeneous suspension for 20 min at 50,000 g and 4°, resuspend the pellet in buffer G to a volume of 10 ml containing 100-200 A280units, and centrifuge a second time. Protein to be applied directly from the hydroxylapatite column must be concentrated to 10 ml on an Amicon PMI0 or YM10 ultrafiltration membrane (Amicon, Danvers, MA). We have not observed any difference in yield between the two membranes, but the PM 10 gives a higher flow rate. Recovery is usually greater than 90%, and the protein is then centrifuged to remove any aggregated material. Profilin: actin is applied to the S-200 HR column at a flow rate of 100 ml/ hr and eluted at the same rate with buffer G. The elution profile of an S-200 HR run on profilin :fl-actin is shown in Fig. 3 and features a small peak (A), running at the void volume, and consisting of actin and several minor components. No profilin is present in this peak. The presence of actin at the void volume is probably a result of an aggregation or polymerization phenomenon. The larger, second peak (PA) contains profilin: actin of very high purity. SDS gel electrophoresis shows the protein composition in this peak to be nearly identical to the composition of the starting material, hydroxylapatite-purified profilin :fl-actin. Table I shows the protein yields and DNase I inhibition activities to be comparable for S-200 HR-purified profilin:actin from spleen and thymus. Relative to the specific inhibition activities of the poly(L-proline)-Sepharose and hydroxylapatite-purified profilin: actin, the activity after S-200 HR chromatography has increased to the theoretical maximum for profilin: actin, ~°even though no significant purification differences are evident by SDS gel electrophoresis. This suggests that relatively minute changes in the protein composition of profilin: actin can affect significantly the behavior of the system.
Column Regeneration Immediately after completion of the buffer E elution, the poly(Lproline)-Sepharose column is washed in 5 vol of buffer A to remove DMSO. If regenerated carefully, the poly(L-proline)-Sepharose matrix is reusable with no apparent loss of profilin: actin binding capacity or deterioration of the quality of protein recovered. The most obvious contaminant on the column appears to be an insoluble off-white material. Although we have not analyzed the material, it appears to be lipid-like in consistency and is more pronounced in spleen than in thymus preparations, and when spleens larger than 150 g are used. Aside from its potential as a contaminant of profilin:actin, the material has a tendency to clog the column, thereby reducing flow rates. To remove this material, the column contents
I 14
MYOSIN- A N D ACTOMYOSIN-RELATED
PROTEINS
[10]
1.0-
0.7o Go N
0 z
0u~
0.4-
