Characterization of renatured profilin purified by urea ...

Cell Motility and the Cytoskeleton 14:251-262 (1989)

Characterization of Renat u red Profi I in Purified by Urea Elution From Poly-L-Proline

Agarose Columns

Donald A. Kaiser, Pascal J. Goldschrnidt-Clerrnont, Barry A. Levine, and Thomas D. Pollard

Department of Cell Biology and Anatomy, The Johns Hopkins Medical School, Baltimore (D.A. K., P. J. G. -C., T. D. P.); Inorganic Chemistry Laboratory,

Oxford University, Oxford, England (B.A. L.)

We present evidence that native profilin can be purified from cellular extracts of Acanrhamoeba, Dictyostelium. and human platelets by affinity chromatography on poly-L-proline agarose. After applying cell extracts and washing the column with 3 M urea, homogeneous profilin is eluted by increasing the urea concentration to 6-8 M. Acanrhamoebu profilin-I and profilin-I1 can subsequently be separated by cation exchange Chromatography. The yield of Acunthamoebu profilin is twice that obtained by conventional methods. Several lines of evidence show that the profilins fully renature after removal of the urea by dialysis: 1) dialyzed Acanthamoeba and human profilins rebind quantitatively to poly- L-proline and bind to actin in the same way as native, conventionally purified profilin without urea treatment; 2) dialyzed profilins form 3-D crystals under the same conditions as native profilins; 3) dialyzed Acanthamoeba profilin-I has an NMR spectrum identical with that of native profilin-I; and 4) dialyzed human and Acanthamoeba profilins inhibit actin polymerization. We report the discovery of profilin in Dictyxtelium cell extracts using the same method. Based on these observations we conclude that urea elution from poly-L-proline agarose followed by renaturation will be generally useful for preparing profilins from a wide variety of cells. Perhaps also of general use is the finding that either myosin-11 or alpha- actinin in crude cell extracts can be bound selectively to the poly-L-proline agarose column depending on the ionic conditions used to equilibrate the column. We have purified myosin-I1 from both Acanrhamoebu and Dicfyosrelium cell extracts and alpha-actinin from Acanrhnmoebu cell extracts in the appropriate buffers. These proteins are retained as complexes with actin by the agarose and not by a specific interaction with poly-L-proline. They can be eluted by dissociating the complexes with ATP and separated from actin by gel filtration if necessary.

Key words: Acanthamoeba, affinity chromatography, Dictyostelium, NMR spectroscopy, platelets, myosin

INTRODUCTION

Profilins are small actin monomer binding proteins originally discovered in vertebrate tissues [Carlsson et al., 19761 but now known t o exist in many, if not all, cells [reviewed by Pollard and Cooper, 19861. The ob- servation that profilins are inhibited by acidic phospho- lipids [Lassing and Lindberg, 198.51 and that profilin is probably necessary for cellular viability [ Magdolen et

0 1989 Alan R. Liss. Inc.

al., 19881 has added further to their importance. At the present time a variety of detailed biochemical [Pollard

Received March 7, 1989: accepted May 25, 1989.

Address reprint requests t o Donald A. Kaiser. Department of Cell Biology and Anatomy. The Johns Hopkins Medical School. 725 N . Wolfe Street. Baltimore. MD 21205.

252 Kaiser et al.

and Cooper, 1984; Kaiser et al., 1986; Lindberg et al., 1988; Vandekerckhove et al., 19891, molecular biologi- cal [Kwiatkowski and Bruns, 1988; Magdolen et al., 19881, physiological [Lind et al., 19871, and biophysical [Magnus et al., 19861 studies are being done with profilins in a number of laboratories.

Tanaka and Shibata [ 19851 discovered that profilin binds to poly-L-proline immobilized on agarose beads. The association is strong since at least 6 M urea is required to elute the bound profilin from the column. This property of profilin has already been used as an assay for profilin in crude extracts [Lind et al . , 19871. These results suggested affinity Chromatography on immobilized poly-L-proline would be a valuable method for purifying profilin directly from crude extracts providing that the profilin eluted from poly-L-proline with urea could be renatured.

Here we show that Acanthamoeba profilins-I and -11, Dictyostefium profilin, and human platelet profilin can be purified by affinity chromatography on poly-L- proline agarose. The profilins are eluted from the colzmn with 6-8 M urea, renatured by dialysis against a low ionic strength buffer without urea and, in the case of Acanthamoeba, separated into the isoforms by cation exchange chromatography. The yield is higher than conventional methods by a factor of about 2, and the dialyzed profilins have native structure by several criteria. Independently, Lindberg et al. [ 19881 showed that affinity chromatography on poly-L-proline-Sepharose with dimethylsulphoxide elution can be used to purify a mixture of spleen profilin and profilactin complex. We have found that in addition to profilin, the poly-L-proline agarose column also binds myosin-I1 and alpha-actinin from crude extracts of Acanthamoeba and myosin-I1 from Dic- tyostefium cell extracts. These proteins are retained as complexes with actin, and it is the agarose beads rather than the poly-L-proline that traps them. They can be eluted by dissociating the complexes with ATP.

MATERIALS AND METHODS Cell Extracts

Acanthamoeha cell extracts were prepared by cen- trifuging homogenates of whole cells in 0.34 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, and 10 mM imida- zole-HCI, pH 7.0, with or without 1 mM ATP at 100,000g for 90 min at 4°C [Tseng et al., 19841. Dicfy- ostelium Ax-3 (wild-type) cells in 0.20 M sucrose, 0.2 mM EDTA, and 10 mM tris-HCI, pH 8.0, were lysed by forcing through a 5 pm filter and centrifuged at 100,000g for 60 min at 4°C [Devreotes et al., 19871. Cells were developed 4-5 h prior to lysis. Outdated human platelets (7-9 days post-phlebotomy) were provided by the American Red Cross in Baltimore, Maryland.

Platelets were washed by three successive centrifuga- tions ( I , 0 0 0 ~ for 30 min at 22°C) in 135 mM NaCI, 8.6 mM trisodium citrate, 5.3 mM citric acid, and 1 1 . 1 mM glucose, 10 mM Tris, pH 6.5. After the last wash the pellet was resuspended in 5 volumes of ice-cold lysing buffer containing 2 mM Tris (pH 7.2), 0.1 mM ATP, 0.5 mM dithiothreitol, 1 % Triton X-100, 1 % dimethyl sul- foxide, and 50 Fgiml each of chymostatin, leupeptin, antipain, and pepstatin. After sonicating for 60 s, the extract was clarified by centrifugation at 12,000~ for 15 min at 4°C.

Monoclonal Antibodies

Mouse monoclonal antibodies to myosin-I, myosin-11, alpha-actinin, profilin-I, and profilin-I1 from Acanthamoeba were prepared as previously described [Kiehart et al., 19841. A mouse monoclonal anti-actin antibody, c4D6, was kindly provided by Dr. James Lessard [Lessard, 19881.

Reagents

I-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) were purchased from Pierce Chemicals (Rockford, IL). Both EDC and NHS were dissolved at a concentration of 50 mM in 2 mM potassium phosphate, pH 7.5, immediately prior to use. The pH of the NHS was adjusted with NaOH. Cyanogen bromide and dimethylsulfoxide were from Fisher Chemicals, (Pittsburgh, PA). Sepharose-4B was from Pharmacia (Piscataway , NJ). Poly-L-proline (MW 14,200), glycine , MES (2- [ N-morpholino]ethano- sulfonic acid), tris base, ATP, EDTA, dithiothreitol, sodium dodecylsulphate, beta-mercaptoethanol, phenylenediamine, Triton X- 100, chymostatin, leupeptin, antipain, and pepstatin were from Sigma Chemical Co. (St. Louis, MO). Sucrose was obtained from J.T. Baker Co. (Phillipsburg, NJ). Urea and guanidine-HCI were from Schwarz-Mann Biotech (Cleveland, OH). Car- boxymethylcellulose ion exchange resin (CM-CM) was purchased from Amicon Corp. (Danvers, MA).

Poly-L-Proline Agarose Columns

One gram of poly-L-proline was dissolved in 100 ml of distilled water by stirring at 4°C for 2 days and then coupled to 50 ml of Sepharose-4B activated with cyanogen bromide by stirring for 20 h at 4°C [Tuderman et al., 19751. After each use the beads were washed with 6 or 8 M urea or 5 M guanidine for 1-2 h to remove all bound material. The columns have been used more than 20 times without loss of activity or specificity.

Biochemical Methods

Proteins were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate [Laenimli,

Purification and Renaturation of Profilin 253

19701 and either stained with Coomassie blue or transferred to nitrocellulose paper as previously described [Kiehart et al., 1984; Towbin et al., 19791. Protein concentrations were measured by Coomassie blue dye binding [Bradford, 19761 using ovalbumin as a standard.

lmmunochemical Methods

Proteins were detected on nitrocellulose paper with monoclonal antibodies followed by reaction with goat anti-mouse second antibodies which were labeled with either horseradish peroxidase (Hy-clone Laboratories [Logan, UT]) or iodine- 125 (Cappel Laboratories [Bur- lingame, CAI) [Kiehart et al., 19841. For semi-quantitative analysis, fractions were diluted in 10 mM imida- zole-HCI, pH 7.0, and dried onto polyvinyl-chloride 96- well plates for ELISA with mouse monoclonal antibodies and goat anti-mouse second antibodies labeled with horseradish peroxidase (Tago Inc., Burlingame, CA). The substrate O-phenylenediamine was used to detect antibody binding by measuring absorbance at 490 nm in a Biotech EL-310 plate reader.

' H-NMR Spectral Characterization

For NMR studies Acuntharnoeba profilin-I purified by conventional methods [Kaiser et al., 19861 or by elution from poly-L-proline with 8 M urea were concentrated by precipitation with 0.4 giml ammonium sulphate and dialyzed against 10 mM NaHC03, 20 mM NaCl in 'H20, pH 7.4. The proteins were further concentrated and exchanged into 5 mM NaHC03 in 'H20 by ultrafil- tration on an Amicon 8MC membrane to a final concentration of approximately 10 mg/ml. 'H-NMR spectra were obtained at 500 and 600 MHz at an ambient probe temperature of 303°K. Two-dimensional correlated (COSY) spectra were recorded using the TPPI method [Bodenhausen et al., 19801 to produce phase-sensitive spectra with irradiation of the residual OH resonance during the relaxation time. Spectra were recorded with a sweep width of 6,024 Hz in both dimensions with 192 scans per T I increment. The resulting data matrix (512 x 4K) was filtered by shifted sine bell multiplication and zero filled to 2K in T, before complex Fourier trans- formation. One dimensional nuclear Overhauser enhancement (NOE) measurements were carried out using an 0.8 s irradiation pulse with the decoupler in the HG mode.

RESULTS Affinity Chromatography of Crude Extracts of Acanthamoeba on Poly-L-Proline

Profilin, myosin-11, alpha-actinin, and actin bind to columns of poly-L-proline immobilized on agarose beads. A substantial amount of the myosin-I1 in the cx-

tract binds in a high salt buffer (Fig. IA) but little or none binds in high salt buffer with ATP or in a low salt buffer (Fig. IB). In low salt, a substantial amount of alpha- actinin is retained and little or no myosin binds to the column (Fig. IA,B). Profilin binds quantitatively in both buffers. Only a small fraction of the total actin binds in either buffer (Fig. IA). Control experiments showed that myosin-I1 (Fig. 2) is retained by the agarose beads alone, but that the profilin binds to the poly-L-proline. Purified myosin-I1 is not retained by the agarose unless actin is present under conditions which favor the formation of actomyosin (not illustrated).

The myosin-I1 and alpha-actinin retained on columns of agarose or poly-L-proline-agarose are eluted quantitatively by 5 mM MgATP (Figs. 1-4). Depletion of the endogenous ATP in the extract with hexokinase and glucose did not improve the yield of either myosin-I1 or alpha-actinin significantly. However, in extracts where 5 mM ATP or 30 mM pyrophosphate were added prior to chromatography, neither myosin-I1 nor alpha-actinin bound (data not shown). The bound actin is eluted partially by MgATP and completely by 3 M urea which denatures the actin irreversibly. Profilin is not necessary for retention of actin and myosin on either column, since actin and myosin-I1 can be purified from extracts depleted of profilin (not illustrated). The profilin is quantitatively retained on poly-L-proline agarose in 3 M urea, but is all eluted by 6 M urea (Figs. 1, 3) or 5 M guanidine (not illustrated). The profilin elutes in a sharper peak with 8 M urea, presumably due to some weak interaction with the column even in 6 M urea.

The myosin-I1 is the major protein eluted from the high salt column with MgATP (Figs. lA, 2, 3). The contaminating actin is easily removed from the myosin-I1 by gel filtration using standard methods [Pollard et al., 19781. This highly purified myosin-I1 has normal Ca" ATPase activity. The yield of myosin-I1 from 1 g of cells is about 0.25 mg, about 30% of the total of 2 pmol/kg [Pollard et al., 19781. This yield is about the same as that obtained by conventional methods.

The alpha-actinin bound to the column in low salt buffer elutes with 5 mM ATP (Figs. lB, 4). It has the same complex electrophoretic pattern of multiple bands as Acantharnoeba alpha-actinin purified in other ways [Pollard et al., 19861. It is positively identified as alpha-actinin by binding to anti-alpha-actinin monoclonal antibodies (Fig. I , 4). The yield is about 0.38 mg per g of cells, about 4 times higher than conventional methods [Pollard et al., 19861. We presume that like myosin-11, alpha-actinin is retained as a complex with actin. We do not yet understand why this association of alpha-actinin with actin occurs more favorably in the low salt buffer.

Column A

Acanthamoeba extract in high salt

2.0

1.6

1.2

0.8

Column B

Acantharnoeba extract in low salt

- a I- - s 3 n g 2

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0.3 -

0.2 - 0.1 -

0.0 0 10 20 30 40 50 60

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0.5 I 1

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0.5 s

Fract ion Nm&r


3 to 4 M urea, while the elution of the profilin requires 6 M urea (Fig. 5 ) . Although actin was irreversibly de- natured by this treatment, purified profilin was renatured by dialysis against 0.2 mM ATP, 0.1 mM CaCI2, 0.5 mM dithiothreitol, 2 mM Tris pH 7.2 for 48 h. The yield is about 0.5 mg profilin per gram of packed platelets. Renatured profilin has normal functional properties including binding to actin, inhibition of actin polymerization, and binding to poly-L-proline-agarose.

Affinity Chromatography of Crude Extracts of Dicfyosfelium discoideum on Po I y- L- Pro1 i n e

Chromatography of Dictyostelium extracts on poly- L-proline equilibrated with high salt gives the same results as the Acanthamoeba extracts (Fig. 6 ) ; both myosin-I1 and a polypeptide the size of profilin bind and are eluted with MgATP and 6 M urea, respectively. The myosin-I1 was positively identified by its cross-reactivity with 2 monoclonal antibodies to Acanthamoeba myosin- I1 (Fig. 6). The polypeptide eluting in a highly purified form with 6 M urea coelectrophoreses with Acan- thamoeba profilin and is positively identified as Dictyo- stelium profilin by cross-reactivity in ELISA with 2 of our 14 monoclonal antibodies to Acanthamoeba profilins. None of these antibodies bound to the Dictyostelium protein on immunoblots after gel electrophoresis in SDS (data not shown).

Large-Scale Purification of Acanthamoeba Profilins by Affinity Chromatography

Substitution of poly-L-proline affinity chromatography for hydroxylapatite and gel filtration chromatography [Reichstein and Korn, 19791 results in improved yield and purity of Acanthamoeba profilins. Crude extracts can be applied directly to the affinity column (Figs. I , 3), but we usually start with a large DEAE-cellulose column, since it is the initial step in the purification of several other proteins including actin and myosin-I1 [Tseng et al., 19841. The flow-through from the DEAE- column containing the profilins is run on the affinity column. The capacity of the poly-L-proline agarose column is 5-10 mg of profilin per ml, so that the 1.5 x 15 cm column is large enough for 1.3 kg of cells. After washing with buffer and 3 M urea, highly purified profilins are eluted with 6 or 8 M urea (Fig. 7). The profilins are renatured by dialyzing out the urea and the two isoforms separated by cation exchange chromatography (Fig. 7). The yield of total profilin is about 0.1 mg per gram of cells compared with 0.05 mg by the Kaiser et al. [ 19861 modification of the method of Reichstein and Korn [ 19791. The ratio of purified profilin-I to profilin-I1 is about 2 to 1 compared with 4 to 1 by the method of Kaiser et al. [ 19861. In the conventional method, part of the profilin-I1 is lost during hydroxylapatite chromatog-

t- u [r t- X W

a

5mM MgATP

1 2 3 4 5 6

2 0 0 -

68 - 60- 55 - 43 -

29 -

14-

Fig. 2. Retention of Acunthumoebu myosin-I1 and actin on a control column of agarose without poly-L-proline. Twenty milliliters of extract containing 57 1 mg protein was diluted into 20 ml high salt buffer and applied to a 2 .0 X 1.5 cm column of Sepharose 4B. The column was rinsed with 1 L of high salt buffer. Samples were analyzed by SDS-polyacrylamide gel electrophoresis. Shown are the extract sample applied to the column (lane l), the unbound fraction (lane 2), and fractions eluted with 5 mM MgATP in high salt buffer (lanes 3-6). Approximately 6.5 mg of actomyosin was recovered in this experi- ment.

Affinity Chromatography of Extracts of Human Platelets on Poly-L-Proline

Chromatography of extracts of human platelets on poly-L-proline-agarose retains virtually all detectable profilin present in the extract and a small fraction of the actin (corresponding to approximately one third of the profilin concentration, Fig. 5 ) . The actin was eluted with

~~

Fig. I . Column A: A 3 ml extract of A~~~rr,~tlztrtnorbtr containing 2 0 mg o f protein was applied to a 5 X 0.75 cm column of poly-L-proline agarose equilibrated in high salt (100 mM NaCl, 100 mM glycine, 1 mM dithiothreitol. and 10 mM Tris-HCI. pH 8 .0) . Column B: 4 ml of Acuiithumoohli extract containing 35 mg of protein was applied to a 15 x I .5 cm column ofpoly-L-proline agarose equilibrated in low salt (0.34 M sucrose. I .O mM EDTA, I .O mM dithiothreitol. and 10 mM imidarole, pH 7.0). Unbound protein was eluted with column buffer and bound material was eluted successively with 5 mM MgATP. 3 M urea, and 6 M urea all in column buffer. The top panels show the protein concentration in the fractions. The lower panels show ELISA assays for profilin. actin. myosin-Il. and alpha-actinin

256 Kaiser et al.

COOMASSIE BLUE ANTI-PROFILIN ANTI-ACTIN ANTI-MYOSIN II ANTI-ALPHA-ACTININ ANTI-MYOSIN I

200 - 116 - 95 - 68 - 60 - 55 - 43 - 40 -

29 -

18 - 11 -

Fig. 3. Affinity chromatography of Acanrhamoeba extract on poly- L-proline agarose in high salt buffer. Samples were analyzed by SDS- polyacrylamide gel electrophoresis and immunoblotting. Left: A gel stained with Coomassie blue. Right: Autoradiograms of five identical gels transferred to nitrocellulose and reacted with monoclonal antibodies specific for profilin, actin, myoain-If, alpha-actinin and myosin-I. Monoclonal antibody binding was visualized with goat anti-

mouse second antibodies labeled with '"1. The lanes in each panel are extract sample applied to column, unbound fraction, fraction eluting with 5 mM MgATP, fraction eluting with 3 M urea, and fraction eluting with 6 M urea. (The immunoreactive bands migrating ahead of the myosin-I1 heavy chain in the anti-myosin-11 panel are breakdown products of the myosin-11 heavy chain.

raphy where none of the profilin-I, but part of the profilin-11 binds to the column (D.A. Kaiser and T.D. Pol- lard, unpublished observations).

The two Acanrhamoeba profilin isoforms purified by urea elution from poly-L-proline and renatured by dialysis are native by several criteria. In a chemical cross-linking assay (Fig. S), both bind to actin monomers as well as profilins purified without urea [Vandekerck- hove et al., 19891. They also inhibit actin polymerization as described previously [Pollard and Cooper, 19841 and rebind to poly-L-proline. Both renatured profilin isoforms also bind to anti-profilin monoclonal antibodies equally well [Kaiser and Pollard, 19881. The affinity purified preparations also form crystals under the same conditions as conventional preparations [Magnus et al., 19861 (L. Machesky and T.D. Pollard, unpublished observations).

Comparative ' H-NMR Spectral Study of Profilin-l Prepared Using Poly-L-Proline Purification and Conventional Procedures

The spectra of the two profilin-I preparations (Fig. 9) are virtually indistinguishable. The identity in chemical shift position and resonance lineshape for signals that provide a fingerprint for the conformation of the protein proves that the renatured profilin-I adopts the native structure. Some 30 amide hydrogens slow to exchange with solvent (t1,2 > 20 h) are readily resolved (7.5 < 6 < 9.7 ppm, Fig. 9) while downfield-shifted backbone-CalphaH signals, typical of beta-sheet segments [Dalgarno et al., 19831, also show spectral homology. Correlation of these indicators of beta-structure and several of the slowly exchanging -NH resonances is shown by the cross peaks in the identical COSY spectra ob-


COOMASSIE ALPHA-ACTININ ACTIN MYOSIN II 1 2 3 4

200 - 116 - 95 - 68 - 60 = 55

43 - 40 - 29 -

18-

11-

Fig. 4. Affinity chromatography of Acanrharnoeha extract on poly- L-proline agarose in low salt buffer. Samples were analyzed by SDS- polyacrylamide gel electrophoresis and immunoblotting. Left: A gel stained with Coomassie blue. Right: Autoradiograms of 3 identical gels transferred to nitrocellulose and reacted with antibodies to alpha- actinin, actin, and myosin-11. They were prepared as in Figure 2, except that the gel sample of the fraction eluting with 5 mM ATP was not boiled. This accounts for slower niigration of the upper band of alpha-actinin [Pollard et al., 19861.

tained for both profilin-I preparations (Fig. 10). Reten- tion of the native structure by the renatured profilin-I is also indicated by diagnostics of more localized folding characteristics seen from the ring-shifted methyl group signals (0.7 < 6 < I ppm) and the homologous aromatic sidechain spectral region (6.3 < 6 < 7.5 ppm). The conserved proximity of the corresponding groups was verified using nuclear Overhauser enhancement effects.

DISCUSSION

Profilin eluted from poly-L-proline agarose in 6-8 M urea and dialyzed extensively into low salt buffers without urea appears to be native by the following criteria: 1) rebinding to poly-L- proline; 2 ) binding to actin (Fig. 8): 3) formation of 3D crystals [Magnus et al., 19861 (L. Machesky and T.D. Pollard, 1988, unpub-

200 - 116 -

95 - 60 - 55 - 43 -

68 -

29 -

18 -

11 -

Fig. 5 . Affinity chromatography of a human platelet extract on poly- L-proline-agarose. Samples were analyzed by SDS-polyacrylamide gel electrophoresis and staining with Coomassie blue. Lane 1: Triton soluble extract (10 pg protein). Lane 2: Material that did not bind t o the column (10 pg protein). Lane 3: Pooled fraction eluted from the column with 4 M urea (2 pg protein). Lane 4: Pooled fractions eluted from the column with 8 M urea (3 pg protein). This fraction contained a single protein of about 15 kD.

lished observations); 4) structure from NMR spectra (Figs. 9, 10); and 5 ) binding of anti-profilin monoclonal antibodies (Figs. I , 3) [Kaiser and Pollard, 19881.

This work establishes the utility of poly-L-proline affinity chromatography for the simple and rapid purification of profilin in high yield from a variety of cells. Contaminating actin can be removed by a wash with 3 M urea during the affinity chromatography or by prior ad- sorption to DEAE (Fig. 7). In either case the procedures are somewhat simpler than the methods recently described by Lindberg et al. [I9881 since an additional hydroxylapatite column was required to obtain pure profilin. Our yields of profilin from both Acnnthamoeha (see Fig. 3 ) and human platelets are nearly quantitative. Our recovery of 0.5 mg of profilin from 1 g of platelets is in the same range (0.4-0.5 mg per gram of spleen) reported by Lindberg et al. [ 19881, although most of their profilin was bound to actin. Their use of DMSO to elute profilin from the poly-L-proline column has the proven advantage of producing native profilactin.

We have already used the Acunthumoeha and human profilins for a variety of structural and functional studies. Based on our discovery of profilin in Dictyoste- liutn, we expect that the method can be used effectively for many other cell types. I t is still possible that more detailed studies of the mechanism of action or structure

258 Kaiser et al.

COOMASSIE BLUE IMMUNOBLOTS

200 -

116 - 95 - 68 - 60 - 55 -

4 3 - 4 0 -

29 -

1 8 -

11 -

Fig. 6 . Affinity chromatography of DicQosreliurn extract on poly- L-proline agarose in high salt buffer. Left panel: An SDS-polyacrylamide gel stained with Coomassie blue showing the composition of the extract applied to the column. the unbound fraction. the fractions eluting with 5 mM MgATP. 3 M and 6 M urea. Last panel: A mixture of Acanthurnoubu profilin-1 and profilin-11. The irnmunoblots shown to the right show the reaction of samples eluted with 5 mM MgATP

with monoclonal antibodies to Acunthuinorba myosin-I1 and actin. Monoclonal antibody binding was visualized with horseradish peroxidase-labeled goat anti-mouse second antibodies. Only M2.17 and M2.42 cross-react appreciably with myosin-I1 from Dicryostoliurn. None of 14 different monoclonal antibodies to Acanthurnorhu profilin bound t o the polypeptide with the same electrophoretic mobility as Actrnthuinoebu profilin that eluted in 6 M urea (data not shown).

of the profilins will reveal some difference between the profilins recovered from the poly-L-proline columns by denaturation with high concentrations of urea, but our efforts to date have not uncovered any such differences. Consequently, we feel i t safe t o conclude that both human and amoeba profilins are capable of refolding into a native conformation after denaturation with urea o r guanidine.

Insight into the mechanism of binding of the vari- ous profilins to the poly-L-proline will have to await o u r current efforts to determine the structure of Acan- rhamorha profilin [ Magnus et al., 19861 and the complex of Actitithumorha profilin with poly-L-proline by X-ray crystallography. Since all of the known profilins have similar amino acid sequences [Ampe et al., 1985; Ampe and Vandekerckhove, 1987; Ampe et al., 1988; Kwiat-

1 2 3 4 5

200- 116- 95 - 68- 60-

55-

43- 40-


1 2 3 4 5

200 - 116 - 95 - 68 - 60 - 55 - 43 - 40 -

29 -

18 - 11 -

18-

11-

Fig. 7. Large-scale purification of profilins from Acantharnorba by affinity chromatography on poly-L-proline agarose. The samples were analyzed by SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue. Lane 1: The flow-through fractions from DEAE- cellulose chromatography of a low-salt extract of Accor//~amoeba that was derived from 970 g of cells. This material (3 L) was applied t o a 15 X 1.5 cm column of poly-L-proline agarose. The column was washed with 10 volumes of column buffer (100 mM NaCI, 100 mM glycine, I mM dithiothreitol, 10 mM Tris-HCI, pH 8.0) and 1 volume of 3 M urea in column buffer. Lanes 2 and 3: Profilin eluted with 6 M urea. This mixture of 2 profilin isofomis was then fractionated by cation-exchange chromatography on Amicon CM-CM in 10 mM MES, pH 6.0. Lane 4: Purified profilin-1. Lane 5: Purified profilin- 11. A total of 103 mg profilins was obtained with approxirnately twice as much profilin-l as profilin-11.

kowski et al., 19881, i t is not possible to identify unique regions of homology that might endow all of the proteins in this family with the ability to bind to poly-L-proline.

Probably the most interesting question raised by our work and the related studies of Tanaka and Shibata 119851, Lind et al. 119871 and Lindberg et al. I19881 is, Why do profilins bind t o poly-L-proline with such high affinity'? I t is obvious from the conditions required to elute actin and profilin from the affinity column that profilin binds to poly-L-proline much more strongly than

Fig. 8. Comparison by chemical cross-linking of the actin monomer binding activities of profilins purified by a conventional method without urea [Kaiser et al.. 19861 and by urea elution from poly-L-proline. Mixtures of 20 p M actin and 20 p M profilin were dialyzed vs. 2 mm KP, buffer, pH 7.5, and cross-linked with I mM EDC and 1 mM NHS for 30 min at 22°C [Vandekerckhove et al., 19893. Samples were ana lyxd by SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue. Lane 1: Rabbit skeletal muscle actin. Lane 2: Con- ventional profilin-1 plus actin. Lane 3: Poly-L-proline purified profilin-1 plus actin. Lane 4: Conventional profilin-I1 plus actin. Lane 5: Poly-L-proline purified profilin-11 plus actin. The 55 kD covalently cross-linked I : I complex o f profilin and actin is indicated.

to actin. We suspect, but cannot yet document, that there are proteins in cells with segments of poly-L-proline that bind to profilin. Such interactions could modulate the activity of one of the members of the complex or possi- bly serve as an anchor for localizing prof'ilin in the cell.

We have not carefully investigated the mechanism of the binding of alpha-actinin and myosin-I1 to the agarose columns. but we point out the phenomenon here, because the method appears to be very useful for the rapid. high-yield purification of these proteins from crude extracts. We hope that it will be useful to others, particularly where a simple method is required t o sepa- rate these proteins from the bulk of cytoplasmic compo- nents for analytical purposes. Since actin appears to be required for the retention of both proteins by agarose, we suspect that aggregates of the proteins are being trapped

260 Kaiser et al.

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1 ' s ' ' - " ' I " " " ' * I ' . ' ' ' 7 r T 1 1 7 1 z z " ' l " " ' . ' ' I " ' ' ' . " 1 ' ' " " ' l I t ' * x ~ " ' ~ ~ " ~ ~ " ~ ' ~ ~ " ~ x 1 ' 1 1 7 s 1

9.0 8.0 7 . 0 6.0 5.0 4.0 3.0 2 . 0 1 . o 0.0 PPn

agarose. C: A fourfold expansion of the low-field region of spectrum B. Conditions: 303°K. pH 7.4. 10 mgiml protein. The signal marked by an x derives from residual Trk .

Fig. 9. Proton magnetic resonance spectra of A) profilin-I prepared by conventional procedures and B) profilin-I renatured from 6 M urea following purification by affinity chromatography on poly-L-proline

rather nonspecifically. Such a mechanism is also consis- tent with the observed elution of myosin-I1 by ATP, since dissociation of the actinmyosin complex could well release the individual proteins trapped in the column as a large complex.

ACKNOWLEDGEMENTS

This work was supported by NIH research grant GM-26338. The authors are very grateful to Dr. Masa- hiko Sato and Sachiko Karaki for their help in preparing


a i . h . 9 f :

1 1 , r 1 I I , I I I I I I I I I l p p M 9 . 5 9 .0 8.5

PPM

B

A ~ ' " ~ " " 1 ' " ' ~ " " I " " ~ " " I ' ' " r ' " 1 " ' ' j ' ' ' ' I ' ' ' T I I ' " ' " ' ' a I ' ' 1 ' I ' ' - 1 " ' ' I ' " I "

8 . 0 7 . 0 6 . 0 5 . 0 4 . 0 3 . 0 2 . 0 1 . 0 0 . 0 9 . 0 PPM

Fig. 10. A: Filter 'H-NMR spectrum of native profilin-1 showing the slowly exchanging amide proton resonances. B: The aromatic sidechain and downfield C alpha H signals together with C a COSY section depicting NH-C,,,,,H cr-ma-peaks. Conditions as in Figure 9.

monoclonal antibodies to profilins and alpha-actinin and to John Sinard and Dr. Richard Adams for many useful suggestions. We also thank Pamela Lilly and Dr. Peter Devreotes for providing Dicfyosteliurn cell extracts.

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