THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255. No. 11, Iasue of June 10. pp. 5429-5434. 1980 Printed m U.S.A.

New, Rapid Methods for Puriwng a-Actinin from Chicken Gizzard and Chicken Pectoral Muscle*

(Received for publication, October 23, 1979, and in revised form, January 14, 1980)

Barbara G. Langer and Frank A. Pepe From the Department of Anatomy, School of Medicine, University of Pennsylvan.ia, Philadelphia, Pennsylvania 19104

We introduce two new, rapid procedures. One is spe- cifically designed for isolating a-actinin from skeletal and the other for isolating a-actinin from smooth mus- cle. Approximately 20 mg of >95% pure a-actinin can be obtained/100 g of ground chicken pectoral muscle in just 4 days. The smooth muscle protocol yields 2.7 mg of >99% pure a-actinin/lOO g of ground gizzard after just 5 days. Differences in protein contaminants and in the extractability of a-actinin necessitated the development of separate isolation procedures for the two muscle types. Antibody prepared against the pu- rified gizzard a-actinin reacted with a-actinin from skeletal, cardiac, and smooth muscle in immunodiffi- sion. Anti-a-actinin reacted only with a-actinin from crude extracts of skeletal and smooth muscle on Staph A gels. Anti-a-actinin stained Z-bands from skeletal muscle in indirect immunofluorescence microscopy and stress fibers from baby hamster kidney fibroblasts and mouse mammary epithelial cells in the characteristic punctate pattern observed by other workers (Lazar- ides, E., and Burridge, K. (1975) Cell 6,289-298). These two methods for purifying a-actinin from skeletal and smooth muscle represent a significant improvement over that published previously.

The structural protein a-actinin was first described in striated muscle by Ebashl and co-workers in 1964 (2). Fluo- rescein-labeled antibody prepared against crude a-actinin from chicken striated muscle was localized in the Z-band of myofibrils (3). This location of a-actinin in vivo seems com- patible with its abilities to gel and cross-link F-actin in vitro (4). Crude a-actinin accelerated the superprecipitation of syn- thetic actomyosin (5). But, since it is located at the Z-band, a-actinin seems too remote to affect the interaction of actin and myosin filaments in vivo. Similar preparations of crude a-actinin from chicken gizzard displayed similar properties (6) .

These results were confi ied and expanded upon by Goll and co-workers who developed a protocol for producing a more pure form of a-actinin (7-9). This basic protocol has been widely used by other investigators for making a-actinin from striated muscle (10-13), as well as from smooth muscle (14-16). Regrettably, the procedure takes 2 weeks for the extensive extractions and three chromatographic steps. When

* This work was supported in part by United States Public Health Service Grant HL15835 to the Pennsylvania Muscle Institute and by Anatomy Training Grant 5 TO1 GM00281. This work was submitted as partid fulfillment of the Ph.D. degree of B.G.L. Portiorls of this work were presented in preliminary form (1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

used to purify a-actinin from chicken gizzards, the procedure involves two additional chromatographic steps (for a total of five) (15). No method designed specifically for the isolation of a-actinin from smooth muscle has been reported.

We have developed simple and rapid methods for isolating a-actinin of high purity from skeletal or smooth muscle. From skeletal muscle, a yield of 20 mg/100 g of ground muscle can be obtained in 4 days with purity exceeding 95% as determined by PAGE.’.’ From smooth muscle, a yield of 2.7 mg/100 g of ground gizzard can be obtained after 5 days with >99% purity. The speed and ease of our new method for purifying a-actinin from striated muscle establish its usefulness. Our correspond- ingly efficient method for the purification of a-actinin from smooth muscle is the fwst to be reported which is designed exclusively for this tissue,

MATERIALS AND METHODS

Smooth Muscle a-Actinin from Chicken Gizzard DQY I : Extraction of a-Actinin after Washing with Successively

Lower Ionic Strength Solutions-Thirty to fifty fresh chicken giz- zards were cleaned of connective tissue, diced, and then passed twice through a meat grinder a t 4°C. Three hundred grams of ground gizzard was suspended to 1500 ml with Wash Solution One (0.25 M sucrose, 50 mM Tris, 1 m M EDTA, 1 m~ NaN:I, 0.5 mM phenylmeth- ylsulfonyl fluoride or PMSF, pH 8.25), homogenized with four 10-s bursts of a Waring Blendor, and centrifuged 20 min a t 25,500 X g. This wash was discarded. The pellet was resuspended to 1500 ml with Wash Solution One, homogenized with one 10-s burst, and centrifuged as before. This wash was discarded. This pellet was resuspended, rehomogenized in Wash Solution Two (50 mM Tris, 1 m~ EDTA, 1 mM NaNa, 0.5 mM PMSF, pH 7.6). and centrifuged. This wash was discarded. The residue was resuspended in extracting solution (1 mM EDTA, 1 mM NaNa, 0.5 mM PMSF, pH 7.0), rehomogenized, and following centrifugation, the extract containing a-actinin was Filtered through two layers of Kimwipes and set aside. Further extraction was with deionized water. This was repeated four times, homogenizing as before. But as the tissue swelled, centrifugation was extended to 2 h, yielding very voluminous, gel-like pellets, and supernatants of reduced volume. All extracts were brought to 1 mM with NaN-, and to 0.5 mM with PMSF, following centrifugation.

Day 2: First Ammonium Sulfate Fractionations to Isolate Crude a-Actinin-The five extracts were pooled, clarified by centrifugation for 2 h a t 25,500 X g, and precipitated with sequential additions of three ammonium sulfate fractions to separate and concentrate the following: 1) 0 to 16% ammonium sulfate saturation, 9.08 g/100 ml (actin and desmin); 2) 16 to 248, 4.72 g/100 ml (a-actinin); and 3) 24

’ The abbreviations used are: PAGE, polyacrylamide gel electro- phoresis; PMSF, phenylmethylsulfonyl fluoride. ’ To pour the fastest flowing hydroxyapatite columns, a slurry of

equal volumes of buffer and hydroxyapatite was allowed to settle slowly with an outflow rate of 1 drop/:! s, and a bed height of no more than 4 times the bed diameter. Compression from higher height to diameter ratios resulted in greatly reduced flow rates. Columns were repeatedly reused following re-equilibration in low ionic strength buffer. Care was taken to clarify all samples prior to chromatography, particularly those with high actin content, since clogging or reduced flow rates would otherwise result.

5429

5430 Fast New Purifications of Smooth and Skeletal Muscle a-Actinin

to 40% 10.1 g/100 ml (a-actinin and filamin). Each precipitate was centrifuged at 25,500 X g for 30 min. Following centrifugation of the 24 to 40% fraction, primarily actin remained in the supernatant. The pellets were suspended and dialyzed in low ionic strength buffer (20 mM potassium phosphate, 1 m~ NaN?, 0.5 mM PMSF, pH 7.2). Each of these suspensions was clarified by centrifugation for 2%. h at 150,000 x g.

Day 3: Second Ammonium Sulfate Fractionations to Purify a- Actinin-The top three-fourths of each of the clarified supernatants from the fvst ammonium sulfate precipitations was sequentially re- precipitated with an ammonium sulfate solution saturated at 4°C (3.93 M, 1 mM EDTA, pH 7.1): 0 to 16% (0.19 d/ml of supernatant), 16 to 24% (0.11 ml/d of supernatant), and 24 to 40% (0.25 ml/ml of supernatant). Following centrifugation, the small pellets were dis- solved and dialyzed in low ionic strength buffer.

Day 4: Hydroxyapatite Chromatography to Remove Actin-The fractions shown by PAGE to contain the purest a-actinin were clari- fied as above and chromatographed in a short, wide column part idy filled to a bed volume of about 1 ml of packed hydroxyapatite (Bio- Gel HT equilibrated in 50 mM potassium phosphate buffer, pH 7.2) per 1 to 5 mg of protein.’ The protein was eluted with a linear gradient of 0.05 M to 0.50 M potassium phosphate buffer, pH 7.2, with total volume 5 times that of the bed. Fractions shown by PAGE to be most enriched for a-actinin were pooled and dialyzed against half-strength low ionic strength buffer.

D a y 5 Sephacryl Chromatography to Remove Proteins with Chain Weights >100,000-The dialyzed samples were rapidly con- centrated to about 1 ml by laying the dialysis bags directly in dry Sephadex G-200. The concentrated solutions were clarified by centri- fuging 1 h at 100,OOO X g, and then passed over a column (90 X 1.5 cm) packed with Sephacryl S-200 or S-300. The column was equili- brated and eluted with half strength low ionic strength buffer. When relatively pure samples were applied, a single major peak collected after a few hours contained a-actinin fractions of 299% purity on PAGE.

The complete a-actinin purification lasted 5 days. When it was delayed at any step before final purification, the yield of, and even the ability to produce highly purified a-actinin was compromised because a-actinin then became irreversibly aggregated with actin and filamin. The use of chromatography prior to at least one set of ammonium sulfate precipitations resulted in poor separations and substantial losses. a-Actinin reversibly precipitates from pH 5.8 to 5.3,” and it has a molar extinction coefficient of 1.0 f 0.2 M” cm”. a-Actinin was stored in half-strength low ionic strength buffers (or 10 mM imidazole or Tris, pH 7.2, 1 mM NaN:,, and 0.5 mM PMSF) either at 4°C or glycerinated at -20°C in air-tight vials. Crude a-actinin degraded on storage much faster than did pure a-actinin.

Striated Muscle a-Actinin from Chicken Pectoral Muscle In this procedure, the following modifications of the protocol used

for smooth muscle were made: Day I : Removal of a n 85,000-Dalton Polypeptide Contaminant by

Selective Extraction with Sucrose-Striated muscle contains a pro- tein with a chain weight of 85,000 which is not present in smooth muscle. This protein and a-actinin are both soluble in the wash solutions, in extracting solution, and in water. However, a-actinin does not begin to extract until the 85,000-dalton protein is dissolved. To remove the 85,000-dalton protein prior to the extraction of a- actinin, the first two exposures to Wash Solution One were extended to 1 and 2 h, respectively, and a third, %-h wash was added.4 These supernatants were discarded. The residue was further rinsed with Washing Solution Two for 1 h and the supernatant was discarded.

Extraction of a-Actinin--a-Actinin was extracted with extracting solution for 40 min and the suspension was centrifuged. The partially swollen residue was extracted overnight with water by bringing its volume to 2 liters with water and homogenizing briefly in small batches. The fully swollen, gel-like suspension was centrifuged at 25,500 X g for 2 h. Extraction of a-actinin with water was repeated twice for 2 h each,

Day 2: First Ammonium Sulfate Fractionations to Isolate Crude a-Actinin-The four extracts were pooled, clarified, and precipitated sequentially with two ammonium sulfate additions (0-1696 and 16 to

,‘I Ebashi and Ebashi originally reported a slightly higher PI between

‘ Overnight extraction was sometimes used to remove the 85,000-

-~

pH 5.6 and 5.8 for crude a-actinin (5).

dalton contaminant, but some a-actinin was lost.

40% saturated ammonium sulfate) to separate a-actinin from actin and tropomyosin.

Day 3-These two fractions were each refractionated from 0 to 16% 16 to 2496, and 24 to 40% saturation to purify a-actinin.

Day 4: Chromatography of the Purest Ammonium Sulfate Frac- tions using Sephacryl or Hydroxyapatite as the Sole Chromato- graphic Step-Those supernatants whose contaminants were primar- ily of >1OO,OOO chain weight were concentrated to 1 ml and chromat- ographed over S-300. If actin was the primary contaminant they were chromatographed over hydroxyapatite. Chromatography in each case was done as described for smooth muscle. The eluant contained 95 to 99% pure a-actinin on overloaded sodium dodecyl sulfate-polyacryl- amide gels. Altogether, about 20 mg of highly purified a-actinin/100 g of ground muscle was obtained.

Sodium Dodecyl Sulfate Polyacrylamide Electrophoresis Gradient gels of 5 to 15% acrylamide were made according to the

method of Laemmli (17). Destained gels were photographed with translumination from a light box to record fine as well as dense bands, while maintaining high contrast. SO-115 film was exposed through a green Wratten No. 99 filter for 2 s at half-stops about F 8. Negatives were developed for 8 min in a 1:80 dilution of HC-110 (Kodak). With this method, all bands visible to the naked eye were also visible on the negatives.

Preparation of Antibody to a-Actinin from Chicken Gizzard Preimmune sera from four large rabbits were stored frozen. A

solution containing 8 mg of chicken gizzard a-actinin (>95% pure) in 8 ml of 10 mM imidazole, pH 7.1, was emulsified with an equal volume of Freund’s complete adjuvant (Gibco) at 4°C. Two milliliters of the antigen emulsion was injected into each rabbit. Booster shots were given in the same manner as the original injections, using a-actinin (>99% pure) in Freund’s incomplete adjuvant. Serum was obtained weekly. The IgG fraction was purified by the method of Cebra and Goldstein (18). Immunodiffusion of purified immunoglobulin was performed as described elsewhere (19). Staphylococcus aureus PAGE was performed by a modification (21) of the method of Kessler (20).

RESULTS

Chicken Gizzard a-Actinin-Fig. 1 shows PAGE analysis of each step in the extraction of chicken gizzards. Washes contained actin, tropomyosin, myoglobin, and myosin. Little a-actinin was extracted in the wash. This method for purifying a-actinin depends heavily on extracting a-actinin in the ab- sence of as many contaminants as possible. Prolonging the exposure of the gizzard homogenate to Wash Solution One resulted in the loss of much a-actinin to these washes. Con- sequently, the washing steps were limited to a total time of about an hour, which removes some contaminants and low- ered the ionic strength of the muscle homogenate.

a-Actinin was extracted once with Extracting Solution (Fig. I d ) and four times with water (Fig. 1, e to h ) , leaving little in the muscle residue. Co-extracted with a-actinin were actin, fiiamin, a water-soluble 55,000-dalton polypeptide which most likely is a form of desmin, and several polypeptides with chain weights in the range of 100,OOO to 200,000 and 10,OOO to 40,000. Traces of myosin in the extracts disappeared during ammo- nium sulfate fractionation.

Once extracted, the a-actinin was crudely separated from contaminants by sequential ammonium sulfate fractionation. Fig. 2 shows that actin and desmin were removed in the 0 to 16% fraction (Fig. 2b) , that the 16 to 24% fraction was greatly enriched for a-actinin relative to the extracts (Fig. 2c), and that a-actinin and fiiamin were precipitated together in the 24 to 40% fraction (Fig. 2d). Poorer separations resulted with faster additions of ammonium sulfate.

When a single 0 to 40% ammonium sdfate saturation was made from the extract pool instead of making the sequential fractions, repeated centrifugation of large volumes was avoided, but a far smaller yield of a-actinin resulted. However, preparing a 0 to 16% fraction to remove actin, desmin, and

Fast New Purifications of Smooth and Skeletal Muscle a-Actinin 5431

c z . A "

"s.

a-A

85,000 .r

1 1 1 1 1 1 1 1 1 I a b c d e f g h i I

FIG. 1. PAGE analysis of each wash and each extract of smooth muscle. Contaminants were removed in the washes and a- actinin was present in the extracts. a and b, first and second washes in Wash Solution One, respectively; c, third wash in Wash Solution Two; d, first extract in Extracting Solution; e to h, second through fifth extracts in water; i, standards: C, skeletal muscle C protein, 135,000, a-A, a-actinin from skeletal muscle, 94,000; P, phosphorylase, 91,000; BSA, bovine serum albumin, 68,000; and A, actin from skeletal muscle, 45,000, j , standards: M , myosin heavy chain from skeletal muscle, 200,000, a-A, a-actinin from smooth muscle, 94,000; 85,000-to 85,000-dalton polypeptide from striated muscle (provided courtesy of Dr. Prokash Chowrashi). (C protein and myosin standards were kindly provided by Mrs. Barbara Drucker.) About 10 1.11 of each sample was loaded. A gradient of 5 to 15% acrylamide was used.

tropomyosin and a second 16 to 28% fraction to isolate a- actinin with only minor filamin contamination produced a satisfactory yield and required centrifuging only two times5 It is interesting to note that the solubility of major extract proteins in ammonium sulfate seems to be correlated roughly with their increasing chain weights, which has also been observed in the case of serum proteins (22).

Refractionation of the 16 to 24% (Fig. 2, g to i) and 24 to 32% (Fig. 2 j ) fractions resulted in some samples which were greatly enriched for a-actinin and were low in actin or filamin content. Prolonged exposure to ammonium sulfate during refractionation or the subsequent use of ammonium sulfate for the purpose of concentration resulted in the formation of complexes of a-actinin with contaminant proteins that were not separable by hydroxyapatite chromatography or by gel filtration.

Further purifying a-actinin by hydroxyapatite chromatog- raphy removed much actin and filamin (Fig. 3). Contaminant polypeptides with chain weights between 100,000 and 180,000 were characteristically removed in a single fraction prior to the elution of a-actinin (Fig. 3, Fraction 49).

Final purification of the cleanest hydroxyapatite fractions with Sephacryl S-200 or S-300 gel filtration effectively re- moved remaining contaminant polypeptides if the sample applied to the column was of sufficient purity. A yield of approximately 8 mg of the >99% pure sample shown in Fig. 4 was recovered from the Sephacryl column starting with 300 g of muscle. This protein was stored at 4°C for 3 weeks before this PAGE sample was made, which demonstrates the stabil- ity of the purified protein.

' C. Damsky and D. Safer, personal communication.

a-Actinin purified from chicken gizzard had a higher chain weight (94,000) on PAGE than phosphorylase (91,000) (Fig. 1, i and j ) and did not have phosphorylase A or B activity.

a-Actinin from Chicken Pectoral Muscle-Smooth muscle a-actinin began to extract in Wash Solution One after only a 1-h exposure. This solubility of a-actinin differs from that in striated muscle, where extensive washing with Wash Solution One was done before extracting a-actinin in extracting solution and in water (Fig. 5, a to d ) . Striated muscle contains an 85,000-dalton polypeptide not present in smooth muscle. Ex- haustive washing in Wash Solution One dissolves a-actinin after removal of the 85,000-dalton polypeptide. Since a-actinin was extracted after the removal of the 85,000-dalton poly- peptide, and since, unlike smooth muscle, striated muscle contains no filamin, the only major contaminant proteins in the extract were actin and tropomyosin (Fig. 5, e to h) . Purification of this cleaner extract (relative to smooth muscle) required only a single chromatographic step after ammonium sulfate fractionation rather than two steps.

In the absence of many polypeptides over 100,000 in the extract pool, only two ammonium sulfate fractions were needed to isolate a-actinin: the 0 to 16% fraction contained primarily actin, while the 16 to 40% fraction contained pri- marily a-actinin (Fig. 5, i to k ) . Each of these fractions was refractionated to 0 to 16%, 16 to 24%, and 24 to 40% saturation (Fig. 6 a for second refractionation). The cleanest fractions were chromatographed over a column of S-300 (Fig. 6) or hydroxyapatite (not shown), depending on the contaminants which they contained. Effluent fractions from both columns contained a-actinin of >95% purity in good yield: 46 mg were recovered from the hydroxyapatite column while 4 and 14 mg

*-

1 1 1 1 I 1 1 1 1

a b c d e g h i i FIG. 2. PAGE analysis of ammonium sulfate fractionation

and refractionation of the smooth muscle extract pool. a-Ac- tinin was a minor component of the extract pool but was greatly enriched in the first round of ammonium sulfate fractions and was purified to be the principal component when these fractions were refractionated. a, extract pool; b, ammonium sulfate 1, 0 to 16% ammonium sulfate fraction contains actin and desmin; c, ammonium sulfate 2, 16 to 24% fraction is enriched for a-actinin; d, ammonium sulfate 3, 24 to 40% cut contains a-actinin and filamin; e, standards: M, 200,000, C, 135,000; a-A, 94,000; A, 45,000, f, ammonium sulfate 1, 24 to 40% refractionation cut; g, ammonium sulfate 2.0 to 16% cut; h, ammonium sulfate 2, 19 to 24% fraction contains relatively pure a- actinin; i, ammonium sulfate 2, 24 to 40% fractions; j , ammonium sulfate 3, 0 to 16% cut.

5432 Fast New Purifications of Smooth and Skeletal Muscle a-Actinin

E C

cv

36 45 51-61 70 80 I I .

I I I I

3b 49

3.0-

2.5-

2 .o 1.5-

1 .o

0.5

0

I! $4

FRACTION NO. FIG. 3. Hydroxyapatite chromatography and PAGE analysis

of the hydroxyapatite purification of smooth muscle a-actinin from a crude sample. Hydroxyapatite chromatography produced fractions greatly enriched for a-actinin that were largely freed of contaminating actin and fdamin. SA, sample: 385 ml of this 3.5 mg/ ml sample was applied to the column. The protein was eluted with a linear potassium phosphate gradient of 550 ml each. Fractions 51 to 61 from the second peak were pooled giving 225 mg of partially purified a-actinin in 180 ml.

were recovered from two separate S-300 columns starting with 300 g of muscle.

Anti-chicken Gizzard a-Actinin-Anti-chicken gizzard a- actinin formed a single precipitin line with crude and pure a- actinins from chicken gizzard and from chicken pectoral mus- cle (Fig. 7, b and c, respectively). Antibody specificity for a- actinin from both smooth and striated muscle was confirmed on Staph A PAGE (not shown). Only a-actinin was precipi- tated from extracts of both muscles. Immunoglobulin G from preimmune sera did not precipitate any protein from either muscle either in immunodiffusion (Fig. 7) or on Staph A gels

2 .o

E 0 ' C

co P4

z 1.0

Z Q r n .

8 2 r n '

0

FRACTION NO. FIG. 4. PAGE analysis of S-200 chromatography for final

purifications of samples of smooth muscle a-actinin partially purified by hydroxyapatite. A 2-ml sample containing 27 mg of partially purified a-actinin was applied to a column (1.5 X 90 cm). The sample was eluted with 10 mM imidizole, pH 7.1. Eight milligrams of the >99% pure a-actinin was recovered. To visualize any contami- nants, 20 and 40 pg were loaded in the two right-hand wells, respec- tively. The standards PM, paramyosin, 100,000; BSA, bovine serum albumin, 68,000; and A, actin, 45,000 were loaded into the left-hand wells.

85,000

1 1 1 1 1 1 1 1 1 1 1 1 a b c d e f g h i i k I

+a-A

FIG. 5. PAGE analysis of each wash and each extract of a- actinin from striated muscle. An 85,000-dalton polypeptide was removed with other contaminants prior to the extraction of a-actinin. a to c, first three washes in Wash Solution One (Prolonged exposure to Wash Solution One results in solubilization of a-actinin (a-A) after the 85,000-dalton polypeptide is removed, not shown.); d, fourth wash in Wash Solution Two; e, first extract contains a-actinin in Extracting Solution; f to h, second through fourth extracts of a-actinin in water. PAGE analysis of ammonium sulfate fractionation of the striated muscle extract pool. a-Actinin was enriched and actin contamination was reduced with two cuts; i, extract pool;j, 0 to 16% cut, ammonium sulfate 1, enriched for actin; k , 16 to 40% cut, ammonium sulfate 2, enriched for a-actinin; I , standard a-actinin, 94,000.

Fast New Purifications of Smooth and Skeletal Muscle 0-Actinin 5433

E 0.80- c 2

0.60- z 5 0.40- Z

0" I -\ I

I 57

FRACTION NO.

T 613

FIG. 6. S-300 gel filtration and PAGE analysis of the final purification of selected ammonium sulfate fractions contain- ing skeletal muscle a-actinin. a-Actinin was recovered to >95% purity in this step. a, 36.8-mg sample in 3.2 ml was applied to a column (90 X 1.5 cm); b, Fraction 7 was eluted with half-strength low ionic strength buffer; c, standard a-actinin, 94,000.

FIG. 7. Immunodiffusion of anti-chicken gizzard a-actinin against a-actinin isolated from smooth, striated, and cardiac muscle. Immunofluorescence staining of anti-gizzard a-actinin in myofibrils. a, preimmune immunoglobulin G control (pi) (0.146 mg) diffused against serial dilution of a-actinin from skeletal muscle ( S ) (10 pg) and smooth muscle (not shown) did not give a precipitin line: b, anti-a-actinin (22 pg) diffused against a serial dilution of gizzard a-actinin ( G ) (22.8 pg) gave a single precipitin line. c, extracts of skeletal muscle ( E ) ( 3 4 pg) containing a-actinin and purified skeletal muscle a-actinin ( S ) (10 pg) gave a single line of identity with the antibody ( a b ) (63.5 pg). d, serial dilution of a-actinin from cardiac muscle (C) (0.5 pg) against anti-a-actinin (180 pg) gave a single precipitin line. e, myofibril from chicken pectoral muscle reacted with 0.635 mg of anti-a-actinin and then indirectly stained with 0.33 mg of fluorescein-labeled goat-anti-rabbit IgG. Typical staining of Z-bands resulted. Preimmune immunoglobulin G gave no staining (not shown).

FIG. 8. Indirect staining of cultured non-muscle cells with anti-a-actinin gives a characteristic punctate pattern in stress fibers, along the membrane, in cytoplasmic extentions, and at cell-cell contacts. a, mouse epithelial cells; 6, baby hamster kidney fibroblasts. (Micrographs through the courtesy of Dr. Caroline Dam- sky.)

(not shown). The antibody cross-reacted in immunodiffusion with purified a-actinin from pig heart (Fig. 7d) . The antibody specifically and brilliantly stained Z-bands in chicken myofi- brils when used in conjunction with fluorescein-labeled goat- anti-rabbit immunoglobulin in the indirect fluorescence tech- nique (Fig. 7 e ) . The antibody also stained mouse epithelial cells and baby hamster kidney fibroblasts in a characteristic punctate pattern (Fig. 8, a and b ) (23).

DISCUSSION

The major differences and improvements of these proce- dures over that previously published (9, 16) for purifying a- actinid are that (a) we have shown differences in the extract- ability of a-actinin from smooth and skeletal muscle and have adapted the procedures for isolation and purification to these differences, ( b ) we have extensively simplified the washing and extracting steps in isolating a-actinin, shortening them by 3 days, (c ) we have fractionated the crude a-actinin from contaminant proteins in three ammonium sulfate cuts instead of bringing all the a-actinin and contaminants down in one wider cut, ( d ) we have reduced the amount of chromatogra- phy required for purification of a-actinin from both skeletal and smooth muscle by more than half, ( e ) we have found that to get a highly purified final product, it is important to minimize exposure of the crude a-actinin to the high ionic strength conditions resulting from the necessary ammonium sulfate fractionations and the hydroxyapat.ite chromatogra- phy, and ( f , we have developed protocols which are substan- t i d y more simple and rapid than the earlier method.

Table I summarizes the yield of a-actinin a t each step in a typical isolation and purification from skeletal muscle. After

Although a simplified method for purifying a-actinin from cardiac muscle has been reported (24). it has not been adopted for skeletal and smooth muscle. If the extractability of a-actinin from cardiac muscle is similar to skeletal muscle, then, using the procedure de- scribed here, only one chromatographic column would be required instead of two. Other major differences are that in that method: ( a ) the extraction of crude a-actinin is done over a 3-day period instead of overnight and ( b ) one wide ammonium sulfate cut is used instead of sequential fractionation.

5434 Fast New Purifications of Smooth and Skeletal Muscle a-Actinin

TABLE I Typical protein yields in the purification of skeletal muscle a-

actinin Protein concentrations were determined bv the Biuret method.

1. Wash solution 1 supernatant No. 1 2. Wash solution 1 supernatant No. 2 3. Wash solution 1 supernatant No. 3 4. Wash solution 2 supernatant 5. Extracting solution supernatant 6. Water supernatant No. 1 7. Water supernatant No. 2 8. Water supernatant No. 3 9. Extract pool

10. Ammonium sulfate 0-16% precipitate ( 1 ) 11. Ammonium sulfate 16-40% precipitate (2) 12. Ammonium sulfate 1: 0-16% precipitate 13. Ammonium sulfate 2: 0-16% precipitate 14. Ammonium sulfate 1: 16-24% precipitate 15. Ammonium sulfate 2: 16-248 precipitate 16. Ammonium sulfate 1: 24-40% precipitate 17. Ammonium sulfate 2: 24-40% precipitate 18. Sample onto S-300 19. a-Actinin off 55-300 20. Sample onto hydroxyapatite 21. a-Actinin off hydroxyapatite 22. >95% Dure a-actinin

mg/ml 19.4 4.30 2.30 1.10 0.90 2.50 3.70 2.30 2.13

11.5

25.5 0.71

1.41

mg 23,100 4,500 2,100 1,100

720 1,170 1,850

345 4,090

284 75.6

28.5 85.0 18.8

17.5 19.3 36.8 14.2

46.2 60.4

145

145

a single chromatographic step, a final yield of approximately 20 mg of a-actinin of >95% punty was obtained/100 g of ground chicken pectoral muscle. There is a 7-fold greater yield of a-actinin from skeletal muscle over that from smooth muscle. This is consistent with what appears to be a higher content of a-actinin in skeletal muscle and in extracts of it.

The previously published method for isolating a-actinin has been generally applied to all muscle tissues (skeletal, cardiac, and smooth) (9). Thus, important differences in protein com- position and in the extractability of a-actinin from smooth and striated muscle were not distinguished in that protocol. Of particular importance are differences in protein contami- nants extracted along with a-actinin from the two muscle types, since these different contaminants have significantly different effects on the ease and means by which a-actinin can be purified.

We have found that skeletal muscle contains an 85,000- dalton polypeptide which does not seem to be present in smooth muscle. This protein appears to make a considerable difference in the extractability of a-actinin from striated mus- cle compared with smooth muscle. From skeletal muscle, a- actinin does not begin to extract until the 85,000-dalton pro- tein has been thoroughly washed away after several hours in Wash Solution One. From smooth muscle, which lacks this protein, a-actinin begins to dissolve after just an hour of washing, while still in Wash Solution One. From these differ- ences in solubility, it seems that striated muscle a-actinin is protected from extraction by the 85,000-dalton polypeptide. The protective effect of the 85,000-dalton polypeptide may be related to recent evidence that it is a component of the Z- band of skeletal muscle myofibrils.’ Since the 85,000-dalton polypeptide and a-actinin cannot be separated once they are together in solution, it is essential to take advantage of their different extractabilities when preparing a-actinin, completely washing away the former prior to the extraction of the latter from the muscle residue.

IR devising the procedures for purifying a-actinin from

’ P. Chowrashi and F. Pepe, personal communication.

smooth and striated muscle presented here, particular atten- tion was paid to the following: (a) the protein contaminants characteristic for each tissue, ( 6 ) removal of as many contam- inating proteins as possible prior to the extraction of a-actinin without loss of Lu-actinin, ( e ) keeping the time during which the a-actinin is in contact with contaminants to a minimum, and ( d ) keeping the time during which cy-actinin and contam- inants are exposed to high ionic strength to a minimum. These guidelines have led to a rapid procedure for the purification of a-actinin from skeletal muscle in high yield and purity. Ad- ditionally, we report here for the first time a protocol exclu- sively designed for purifying a-actinin from smooth muscle in high yield and of high purity.

Acknowledgments-We are very grateful to Theodore Gross Gour- met Foods of Doylestown, Pa., for generously donating all the gizzards used in this study. B.G.L. wishes to thank Dr. Prokash Chowrashi for many enjoyable and valuable discussions. We are also very grateful to Dr. Caroline H. Damsky for micrographs of stained fibroblasts and epithelial cells presented in this report.

Note Added in Proof-Since going to press, another rapid method for purifying a-actinin has become available (Feramisco, J. R., and Burridge, K. (1980) J. Bwl. Chem., 255, 1194-1199).

REFERENCES 1. Langer, B. G., and Pepe, F. A. (1979) J. Cell Biol. 83,388a 2. Ebashi, S., Ebashi, F., and Maruyama, K. (1964) Nature 203,

3. Masaki, T., Endo, M., and Ebashi, S. (1967) J. Biochem. (Tokyo)

4. Maruyama, K., and Ebashi, S. (1965) J. Biochem. (Tokyo) 58,

5. Ebashi, S., and Ebashi, F. (1965) J. Biochem. (Tokyo) 58, 7-12 6. Ebashi, S., Iwakura, H., Nakajima. H., Nakamura, R., and Ooi, Y.

(1966) Biochem. Z. 345,201-211 7. Arakawa, N., Robson, R. M., and GoU, D. E. (1970) Biochim.

Biophys. Acta 200,284-295 8. Robson, R. M., Goll, D. E., Arakawa, N., and Stromer, M. H.

(1970) Biochim. Biophys. Acta 200,296-318 9. Suzuki, A,, Goll, D. E., Singh, I., Allen, R. E., Robson, R. M., and

Stromer, M. H. (1976) J. Biol. Chern. 251,6860-6870 10. Podlubnaya, Z . A,, Tskhovreboca, L. A., Saalishvilli, M. M., and

Stefanenko, G. A. (1975) J. Mol. Biol. 92, 357-359 11. Reddy, M. K., Etlinger, J . D., Rabinowitz, M., Fischman, D. A.,

and Zak, R. (1975) J. Biol. Chem. 250,4278-4284 12. Jockusch, H., Jockusch, B. M., and Burger, M. M. (1979) J. Cell

&of. 80,629-641 13. Puszkin, S., Puszkin, E., Maimon, J., Rouault, C., Schook, W.,

Ores, C., Kochwa, S., and Rosenfield, R. (1977) J. Biol. Chem. 252,5529-5537

14. Schollmeyer, J. E., Furcht, L. T., Goll, D. E., Robson, R. M., and Stromer, M. H. (1976) in Cell Motility (Goldman, R. D., Pollard, T., and Rosenbaum, J., eds) pp. 361-388, Cold Spring Harbor Laborabory, Cold Spring Harbor, N. Y.

645-646

62,630-632

13-19

15. Craig, S. W., and Pardo, J . W. (1979) J. Cell Biol. 80, 203-210 16. Fuiiwara. K.. Porter, M., and Pollard, T. D. (1978) J. Cell Biol.

i 9 , 268-275 17. Laemmli, U. K. (1970) Nature 227,680-685 18. Cebra, J., and Goldstein, G. (1965) J. Zmmunol. 95, 230-245 19. Pepe, F. A. (1976) in Cell Motility (Goldman, R. D., Pollard, T.,

and Rosenbaum, J., eds) pp. 337-346, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.

20. Kessler, S. W. (1976) J. Zmmunol. 117, 1482-1490 21. Wylie, D. E., Damsky, C., and Buck, C. A. (1979) J. Cell Biol. 80,

22. Alexander, P., and Block, R. J., eds (1961) A Laboratory Manual of Analytical Methods of Protein Chemistry I , pp. 9-13, Per- gamon Press, N. Y.

385-402

23. Lazarides, E., and Burridge, K. (1975) Cell 6,289-298 24. Singh, I., Goll, D. E., Robson, R. M., and Stromer, M. H. (1977)

Biochim. Biophys. Acta 491,29-45