THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 245, No. 4, Issue of February 25, PP. 669-673, 1970

Printed in U.S.A.

Isolation and Crystallization of Acyl Phosphatase from Rabbit Muscle*

(Received for publication, February 24, 1969)

HIROYUKI SHIOKAWA$ AND L. NODA

From the Department of Biochemistry, Dartmouth Medical School, Hanover: New Hampshire 03755

SUMMARY

A crystalline acyl phosphatase has been obtained from rabbit muscle. The enzyme was homogeneous by ultra- centrifugation (s%~,~ = 2.1) and disc electrophoresis on polyacrylamide gel at pH 4.5. The crystals had a specific activity of 860 units per mg of protein which was a 390-fold purification over the initial acid extract of rabbit muscle.

Acyl phosphate phosphohydrolase (EC 3.6.1.7)) commonly called acyl phosphatase, was first found by Lipmann (2) during the course of his studies on the role of acetyl phosphate. Several workers have purified the enzyme from various sources. Kosh- land (3) attained about 37-fold purification from horse muscle, and from the same source Harary (4) reported a 635-fold purifi- cation of the enzyme over the initial water extract. Raijman, Grisolia, and Edelhoch (5) were able to purify the enzyme from bovine brain 1760-fold over the water homogenate, and the preparation showed a single peak in the ultracentrifuge. Guer- ritore, Ramponi, and Baccari (6) prepared acyl phosphatase from horse muscle which had a specific activity of about 2500 units, expressed as micromoles of benzyl phosphate split per min per mg of protein, at 25” and pH 5.3. Pechere (7) purified acyl phosphatase from chicken muscle and obtained three fractions with enzymatic activity.

We have crystallized acyl phosphatase isolated from rabbit muscle. The present paper reports the details of the purifica- tion procedure and some properties of the enzyme.

EXPERIMENTAL PROCEDURE

Rabbit Muscle-Fresh muscle from rabbits procured locally (yield of enzyme may be 10 to 25% lower than from frozen muscle) and frozen rabbit muscle purchased from Pel-Freeze Company, Rogers, Arkansas, were used as starting material. Frozen muscles received in 2-pound packages were thawed in a cold room (2-5”) for 24 to 48 hours before use.

* This investigation was supported by Grant 5ROl-03599 from the United States Public Health Service. A preliminary report of this work was presented at the 50th Annual Meeting of the Federation of American Societies for Experimental Biology in Atlantic City, April, 1966 (1).

$ Present address, Research Institute for Tuberculosis, Hok- kaido University, Sapporo, Hokkaido, Japan.

Carboxymethyl Cellulose-Carboxymethyl cellulose used for adsorption of enzyme in Step 2 (see “Purification of Enzyme”) was obtained from Bio-Rad Laboratories, Richmond, California. In our initial preparations a mixture of three different lots (Lots 2770, 2439, and 3191; exchange capacities, 0.71, 0.7, and 0.80 meq per g, respectively) was used as the H+ form after washing with 0.5 M NaOH-0.5 M NaCl, water, 0.5 M HCl, and then thoroughly with distilled water. Cm-cellulose1 used for chromatography in Step 4 (see “Purification of Enzyme”) was obtained from Carl Schleicher and Schuell Company, Keene, New Hampshire (Selectacel Cm type 40, Lot 1338, exchange capacity, 0.6 meq per g), and washed with a mixture of 0.5 M

HCl and distilled water. In Step 2 involving large volumes a high flow rate is advantageous while in Step 4 resolution is critical. Other lots of Selectacel Cm-cellulose “Standard” in Step 2 and type 40 in Step 4 also have been used.

Dilithium Acetyl Phosphate-This was synthesized (8) or purchased from Sigma and recrystallized according to Stadtman and Lipmann (9).

All other reagents used were of analytical grade. Glass-dis- tilled water was used throughout.

Protein-This was determined by the method of Lowry et al. (10) with bovine serum albumin as standard, except in chroma- tography, in which the amounts of protein were calculated by measuring absorbance at 280 rnp and assuming a factor of 1 to convert to milligrams of protein per ml.

Ammonium Sulfate Precipitation-The weight of ammonium sulfate in grams to be added to attain saturation SZ (expressed as a decimal) from saturation X1 and volume in milliliters, ~1, was calculated by the formula, w = 0.515 ~1 (SZ - 81) /(l - 0.272 8~). When significant and necessary, corrections were made for the ammonium sulfate solution associated with precipitated protein. The increase in volume over the volume of solvent used was assumed as equal to the ammonium sulfate solution at which initial precipitation was carried out (i.e. the volume of protein is neglected).

Acyl Phosphatase Activity-Acetyl phosphate (50 ~1 of a 0.10 M stock solution in 0.10 M acetate buffer, pH 5.4) and 50 ~1 of enzyme suitably diluted in 0.10 M acetate buffer, pH 5.4, were incubated for 10 min at 25”. Residual acetyl phosphate was determined by the method of Lipmann and Tuttle (11). One unit of the enzyme is defined as the amount of enzyme which hydrolyzes 1 pmole of acetyl phosphate in 1 min, in a calculated volume of 1 ml under the above conditions. To a maximum of

1 The abbreviation used is: Cm-, carboxymethyl-.

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670 Rabbit Muscle Acyl Phosphatase Vol. 245, No. 4

about 30% of the initial substrate, the hydrolysis is proportional to the amount of enzyme. Specific activity is expressed as units per mg of protein.

Chromatography on Cm-cellulose-This was carried out accord- ing to the method of Peterson and Sober (12).

Disc Electrophoresis-Disc electrophoresis on polyacrylamide gel was performed at 2” with a 15’% gel by the modified method of Reisfeld, Lewis, and Williams (13) for basic proteins.

UZtraeentti@gation-Ultracentrifugal runs were carried out on a Spinco model E ultracentrifuge.

Amino Acid Analysis-Samples of the enzyme (5.40 mg) were hydrolyzed in 6 N HCl at 110” in evacuated hydrolysis tubes for 24 or 72 hours. After hydrolysis, the HCl was removed by evaporation under reduced pressure, and the residue was dis- solved in 5.0 ml of 0.2 N citrate buffer, pH 2.2, for storage. On analysis, a l.O-ml portion of suitably diluted stored hydrolysate solution was applied and analyzed in a JEOL-3BC ammo acid analyzer (Japan Electron Optics Laboratory, Ltd., Tokyo), ac- cording to the procedure of Moore, Spackman, and Stein (14). Tryptophan was separately determined according to the spec- trophotometric procedure of Goodwin and Morton (15).

RESULTS

Purification of Enzyme

All procedures were carried out in a cold room (2-5’) or in ‘an ice bath, unless indicated otherwise.

Step 1: Extraction of Enzyme-The frozen muscles (Pel-Freeze, Rogers, Arkansas), 22.6 kg in a-pound packages, were partially thawed in the cold room for 2 days, and mechanically ground. The ground muscle was homogenized for 1 min with cold 0.1 M

HCl-0.01 M KC1 solution (3.5 liters per kg of muscle) in a 4-liter Waring Blendor. The pH of the homogenate was usually 3.5 to 3.7, and if necessary, was adjusted to 3.5 with 2 N HCl. After stirring for 10 min, the homogenate was neutralized to pH 6.0 with 2 N NaOH. As the neutralization proceeded, the

0 500 1000 1500 2ooo

EFFLUENT VOLUME (ML)

Fxo. 1. Chromatography of partially purified acyl phosphatase on Cm-cellulose column. Fraction 3 (47 ml, 1950 mg of protein) was applied to a column (2.2 X 26.5 cm) previously equilibrated with acetate buffer (pH 4.8, I’/2 = 0.05). The column was eluted with a linear gradient formed from 1500 ml of acetate buffer (pH 4.8, r/2 = 0.05) and 1500 ml of 0.4 M NaCl in the same buffer. The flow rate was 120 ml per hour, and lo-ml fractions were collected. -, optical density at 280 rnp; - - -, acyl phosphatase activity; -, molar concentration of NaCl.

homogenate became so viscous that mechanical stirring by a small motor was impossible. Mixing was carried out by a wooden paddle operated by hand. After the attainment of pH 6.0, stirring was continued for 15 min. For each kilogram of starting muscle, 200 g of Celite (No. 535 or 545) were added and the mixture was filtered through a special 50-cm polyethylene Buchner funnel prepared with cloth filter and a thin layer of Celite. The filter cake was resuspended with cold water equal to half the volume of extract and filtered. The volume of com- bined filtrates was generally about 128 liters.

Step 2: Adsorption of Enzyme on Cm-cellulose-The pH of the combined filtrates was adjusted to 4.5 with 2 N HCl and a small amount of fine precipitate was removed by filtration with the aid of Celite. The filtrate was passed through a Cm-cellulose column (12-cm diameter by lo-cm height, Lucite column). The flow rate was adjusted to 130 to 150 ml per cm2 per hour with a peristaltic pump. During the adsorption, red colored substances which served as an indicator for the enzyme formed a red top layer of about one-fifth of the height of the column. Channeling caused by shrinking of the cellulose was troublesome. About one-third of the Cm-cellulose cake from the top of the column was removed and washed twice (each washing 0.5 liter per kg of muscle) by stirring for 10 min in cold water and filtering off the reddish colored cellulose. Inert protein was further removed by repeated washing with cold water (0.5 liter per kg of muscle) after adjusting the suspension to pH 9.5 with 2 N NaOH and stirring for 10 min before filtering. The washing at pH 9.5 was repeated until the filtrate was colorless and a portion gave no precipitate when tested with trichloracetic acid. The washed Cm-cellulose cake was suspended in 0.05 M acetate buffer, pH 5.4 (300 to 400 ml per kg of muscle). The pH of the suspension was adjusted to pH 5.4 with 5 N acetic acid. The suspension was transferred to a glass column (7 x 40 cm). After the Cm- cellulose had settled down, 0.6 M NaCl-0.1 M acetate buffer, pH 5.4, was applied to elute the enzyme. The eluate was col- lected in 500-ml portions. The enzymatic activity and the

FIG. 2. Photomicrograph by polarized light of once recrystal- lized acyl phosphatase (courtesy of Dr. Hidemi Sato, Department of Biology, University of Pennsylvania).

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Issue of February 25,197O H. Shiokawa and. L. Noda

TABLE I

Purification of acyl phosphatase from rabbit muscle

Total activity

ml rrdts

1. Extraction..................................... 128,000 375,000 2. Adsorption on Cm-cellulose. . . . . . . . . . . . 2,620 340,000 3. First ammonium sulfate fractionation. . . . . . . . 47 224,000 4. Cm-cellulose column. . . . . . . 250 96,600 5. Second ammonium sulfate fractionation. . . . . . . 7.1 95,900 6. Crystallization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 37,200

absorbance at 280 rnp of each fraction were determined, and the fractions (generally about five) which had a specific activity of more than 25 units per mg of protein were pooled (Fraction 2).

Step 3: First Ammonium sulfate Fracsctionation-The pooled eluates from the Cm-cellulose column were adjusted to pH 3.0 with 5 N HzSOI and the volume was measured (about 2.6 liters). Solid ammonium sulfate was added to 50% saturation. After standing in the cold for 2 hours, the precipitate was removed by filtration through a pad of 200 g of Celite on an l&cm Buchner funnel. The filter cake was washed twice with about 200 ml of 50% saturated ammonium sulfate solution adjusted to pH 3.0. To the combined filtrates and washings (3500 ml) was added solid ammonium sulfate (955 g) to 90% saturation. The solu- tion was kept in the cold room overnight. The precipitate was collected by centrifugation and dissolved in 200 ml of 0.1 M

acetate buffer, pH 5.4. The volume was measured and excess over 200 ml was assumed to be 90% saturated ammonium sulfate. Insoluble material was centrifuged off. Solid ammo- nium sulfate was added to the solution to 90% saturation. After standing for several hours in the cold room, the precipitate was collected by centrifugation, dissolved in about 40 ml of acetate buffer, pH 4.8, I’/2 = 0.05, in the cold, and dialyzed against four 2-liter portions of the acetate buffer.

FIG. 3. A, disc gel electrophoresis of crystalline acyl phospha- tase on polyacrylamide gel. Amount of acyl phosphatase corre- sponding to 100 pg of protein was layered onto 15% polyacrylamide gel column (0.5 X 7.0 cm). Electrophoresis was carried out at 5 ma per tube for 90 min in 0.35 M b-alanine-acetic acid buffer system (pH 4.5). The direction of electrophoresis is from top (+) to bottom (-). Protein was detected by staining with 1.0% Amido black 10B in 7% acetic acid followed by electrophoretic destaining in 7yo acetic acid. B, sedimentation velocity pattern of crystal- line acyl phosphatase. Photo was taken 152 min after attainment of full speed, 50,740 rpm. Centrifugation was performed at 13” in 20 mM acetate buffer, pH 5.4, at a protein concentration of 9.4 mg per ml.

T

.-

-

Protein Specific activity Yield Purification

w uds/mg % -fold

169,000 2.2 100 1 11,290 30.2 90.6 13.5 1,950 115 60 51.8

322 310 26 140 158 607 25 276 43.2 860 10 391

671

Step 4: Chromatography on Cm-cellulose-The dialyzed solu- tion was centrifuged to remove precipitated proteins and applied to a Cm-cellulose column (2.2 x 26.5 cm), which had been equili- brated with acetate buffer pH 4.8 and P/2 = 0.05. The column was washed with the acetate buffer until the absorbance of the effluent at 280 rnp was almost zero, and then a linear gradient of NaCl was started. Twin Lucite cylindrical reservoirs, 11.2 cm internal diameter, contained 1500 ml of 0.4 M NaCl in acetate buf- fer (pH 4.8, I’/2 = 0.05) in one cylinder and 1500 ml of acetate buffer in the mixing cylinder. Fractions of 10 ml were collected with a flow rate of 120 ml per hour. The chromatographic pattern is shown in Fig. 1. Major enzymatic activity was contained in Peak B (effluent volume, 1020 to 1210 ml; about 43% recovery). Total activity recovered from the column was about 89 To.

justed to 5.4, and solid ammonium sulfate was added to the supernatant to bring the solution to 60% saturation. The precipitate was collected by centrifugation after standing for 2 hours, dissolved in a small amount of 0.1 M acetate buffer, and dialyzed against 0.1 M acetate buffer, pH 5.4, containing 2 x lo+ M EDTA.

Step 5: Second Ammonium Sulfate Fractionation-The pH of Step 6: Crystallization-After the pH of the dialyzed solution the pooled fraction (Peak B) was adjusted to 5.4 with 5 N had been adjusted to pH 3.5 with 5 N H&04, the solution was NHdOH. Solid ammonium sulfate was added to the pooled brought to 42% saturation in ammonium sulfate. A trace of solution to bring the saturation to 90%. The precipitate was precipitate was centrifuged off. To the supernatant solution collected by centrifugation after standing in the cold overnight, ammonium sulfate was added slowly to 60% saturation. The dissolved in 0.025 M acetate buffer, pH 5.4, and dialyzed against precipitate formed was collected by centrifugation and dissolved the same buffer. The pH of the dialyzed solution was adjusted with 2.4 ml of acetate buffer (0.1 M, pH 5.4), taking note of the to 3.0 with 5 N H&04. Solid ammonium sulfate was added to increase in volume for the purpose of estimating ammonium bring to 42% saturation. A small amount of precipitate was sulfate saturation. Weighed ammonium sulfate was slowly removed by centrifugation, the pH of the supernatant was ad- added with magnetic stirring in an ice bath until the solution

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672 Rabbit Muscle Acyl Phosphatase Vol. 245, No. 4

TABLE II Amino acid composition of acyl phosphatase

Amino acid residues

Lysine . Histidine . Arginine . . . Aspartic acidb.. Threoninec.. Serinec.. . . . . Glutamic acidb. Proline. . . Glycine......... Alanine......... Valine . Methionine . Isoleucine . . Leucine. . . Tyrosine. . Phenylalanine.. . Tryptophand .

. . .

. .

. . . . . .

. .

.

No. of residues”

20.7

0.75 10.1 16.9

14.9 23.6 16.7

7.3 20.3

5.5

21.5 4.1 8.1

6.9 7.8 7.9 2.7

Nearest integer

21 1

10 17

15 24 17

7 20

6 22

4 8

7 8 8 3

r Residues

/IO0 g protein 11.29

0.44 6.70 8.26

6.68 8.73

11.07 3.01

4.93 1.66 9.06 2.27

3.91 3.33 5.40 4.48

2.15

a Calculated by assuming a molecular weight of 23,500. b Includes both free and amidated residues of these amino

acids; amide ammonia was not determined. c Obtained by extrapolation to zero time of hydrolysis. d Determined spectrophotometrically by the method of Good-

win and Morton (15).

showed a very slight cloudiness. The calculation for 42yo satura- tion serves as a guide for the approximate quantity of ammonium sulfate required. The faintly turbid solution after standing even less than an hour generally shows a sheen on swirling. If this evidence of crystallization is not apparent the solution is clarified by centrifugation and further ammonium sulfate is added a few crystals at a time until the solution again shows faint turbidity. The process may be repeated. Crystals have been allowed to grow for 1 or 2 days before harvesting. Fig. 2 is a photomicrograph of needle forms obtained. Recrystallization was performed in a similar manner. Table I summarizes the purification procedure.

Properties of Enzyme

Polyacrylanaide Gel Electrophoresis-The purity of the once recrystallized enzyme was examined by electrophoresis on poly- acrylamide gel according to the method of Reisfeld et al. (13).

The electrophoretic pattern (Fig. 3A) indicates a single band without any trace of contamination.

Ultracentrifugation-Sedimentation of the crystalline enzyme in acetate buffer (0.02 M, pH 5.4) was carried out in the analytical ultracentrifuge, Spinco model E. As shown in Fig. 3B, a single peak was observed in the run. The sedimentation coefficient szO,zu was calculated to be 2.1.

Stability-The enzymatic activity of crystalline rabbit en- zyme was stable at acidic and neutral pH, but somewhat unstable at alkaline pH. At pH 3 and 5.4 the loss of the activity was almost negligible after storage for 12 hours at 37”, while at pH 9 about 30% of the activity was lost when the enzyme was kept at 2” for 1 hour. The enzyme was also unstable to methanol and ethanol even at low temperature (10’) like acyl phosphatase from bovine brain (5), while 70% acetone (v/v) at -10” for 3

hours had no effect on the activity. The crystalline rabbit enzyme when kept as a suspension in a solution of ammonium sulfate of 42% saturation at - 10” is quite stable for long periods; for example one sample was stored for 1 year with no loss of activity.

Amino Acid Composition-The amino acid composition of acyl phosphatase was determined following acid hydrolysis of samples for 24 or 72 hours. The results are shown in Table II. The number of residues per molecule of protein was calculated on the basis of the molecular weight of 23,500, determined by sedimentation equilibrium.2 It is noteworthy that only 1 histidine residue is present in the molecule.

DISCUSSION

By the use of chromatography at pH 4.8 on Cm-cellulose in the purification procedure, the enzymatic activity of acyl phos- phatase was separated into three peaks. The specific activities of two peaks, B and C, Fig. 1, eluted at about 0.16 M and 0.25 M NaCl, respectively, were almost the same, while the specific activity of the other activity peak, A, eluted at about 0.1 M

NaCl was about half that of the above two peaks. The crystals were obtained from Peak B, which contained most of the ac- tivity. In early studies attempts to separate the enzyme by Cm-cellulose chromatography at several pH values between 7 and 9, did not yield any clear-cut chromatogram in which the protein concentrations paralleled the enzymatic activities. This might possibly be due to instability of the enzyme, and chroma- tography at acidic pH (at pH 4.8 in this paper) may be a factor which enabled us to get the reproducible and distinct peaks. We have not determined whether the activities represented by Peaks A and C are isozymes of the crystalline enzyme or arti- facts arising during the purification procedure.

The enzyme has been crystallized in 10% over-all yield from the acid extract of rabbit muscle after 390-fold purification. The crystalline preparation was homogeneous, as judged by ultra- centrifugation and electrophoresis on polyacrylamide gel. The sedimentation coefficient of rabbit enzyme, 2.1, is larger than that of bovine brain enzyme (sZO,, = 1.25) obtained by Raijman et al. (5).

The molecular weight by sedimentation equilibrium, assuming a partial specific volume of 0.75, gave the value of 23,500 for the rabbit enzyme. This value is higher than the molecular weight of 13,200 reported for the enzyme from bovine brain (5) and 9,750 for horse muscle enzyme (16). Some further studies on the properties of the crystalline rabbit enzyme are in progress.

REFERENCES 1. SHIOKAWA, H., AND NODA, L., Fed. Proc., 26,754 (1966). 2. LIPMANN. F.. Advan. Enzumol.. 6,231 (1946). 3. KOSHLAN~, h. E., JR., in S. P: COLO&K AND N. 0. KAPLAN

(Editors), Methods in enzymology, vol. II, Academic Press, New York, 1955, p. 555.

4. HARARY, I., in S. P. COLOWICK AND N. 0. KAPLAN (Editors), Methods in enzymology, Vol. VI, Academic Press, New York, 1963, p. 324.

5. RAIJMAN, L., GRISOLIA, S., AND EDELHOCH, H., J. Biol. Chm., 236, 2340 (1960).

6. GUERRITORE, A., RAMPONI, G., AND BACCARI, V., Abstracts of the First Meeting of the Federated European Biochemical Societies, London, 1964, p. 18, A-20.

7. PECHERE, J.-F., Bull. Sot. Chim. Biol., 49, 897 (1967). 8. AVISON. A. W. D.. J. Chem. Sot.. 732 (1955).

2 Unpublished results.

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9. STADTMAN, E. R., AND LIPMANN, F., J. Biol. Chem., 186, 549 (1950).

13. REISFELD, R. A., LEWIS, U. J., AND WILLIAMS, D. E., Nature, 196, 281 (1962).

10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem., 193, 265 (1951).

14. MOORE, S., SPACKMAN, D. H., AND STEIN, W. H., Anal. Chem., 30, 1185 (1958).

11. LIPMANN, F., AND TUTTLE, L. C., J. Biol. Chem., 169,21 (1945). 12. PETERSON, E. A., AND SOBER, H. A., in S. P. COLOWICK AND

15. GOODWIN, T. W., AND MORTON, R. A., Biochem. J., 4.0, 628 (1946).

N. 0. KAPLAN (Editors), Methods in enzymology, Vol. V, Academic Press, New York, 1962, p. 3.

16. RAMPONI, G., TREVES, C., AND GUERRITORE, A., Arch. Bio- them. Biophys., 120, 666 (1967).

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Hiroyuki Shiokawa and L. NodaIsolation and Crystallization of Acyl Phosphatase from Rabbit Muscle

1970, 245:669-673.J. Biol. Chem. 

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