ANALYTICAL
BIOCHEMISTRY
86,
378-385 (1978)
A Simple Procedure for the Preparation of Highly Purified (Sodium + Potassium) Adenosinetriphosphatase from the Rectal Salt Gland of Squalus acanthias and the Electric Organ of Electrophorus electricus JOHN F. DIXON Department
of Pharmacology, Madison.
AND LOWELL
E. HOKIN
University of Wisconsin Wisconsin 53706
Medical
School,
Received April 6, 1977; accepted December I, I977 A simple purification procedure for the Na,K-ATPase from membranes of the rectal gland of Squalus acanthias or crude microsomal fractions from the electric organ of Hectrophorus electricus is presented here. The purification procedure consists of solubilization of the Na,K-ATPase with the nonionic detergent, Lubrol WX, chromatography of the diluted Lubrol extract on aminoethyl cellulose, and ammonium sulfate fractionation (1) of the concentrated eluate from the aminoethyl cellulose column. The yields of final purified enzyme are comparable to the earlier purification (l-4) involving the expensive and cumbersome zonal centrifugation step. The purity of the final enzyme. as attested to by specific activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is as great or greater than that previously reported for the enzyme purified by the procedure involving zonal centrifugation. The simplicity of the present procedure, coupled with the ready commercial availability of electric eels which are quite hardy on shipment. makes purification of the Na,K-ATPase widely available to workers in the field.
A purification scheme has been developed in this Laboratory which leads to Na,K-ATPase2 (E.C. 3.6.1.3) preparations of 50, 95, and 95% homogeneity from beef brain (l), rectal salt gland of Squafus acanrhias (2), and the electric organ of Electruphorus electricus (3,4), respectively. The purification procedure consists of solubilization of membrane fractions with the nonionic detergent, Lubrol WX. centrifugation in a continuous sucrose gradient in a zonal rotor, and a novel ammonium sulfate fractionation procedure. The procedure has the virtue of very high yields [up to 100 mg per preparation (4)] and no denaturation during purification. Its main drawback is the rather expensive and cumbersome zonal ’ This work was supported by grants from the National Institutes of Health (HL 16318) and the National Science Foundation (PCM 76-20602). 2 Abbreviations used: Na,K-ATPase. (sodium + potassium)-activated adenosine triphosphatase; SDS. sodium dodecyl sulfate. 0003-2697/78/0862-0378$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
378
SIMPLE PURIFICATION
OF Na,K-ATPase
379
centrifugation step. We present here a modified purification procedure in which chromatography of the Lubrol extract on aminoethyl cellulose (5) takes the place of zonal centrifugation. The yields and specific activity of enzyme (based on original starting material) and SDS-polyacrylamide gel patterns are essentially the same or better than we previously reported for the procedure involving zonal centrifugation. Since live electric eels are commercially available and ship quite well, purification from this source by the method described here makes available a simple purification procedure for an important membrane enzyme from a readily available source. In this paper we show data on the purification by this new procedure of the rectal gland enzyme and the electric organ enzyme with essentially identical results. MATERIALS
AND METHODS
Materials. Aminoethyl cellulose was obtained from Accurate Chemical and Scientific Corp., Hicksville, New York. Lubrol WX was obtained from Supelco, Inc., Bellefonte, Pennsylvania. Disodium ATP was obtained from P-L Biochemicals, Milwaukee, Wisconsin. Sodium Verona1 was obtained from Fisher Scientific Company, Chicago, Illinois. Solubilizution of Na,K-ATPase. Main organ electroplax tissue was dissected from cross-sectioned slices of the tails of electric eels immediately after sacrifice (3) and stored indefinitely at -70°C. These slices were used in the preparation of crude microsomal pellets (4). After overnight storage at 4°C the pellets were combined in a small beaker. Sixteen percent Lubrol WX, 0.1 M Na,ATP (pH 7.0), and water (so as to make the final Lubrol concentration 3.2% and the final Na,ATP concentration 1 mM) were pipetted directly onto the pellets. The pellets were broken up so as to form a fibrous, pastelike suspension. The suspension was further mixed by two up and down strokes in a glass homogenizer. All of the mixture was transferred into IO-ml thick wall uncapped centrifuge tubes and centrifuged for 30 min at 105,OOOg. The supernatant was further clarified by two 60-min centrifugations at the same force. The clarified supernatant was the eel Lubrol extract. Crude membrane fractions from shark rectal glands were prepared as before (2) with the following modifications. The pH of the initial sucrose buffer was lowered to 8.0. The loose white top layer of the 19,600g pellet was centrifuged without dilution for 60 min at 78,500g. The resultant pellets were homogenized in 1 mM Tris-EDTA (pH 7.0) at a protein concentration of 20 mg of protein/ml, and the homogenized membranes were stored indefinitely at -70°C. Preparative to chromatography the crude membrane fraction was thawed and homogenized in 1.6% Lubrol at 7 mg of protein/ml.
380
DIXON
AND
HOKIN
After a 15min incubation on ice the suspension was centrifuged for 60 min at 105,OOOg. The clarified supernatant was the rectal gland Lubrol extract. Chromatography
of Na,K-ATPase
on aminoethyl
cellulose
columns.
This was similar to the procedure of Nakao et al. (5), except that the Lubrol was washed from the column at low ionic strength followed by elution of the Na,K-ATPase at high ionic strength, while Nakao et al. (5) eluted the Lubrol and the Na,K-ATPase off the column together at the higher ionic strength. The columns were prepared as follows. Thirty grams of aminoethyl cellulose powder were stirred into 500 ml of 0.5 N NaOH. The suspension was filtered by suction over filter paper in a Buchner funnel. The filter cake was treated with another 500 ml of 0.5 N NaOH, filtered, and washed repeatedly by suspension in water until the filtrate was between pH 7 and 8. Similar treatment with 0.5 N HCl was followed by washing until the pH was 5. Fine particles were removed by decantation in several water washes. For small-scale purification the desired amount of aminoethyl cellulose was poured from a 50% slurry at 4°C into a 2.5 (i.d.) x loo-cm column, the hydrostatic pressure being gradually increased to 20 to 30 cm. The column was equilibrated with “wash buffer” consisting of 14 mM sodium Verona1 acetate buffer, pH 6.6, and 1 mM Na,EDTA. The Lubrol extract was diluted with wash buffer so that the final Lubrol concentration was 0.4% (i.e., 1 vol of eel Lubrol extract + 7 vol of wash buffer or 1 vol of dogfish Lubrol extract + 3 vol of wash buffer). A volume of diluted Lubrol extract equal to or less than the void volume of the column was applied to the column. A flow rate of 0.5 to 2 ml/min was maintained throughout the column run. After one void volume had passed through, the column was further eluted with 1 column of wash buffer. One and one-half column volumes of wash buffer containing 100 mM NaCl and 1 mM Na,ATP were then passed through the column. The single protein peak eluting approximately 1 void volume after the addition of this solution (see Fig. 1) was pooled and concentrated to a protein concentration of 5 mg/ml or more, using a Diaflo ultrafiltration cell with an XM-50 membrane. The concentrate was further purified by the ammonium sulfate fractionation procedure of Uesugi et al. (1). When large bed volumes were used (150 to 300 ml) it was necessary to use a peristaltic pump at the outlet to maintain and increase the flow rate (2 ml/min). The cellulose was regenerated on the large columns with approximately one bed volume of 0.5 N NaOH, a water wash (until the pH was 8.0), followed by washing with approximately one bed volume of 0.5 N HCl and a water wash to pH 5.0. The aminoethyl cellulose was not in contact with the acid or base for more than 2 hr. Determination of Lubrol. Lubrol was extracted into chloroform by the extraction procedure of Bligh and Dyer (6). The chloroform extract was taken to dryness under a stream of nitrogen gas, redissolved in 50% ethanol, and assayed by the method of Garewal (7). There is no interference by phospholipid with this method.
SIMPLE
PURIFICATION
381
OF Na,K-ATPase
Av S.A. 720 u/mg
! ! Av S.A. Zero u/mg
Av S.A. 700dmg
x B
H
”
P .
2 6 z”
1z
\
50
I
I Fraction Number
I50
260
FIG. 1. Eighteen milliliters of Lubrol extract of electric organ crude microsomal fraction were diluted to a final volume of 144 ml with wash buffer and applied to an aminoethyl cellulose column with a 1%ml void volume. See Materials and Methods for details. Six-milliliter fractions were collected from the column. This run was the same as that shown in Table 1 for the “large-scale” aminoethyl cellulose chromatography step in the full purification of the electric eel enzyme. Lubrol was qualitatively located by its formation of a precipitate in the protein assay.
RESULTS Aminoethyl
Cellulose Chromatography of Lubrol Extracts.
When a diluted Lubrol extract containing the Na,K-ATPase from either the rectal gland or the electric organ was applied to aminoethyl cellulose as described under Materials and Methods 95% of the protein bound to the column. A band of free Lubrol equal in volume to that of the applied sample eluted without retardation as seen in Figs. 1 and 2. Five percent of the applied protein eluted in the same area. The columns could be washed with many column volumes of wash buffer without further elution of protein. Forty percent of the bound protein was eluted as a sharp peak when 100 mM NaCl and 1 mM Na,ATP were added to the wash buffer. Samples from various regions of the peak had the same specific activity of Na,K-ATPase, and this was 50% higher than that of the applied sample. Ninety to ninety-five percent of the applied protein could be accounted for after elution with 0.5 N NaOH from the column. Effect of Sample Size
When the volume of the Lubrol extract applied to the aminoethyl cellulose column exceeded the void volume of the column, enzyme eluted
382
DIXON AND HOKIN C Free
Lubrol
+
++
0.81
lb
$0
Fraction Number FIG. 2. Eight milliliters of a Lubrol extract of rectal gland membranes were diluted with wash buffer to a final volume of 32 ml and applied to an aminoethyl cellulose column with a 6-ml void volume. Protein concentration and presence of Lubrol in I-ml fractions are shown.
in the free Lubrol band (Fig. 2). Under these conditions over 85% of the applied protein had eluted after application of the wash buffer. Effect of Dilution of Lubrol Extracts Undiluted extracts of enzyme from both species did not bind as well as the diluted extracts. Fifty percent of the protein of the undiluted extracts from the rectal gland eluted with the free Lubrol and 80% of the undiluted extract from the electric organ eluted with the free Lubrol. In both cases the specific activity of the unbound material was 20% higher than the specific activity of the applied sample. PuriJication
Procedure
Table I shows the purification of Na,K-ATPase from both species, utilizing aminoethyl cellulose chromatography, and compares these purifications with purification utilizing zonal centrifugation as reported previously (l-4). Ammonium sulfate treatment of the enzyme which eluted from the aminoethyl cellulose columns resulted in yields comparable to those achieved by purification involving zonal centrifugation. The specific activities of the enzymes purified with the column method were as high or higher than those purified by the zonal method. The data on purification involving zonal centrifugation are average values. The data on purification involving aminoethyl cellulose chromatography are typical values. There is some variability in the final specific activities by both methods, and specific activities as high as 2100 Fmol of Pi/mg of protein/hr have been obtained. However, the vast majority of values cluster around those shown in Table 1. Bound Lubrol The amount of Lubrol bound to the purified enzyme was the same whether the zonal method or the aminoethyl cellulose method was used.
SIMPLE
PURIFICATION TABLE
PURIFICATION
OF THE Na,K-ATPAsE
FROM
1
THE ELECTRIC
ORGAN
AND
Rectal gland
Total Na,K-ATPase (pmol/hr) Zonal Chrom. (large) (small)
THE RECTAL
GLANDS
Eel
Lubrol extract
Concentrate
(NH&SO,
75,300 87,300
54,500 60.400 8,400
50.000
15.800
383
OF Na,K-ATPase
Lubrol extract
Concentrate
(NH&SO, PPt.
6,600
382,500 100,500 30.600
150,200 55,400 19.100
121,000 47,800 15,700
PPt.
60,100
Total protein (mg) Zonal Chrom. (large) (small)
167 172 29.1
78 69.3 9.01
41 42.9 5.50
617 150 50.0
203 77.0 26.5
90.0 36.4 12.3
Specific activity (pmoli mg protein/hr) Zonal Chrom. (large) (small)
451 508 543
699 872 932
1.220 I .400 1,200
620 670 612
740 719 721
1,350 1,310 1.280
Quantity of tissue Zonal Chrom. (large) (small)
110 rectal glands I10 rectal glands I8 rectal glands
400 g of electroplax tissue 120 g of electroplax tissue 40 g of electroplax tissue
” Purification of Na,K-ATPase by the method presented in this paper is shown on two scales, and purification by the zonal method is shown on the large scale which has been routinely used. “Concentrate” refers to a concentrate of the peak eluting from the aminoethyl cellulose column after application of 100 mM NaCl and 1 mM Na,EDTA in Verona1 buffer or to a concentrate of the Na,K-ATPase peak from zonal centrifugation. as reported previously (l-3).
Three-tenths milligram of Lubrol was bound per milligram of rectal gland enzyme protein and 0.37 mg of Lubrol was bound per milligram of eel enzyme protein with either method of purification. This is comparable to the amount of Lubrol bound to the partially purified beef brain enzyme previously reported (1). Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis
Equal amounts of protein from the three stages of purification were analyzed by SDS-gel electrophoresis (Fig. 3). Increase in purity is indicated by the disappearance of bands other than the two subunit bands of the Na,K-ATPase and the increase in size of these latter bands. By this criterion the purity of the enzyme prepared by the present procedure is comparable to that of the enzymes prepared by the zonal method. It should be noted that a small amount of protein ran in the position of the tracking
384
DIXON
I
AND HOKIN
2
3
3. Ten micrograms of (a) an eel Lubrol extract, (b) the eluate from an aminoethyl cellulose column, or (c) an ammonium sulfate precipitate of the column eluate were dissolved in SDS and submitted to analytical SDS-polyacrylamide gel electrophoresis, and the gels were stained with Coomassie blue, as described previously (2). FIG.
dye. This band accounted for less than 5% of the protein applied to the gel. It probably corresponds to a band previously seen running just above the tracking dye position (2). DISCUSSION
Highly purified preparations of Na,K-ATPase have been obtained from the mammalian kidney #-lo), the rectal salt gland of Squalus acanfhias (2), and the electric organ ofElectrophorus electricus (3-4). The principle of purification of the kidney enzyme involves what may be termed purification by selective extraction of impurities, i.e., protein impurities are extracted with anionic detergents from membranes from the outer renal medulla of the kidney and the enzyme is further purified by various centrifugation techniques, and in some cases other fractionation techniques, leaving behind a membrane skeleton which contains the Na,K-ATPase as essentially its only protein. The advantage of this technique is that mammalian kidneys are readily available. The disadvantages of this technique are that dissection of highly active outer renal medulla fractions is difficult and tedious, the yields are often low, and in some procedures there is considerable denaturation of the Na,K-ATPase during purification. The principle which we have employed for purification of the fish enzymes in the past involves what may be termed purijication
SIMPLE
PURIFICATION
OF Na.K-ATPase
385
after solubilization, i.e., the Na,K-ATPase is solubilized with the nonionic detergent, Lubrol WX, and the solubilized enzyme is purified by zonal centrifugation and a novel ammonium sulfate fractionation scheme. The advantages of this technique are that the yields are high and there is no denaturation during purification. The disadvantages of this technique are that zonal centrifugation is expensive and cumbersome, and rectal glands from Squalus acanthias are not readily available. The method presented here appears to maximize the advantages and to minimize the disadvantages. Electric eels are readily available commercially and are quite hardy on shipment. The aminoethyl cellulose chromatography step can be substituted for the zonal centrifugation step, so that high yields and as high or higher specific activities of the final purified Na,K-ATPase and equally good gel patterns are obtained with no attendant denaturation during purification. The procedure presented here should thus be widely available to workers in the field of Na,K-ATPase with minimal difficulty in its execution. The aminoethyl cellulose chromatography used here is a modification of a chromatography procedure used by Nakao et al. (5). Under their conditions of chromatography, Nakao et al. (5,ll) obtained three peaks of Na,K-ATPase ((.u, /I, and y peaks). They claimed that the glycoprotein subunit was absent from the enzyme in their /3 peak. This is the only report in the literature of a purified Na,K-ATPase which does not contain the glycoprotein (l-4,810). We repeated the method of Nakao et al. (4) with the rectal gland and the electric organ. We observed the usual proportion of glycoprotein in all of the Na,K-ATPase peaks eluting from the column; the glycoprotein could not be separated from the Na.K-ATPase after ultrafiltration with a Diaflo XM-300 membrane, as reported by Nakao et a/. (11). REFERENCES 1. Uesugi. S., Dulak. N. C.. Dixon, J. F.. Hexum. T. D., Dahl, J. L., Perdue, J. F., and Hokin, L. E. (1971)J. Bio/. Chem. 246, 531-543. 2. Hokin. L. E., Dahl, J. L., Deupree, J. D., Dixon, J. F., Hackney. J. F., and Perdue, J. F. (1973) J. Biol. Chem. 248, 2593-2605. 3. Dixon, J. F. and Hokin, L. E. (1974) Arch. Biochem. Biophys. 163, 749-758. 4. Perrone. J. R., Hackney, J. F., Dixon. J. F.. and Hokin, L. E. (1975) J. Bio/. Chem. 250, 4178-4184. 5. Nakao, T., Nakao, M., Nagai. F., Kawai. K., Fujihara, U., Hara, Y., and Fujita, M. (1973) J. Biochem. 73, 781-791. 6. Bligh, E. G. and Dyer, W. J. (1959) Canad. J. Biochem. Physiol. 37, 91 I. 7. Garewal, H. S. (1973) Anal. Biochem. 54, 319-324. 8. Kyte, J. (1971)/. Biol. Chem. 246, 4156-4165. 9. Lane, K.. Copenhaver, J. H.. Lindenmayer, G. E., and Schwartz, A. (1973) J. Biol. Chem. 248, 7197-7200. 10. Jorgensen. P. L. (1974) Biochim. Biophys. Acta 356, 36-Q. 11. Nakao. M., Nakao, T.. Hara. Y., Nagai, F.. Yagasaki, S., Koi, M.. Nakagawa,A., and Kawai, K. (1974) Ann. N. Y. Acud. Sci. 242, 24-33.
BIOCHEMISTRY
86,
378-385 (1978)
A Simple Procedure for the Preparation of Highly Purified (Sodium + Potassium) Adenosinetriphosphatase from the Rectal Salt Gland of Squalus acanthias and the Electric Organ of Electrophorus electricus JOHN F. DIXON Department
of Pharmacology, Madison.
AND LOWELL
E. HOKIN
University of Wisconsin Wisconsin 53706
Medical
School,
Received April 6, 1977; accepted December I, I977 A simple purification procedure for the Na,K-ATPase from membranes of the rectal gland of Squalus acanthias or crude microsomal fractions from the electric organ of Hectrophorus electricus is presented here. The purification procedure consists of solubilization of the Na,K-ATPase with the nonionic detergent, Lubrol WX, chromatography of the diluted Lubrol extract on aminoethyl cellulose, and ammonium sulfate fractionation (1) of the concentrated eluate from the aminoethyl cellulose column. The yields of final purified enzyme are comparable to the earlier purification (l-4) involving the expensive and cumbersome zonal centrifugation step. The purity of the final enzyme. as attested to by specific activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is as great or greater than that previously reported for the enzyme purified by the procedure involving zonal centrifugation. The simplicity of the present procedure, coupled with the ready commercial availability of electric eels which are quite hardy on shipment. makes purification of the Na,K-ATPase widely available to workers in the field.
A purification scheme has been developed in this Laboratory which leads to Na,K-ATPase2 (E.C. 3.6.1.3) preparations of 50, 95, and 95% homogeneity from beef brain (l), rectal salt gland of Squafus acanrhias (2), and the electric organ of Electruphorus electricus (3,4), respectively. The purification procedure consists of solubilization of membrane fractions with the nonionic detergent, Lubrol WX. centrifugation in a continuous sucrose gradient in a zonal rotor, and a novel ammonium sulfate fractionation procedure. The procedure has the virtue of very high yields [up to 100 mg per preparation (4)] and no denaturation during purification. Its main drawback is the rather expensive and cumbersome zonal ’ This work was supported by grants from the National Institutes of Health (HL 16318) and the National Science Foundation (PCM 76-20602). 2 Abbreviations used: Na,K-ATPase. (sodium + potassium)-activated adenosine triphosphatase; SDS. sodium dodecyl sulfate. 0003-2697/78/0862-0378$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
378
SIMPLE PURIFICATION
OF Na,K-ATPase
379
centrifugation step. We present here a modified purification procedure in which chromatography of the Lubrol extract on aminoethyl cellulose (5) takes the place of zonal centrifugation. The yields and specific activity of enzyme (based on original starting material) and SDS-polyacrylamide gel patterns are essentially the same or better than we previously reported for the procedure involving zonal centrifugation. Since live electric eels are commercially available and ship quite well, purification from this source by the method described here makes available a simple purification procedure for an important membrane enzyme from a readily available source. In this paper we show data on the purification by this new procedure of the rectal gland enzyme and the electric organ enzyme with essentially identical results. MATERIALS
AND METHODS
Materials. Aminoethyl cellulose was obtained from Accurate Chemical and Scientific Corp., Hicksville, New York. Lubrol WX was obtained from Supelco, Inc., Bellefonte, Pennsylvania. Disodium ATP was obtained from P-L Biochemicals, Milwaukee, Wisconsin. Sodium Verona1 was obtained from Fisher Scientific Company, Chicago, Illinois. Solubilizution of Na,K-ATPase. Main organ electroplax tissue was dissected from cross-sectioned slices of the tails of electric eels immediately after sacrifice (3) and stored indefinitely at -70°C. These slices were used in the preparation of crude microsomal pellets (4). After overnight storage at 4°C the pellets were combined in a small beaker. Sixteen percent Lubrol WX, 0.1 M Na,ATP (pH 7.0), and water (so as to make the final Lubrol concentration 3.2% and the final Na,ATP concentration 1 mM) were pipetted directly onto the pellets. The pellets were broken up so as to form a fibrous, pastelike suspension. The suspension was further mixed by two up and down strokes in a glass homogenizer. All of the mixture was transferred into IO-ml thick wall uncapped centrifuge tubes and centrifuged for 30 min at 105,OOOg. The supernatant was further clarified by two 60-min centrifugations at the same force. The clarified supernatant was the eel Lubrol extract. Crude membrane fractions from shark rectal glands were prepared as before (2) with the following modifications. The pH of the initial sucrose buffer was lowered to 8.0. The loose white top layer of the 19,600g pellet was centrifuged without dilution for 60 min at 78,500g. The resultant pellets were homogenized in 1 mM Tris-EDTA (pH 7.0) at a protein concentration of 20 mg of protein/ml, and the homogenized membranes were stored indefinitely at -70°C. Preparative to chromatography the crude membrane fraction was thawed and homogenized in 1.6% Lubrol at 7 mg of protein/ml.
380
DIXON
AND
HOKIN
After a 15min incubation on ice the suspension was centrifuged for 60 min at 105,OOOg. The clarified supernatant was the rectal gland Lubrol extract. Chromatography
of Na,K-ATPase
on aminoethyl
cellulose
columns.
This was similar to the procedure of Nakao et al. (5), except that the Lubrol was washed from the column at low ionic strength followed by elution of the Na,K-ATPase at high ionic strength, while Nakao et al. (5) eluted the Lubrol and the Na,K-ATPase off the column together at the higher ionic strength. The columns were prepared as follows. Thirty grams of aminoethyl cellulose powder were stirred into 500 ml of 0.5 N NaOH. The suspension was filtered by suction over filter paper in a Buchner funnel. The filter cake was treated with another 500 ml of 0.5 N NaOH, filtered, and washed repeatedly by suspension in water until the filtrate was between pH 7 and 8. Similar treatment with 0.5 N HCl was followed by washing until the pH was 5. Fine particles were removed by decantation in several water washes. For small-scale purification the desired amount of aminoethyl cellulose was poured from a 50% slurry at 4°C into a 2.5 (i.d.) x loo-cm column, the hydrostatic pressure being gradually increased to 20 to 30 cm. The column was equilibrated with “wash buffer” consisting of 14 mM sodium Verona1 acetate buffer, pH 6.6, and 1 mM Na,EDTA. The Lubrol extract was diluted with wash buffer so that the final Lubrol concentration was 0.4% (i.e., 1 vol of eel Lubrol extract + 7 vol of wash buffer or 1 vol of dogfish Lubrol extract + 3 vol of wash buffer). A volume of diluted Lubrol extract equal to or less than the void volume of the column was applied to the column. A flow rate of 0.5 to 2 ml/min was maintained throughout the column run. After one void volume had passed through, the column was further eluted with 1 column of wash buffer. One and one-half column volumes of wash buffer containing 100 mM NaCl and 1 mM Na,ATP were then passed through the column. The single protein peak eluting approximately 1 void volume after the addition of this solution (see Fig. 1) was pooled and concentrated to a protein concentration of 5 mg/ml or more, using a Diaflo ultrafiltration cell with an XM-50 membrane. The concentrate was further purified by the ammonium sulfate fractionation procedure of Uesugi et al. (1). When large bed volumes were used (150 to 300 ml) it was necessary to use a peristaltic pump at the outlet to maintain and increase the flow rate (2 ml/min). The cellulose was regenerated on the large columns with approximately one bed volume of 0.5 N NaOH, a water wash (until the pH was 8.0), followed by washing with approximately one bed volume of 0.5 N HCl and a water wash to pH 5.0. The aminoethyl cellulose was not in contact with the acid or base for more than 2 hr. Determination of Lubrol. Lubrol was extracted into chloroform by the extraction procedure of Bligh and Dyer (6). The chloroform extract was taken to dryness under a stream of nitrogen gas, redissolved in 50% ethanol, and assayed by the method of Garewal (7). There is no interference by phospholipid with this method.
SIMPLE
PURIFICATION
381
OF Na,K-ATPase
Av S.A. 720 u/mg
! ! Av S.A. Zero u/mg
Av S.A. 700dmg
x B
H
”
P .
2 6 z”
1z
\
50
I
I Fraction Number
I50
260
FIG. 1. Eighteen milliliters of Lubrol extract of electric organ crude microsomal fraction were diluted to a final volume of 144 ml with wash buffer and applied to an aminoethyl cellulose column with a 1%ml void volume. See Materials and Methods for details. Six-milliliter fractions were collected from the column. This run was the same as that shown in Table 1 for the “large-scale” aminoethyl cellulose chromatography step in the full purification of the electric eel enzyme. Lubrol was qualitatively located by its formation of a precipitate in the protein assay.
RESULTS Aminoethyl
Cellulose Chromatography of Lubrol Extracts.
When a diluted Lubrol extract containing the Na,K-ATPase from either the rectal gland or the electric organ was applied to aminoethyl cellulose as described under Materials and Methods 95% of the protein bound to the column. A band of free Lubrol equal in volume to that of the applied sample eluted without retardation as seen in Figs. 1 and 2. Five percent of the applied protein eluted in the same area. The columns could be washed with many column volumes of wash buffer without further elution of protein. Forty percent of the bound protein was eluted as a sharp peak when 100 mM NaCl and 1 mM Na,ATP were added to the wash buffer. Samples from various regions of the peak had the same specific activity of Na,K-ATPase, and this was 50% higher than that of the applied sample. Ninety to ninety-five percent of the applied protein could be accounted for after elution with 0.5 N NaOH from the column. Effect of Sample Size
When the volume of the Lubrol extract applied to the aminoethyl cellulose column exceeded the void volume of the column, enzyme eluted
382
DIXON AND HOKIN C Free
Lubrol
+
++
0.81
lb
$0
Fraction Number FIG. 2. Eight milliliters of a Lubrol extract of rectal gland membranes were diluted with wash buffer to a final volume of 32 ml and applied to an aminoethyl cellulose column with a 6-ml void volume. Protein concentration and presence of Lubrol in I-ml fractions are shown.
in the free Lubrol band (Fig. 2). Under these conditions over 85% of the applied protein had eluted after application of the wash buffer. Effect of Dilution of Lubrol Extracts Undiluted extracts of enzyme from both species did not bind as well as the diluted extracts. Fifty percent of the protein of the undiluted extracts from the rectal gland eluted with the free Lubrol and 80% of the undiluted extract from the electric organ eluted with the free Lubrol. In both cases the specific activity of the unbound material was 20% higher than the specific activity of the applied sample. PuriJication
Procedure
Table I shows the purification of Na,K-ATPase from both species, utilizing aminoethyl cellulose chromatography, and compares these purifications with purification utilizing zonal centrifugation as reported previously (l-4). Ammonium sulfate treatment of the enzyme which eluted from the aminoethyl cellulose columns resulted in yields comparable to those achieved by purification involving zonal centrifugation. The specific activities of the enzymes purified with the column method were as high or higher than those purified by the zonal method. The data on purification involving zonal centrifugation are average values. The data on purification involving aminoethyl cellulose chromatography are typical values. There is some variability in the final specific activities by both methods, and specific activities as high as 2100 Fmol of Pi/mg of protein/hr have been obtained. However, the vast majority of values cluster around those shown in Table 1. Bound Lubrol The amount of Lubrol bound to the purified enzyme was the same whether the zonal method or the aminoethyl cellulose method was used.
SIMPLE
PURIFICATION TABLE
PURIFICATION
OF THE Na,K-ATPAsE
FROM
1
THE ELECTRIC
ORGAN
AND
Rectal gland
Total Na,K-ATPase (pmol/hr) Zonal Chrom. (large) (small)
THE RECTAL
GLANDS
Eel
Lubrol extract
Concentrate
(NH&SO,
75,300 87,300
54,500 60.400 8,400
50.000
15.800
383
OF Na,K-ATPase
Lubrol extract
Concentrate
(NH&SO, PPt.
6,600
382,500 100,500 30.600
150,200 55,400 19.100
121,000 47,800 15,700
PPt.
60,100
Total protein (mg) Zonal Chrom. (large) (small)
167 172 29.1
78 69.3 9.01
41 42.9 5.50
617 150 50.0
203 77.0 26.5
90.0 36.4 12.3
Specific activity (pmoli mg protein/hr) Zonal Chrom. (large) (small)
451 508 543
699 872 932
1.220 I .400 1,200
620 670 612
740 719 721
1,350 1,310 1.280
Quantity of tissue Zonal Chrom. (large) (small)
110 rectal glands I10 rectal glands I8 rectal glands
400 g of electroplax tissue 120 g of electroplax tissue 40 g of electroplax tissue
” Purification of Na,K-ATPase by the method presented in this paper is shown on two scales, and purification by the zonal method is shown on the large scale which has been routinely used. “Concentrate” refers to a concentrate of the peak eluting from the aminoethyl cellulose column after application of 100 mM NaCl and 1 mM Na,EDTA in Verona1 buffer or to a concentrate of the Na,K-ATPase peak from zonal centrifugation. as reported previously (l-3).
Three-tenths milligram of Lubrol was bound per milligram of rectal gland enzyme protein and 0.37 mg of Lubrol was bound per milligram of eel enzyme protein with either method of purification. This is comparable to the amount of Lubrol bound to the partially purified beef brain enzyme previously reported (1). Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis
Equal amounts of protein from the three stages of purification were analyzed by SDS-gel electrophoresis (Fig. 3). Increase in purity is indicated by the disappearance of bands other than the two subunit bands of the Na,K-ATPase and the increase in size of these latter bands. By this criterion the purity of the enzyme prepared by the present procedure is comparable to that of the enzymes prepared by the zonal method. It should be noted that a small amount of protein ran in the position of the tracking
384
DIXON
I
AND HOKIN
2
3
3. Ten micrograms of (a) an eel Lubrol extract, (b) the eluate from an aminoethyl cellulose column, or (c) an ammonium sulfate precipitate of the column eluate were dissolved in SDS and submitted to analytical SDS-polyacrylamide gel electrophoresis, and the gels were stained with Coomassie blue, as described previously (2). FIG.
dye. This band accounted for less than 5% of the protein applied to the gel. It probably corresponds to a band previously seen running just above the tracking dye position (2). DISCUSSION
Highly purified preparations of Na,K-ATPase have been obtained from the mammalian kidney #-lo), the rectal salt gland of Squalus acanfhias (2), and the electric organ ofElectrophorus electricus (3-4). The principle of purification of the kidney enzyme involves what may be termed purification by selective extraction of impurities, i.e., protein impurities are extracted with anionic detergents from membranes from the outer renal medulla of the kidney and the enzyme is further purified by various centrifugation techniques, and in some cases other fractionation techniques, leaving behind a membrane skeleton which contains the Na,K-ATPase as essentially its only protein. The advantage of this technique is that mammalian kidneys are readily available. The disadvantages of this technique are that dissection of highly active outer renal medulla fractions is difficult and tedious, the yields are often low, and in some procedures there is considerable denaturation of the Na,K-ATPase during purification. The principle which we have employed for purification of the fish enzymes in the past involves what may be termed purijication
SIMPLE
PURIFICATION
OF Na.K-ATPase
385
after solubilization, i.e., the Na,K-ATPase is solubilized with the nonionic detergent, Lubrol WX, and the solubilized enzyme is purified by zonal centrifugation and a novel ammonium sulfate fractionation scheme. The advantages of this technique are that the yields are high and there is no denaturation during purification. The disadvantages of this technique are that zonal centrifugation is expensive and cumbersome, and rectal glands from Squalus acanthias are not readily available. The method presented here appears to maximize the advantages and to minimize the disadvantages. Electric eels are readily available commercially and are quite hardy on shipment. The aminoethyl cellulose chromatography step can be substituted for the zonal centrifugation step, so that high yields and as high or higher specific activities of the final purified Na,K-ATPase and equally good gel patterns are obtained with no attendant denaturation during purification. The procedure presented here should thus be widely available to workers in the field of Na,K-ATPase with minimal difficulty in its execution. The aminoethyl cellulose chromatography used here is a modification of a chromatography procedure used by Nakao et al. (5). Under their conditions of chromatography, Nakao et al. (5,ll) obtained three peaks of Na,K-ATPase ((.u, /I, and y peaks). They claimed that the glycoprotein subunit was absent from the enzyme in their /3 peak. This is the only report in the literature of a purified Na,K-ATPase which does not contain the glycoprotein (l-4,810). We repeated the method of Nakao et al. (4) with the rectal gland and the electric organ. We observed the usual proportion of glycoprotein in all of the Na,K-ATPase peaks eluting from the column; the glycoprotein could not be separated from the Na.K-ATPase after ultrafiltration with a Diaflo XM-300 membrane, as reported by Nakao et a/. (11). REFERENCES 1. Uesugi. S., Dulak. N. C.. Dixon, J. F.. Hexum. T. D., Dahl, J. L., Perdue, J. F., and Hokin, L. E. (1971)J. Bio/. Chem. 246, 531-543. 2. Hokin. L. E., Dahl, J. L., Deupree, J. D., Dixon, J. F., Hackney. J. F., and Perdue, J. F. (1973) J. Biol. Chem. 248, 2593-2605. 3. Dixon, J. F. and Hokin, L. E. (1974) Arch. Biochem. Biophys. 163, 749-758. 4. Perrone. J. R., Hackney, J. F., Dixon. J. F.. and Hokin, L. E. (1975) J. Bio/. Chem. 250, 4178-4184. 5. Nakao, T., Nakao, M., Nagai. F., Kawai. K., Fujihara, U., Hara, Y., and Fujita, M. (1973) J. Biochem. 73, 781-791. 6. Bligh, E. G. and Dyer, W. J. (1959) Canad. J. Biochem. Physiol. 37, 91 I. 7. Garewal, H. S. (1973) Anal. Biochem. 54, 319-324. 8. Kyte, J. (1971)/. Biol. Chem. 246, 4156-4165. 9. Lane, K.. Copenhaver, J. H.. Lindenmayer, G. E., and Schwartz, A. (1973) J. Biol. Chem. 248, 7197-7200. 10. Jorgensen. P. L. (1974) Biochim. Biophys. Acta 356, 36-Q. 11. Nakao. M., Nakao, T.. Hara. Y., Nagai, F.. Yagasaki, S., Koi, M.. Nakagawa,A., and Kawai, K. (1974) Ann. N. Y. Acud. Sci. 242, 24-33.
