Purification and Characterization of a High Molecular Weight Type 1

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 262, No. 5, Issue of February 15, pp. 20162024,1987 Printed in U.S.A.

0 1987 by The American Society of Biological Chemists, Inc.

Purification and Characterizationof a High Molecular Weight Type1 Phosphoprotein Phosphatase fromthe Human Erythrocyte* (Received for publication, July 15, 1986)

Peter A. Kienerl, Dennis Carroll, BernardJ. Roth, and EdwardW. Westhead From the Department of Biochemistry, University of Massachusetts, Amherst, Massachusetts 01003

The major Mn2+-activatedphosphoprotein phosphatase of the human erythrocyte has been purified to homogeneity from the cell hemolysate. It is sensitive to both inhibitors 1 and 2 of rabbit skeletal muscle, preferentially dephosphorylates the @ subunit of the phosphorylase kinase, and dephosphorylates a broad range of substrates including phosphorylase a,p-nitrophenyl phosphate, phosphocasein, the regulatory subunit of cyclic AMP-dependent protein kinase, and both = 10 PM) and pyruvate kinase ( K , = 18 spectrin (K,,, MM)purified from the human erythrocyte. The purified enzyme is stimulatedby Mn2+and to a lesser extentby higher concentrationsof M$+. The purification procedure was selected to avoid any change in molecular weight, hence subunit composition, between the crude and purified enzyme. Maintenance of the originalstructure isdemonstrated bynondenaturing gel electrophoresis and gel filtration chromatography. Gel filtration of the purified holoenzyme shows 5 single activecomponent with a Stokes radius of 58 A at a molecular weight position of 180,000. Sedimentation velocity in a glycerol gradient gives a value of 6.1 for s20,w. Together these data indicate a molecular weight of about 135,000. Two bands of equal intensity appear on sodium dodecyl sulfate-gel electrophoresis at molecular weights of 61,700 and 36,300, suggesting a subunit composition of two 36,000 and one 62,000 subunits. The 36-kDa catalytic subunit can be isolated by freezing and thawing the holoenzyme or by hydrophobic chromatography of the holoenzyme. The catalytic subunit shows unchanged substrate and inhibitor specificity but altered metal ion activation.

It has been shown that several cytosolic proteins in the human erythrocyte can be phosphorylated either in vitro or in the intact cell (1, 2) and we have proposed that this may be a mechanism for regulating pyruvate kinase activity in vivo (2). There are also many reports of phosphorylation of the membrane proteins of the cell but the function of these reactions is still not clear (3). If phosphorylation is a mechanism for the regulation of either the function or metabolism of the erythrocyte, there must be a mechanism for reversing the phosphorylation of these proteins. Preliminary reports have described cytosolic phosphopro-

* This work was supported by National Science Foundation Grant DMB 8309306 and by United States Public Health Service Grant HL36704. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. $ Current address: Pharmaceutical Research and Development, Immunology Dept., Bristol Myers Co., Wallingford, CT 06492.

tein phosphatase activity capable of dephosphorylating spectrin (4,5) as well as pyruvate kinase ( 6 ) .A membrane-bound phosphoprotein phosphatase, capable of dephosphorylating membrane-associated proteins has been described by Fischer and co-workers (7). The relationship among these activities is not known. Usui and co-workers (8)have found several forms of phosphoprotein phosphatase in human erythrocyte cytosol. The phosphatases differ in their activity toward various protein substrates andin their molecular weight. Usuiand co-workers have purified 3000-fold, to apparent homogeneity, a 104 kDa phosphatase from the erythrocyte. That enzyme differs in every measured characteristic from the enzyme to be described here (see “Discussion”). Multiple forms of phosphatase activity have been purified from other tissues but it is not clear whether all the forms exist in the tissues or result from breakdown during the purification procedure. In many cases an approximately 30,000-Da subunit can be generated from a larger species by treating the enzyme with ethanol, acetone, or trypsin, or by freezing (9). This appears to be a catalytic subunit common to many of the phosphoprotein phosphatases (9). In the red blood cell, 3 of the 4 reported phosphatases can be converted to an active subunit of 35,000 Da. However, it remains unclear what the relationship is between this catalytic subunit and the noncatalytic peptides associated with the more complex forms of the enzyme. Several reports have suggested that the noncatalytic peptides of phosphoprotein phosphatases function as inhibitors of the enzyme (10, 11)and thatappears well established in at least one case (12). However, the extent of purification has made it uncertain asto whether the inhibitors were truly subunits of the phosphatase or co-purifying proteins. In thispaper we describe the purification and properties of the major Mn2+-activatedphosphoprotein phosphatase in the human erythrocyte which is also the highest molecular weight phosphatase in that cell. The purification procedures reported here were developed to maintain the molecular weight form of the enzyme found in the initial hemolysate as aprerequisite for more detailed studies on the subunit structure and functions. MATERIALS AND METHODS

Ultrogel AcA 34 and Ampholine PAG plates were obtained from LKB, Sephadex G-200, DEAE A-50, and Sephacryls were obtained from Pharmacia P-L Biochemicals, and CM Bio-Gel and Chelex 100 were obtained from Bio-Rad. All other materials were obtained from sources described previously (6). Bujjers-The following buffers were routinely used in theseexperiments: buffer A, 45 mM Hepes,’ 100 mM KCl, 5.7 mMMgC12, 1 mM

* The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; SDS, sodium dodecyl sulfate.

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Erythrocyte Phosphoprotein Phosphatase EDTA, pH 7.4; buffer B, 45 mM Hepes, 100 mM KCl, 14 mM mercaptoethanol, pH 7.4, at 4 "C; buffer C is buffer B + 1mM MnC12; buffer D is buffer B + 0.5 mM MnCL Substrates-Erythrocyte pyruvate kinase was prepared and assayed as described previously (2). Casein was dephosphorylated by the method described by Reimann et al. (13). Spectrin was prepared essentially as described by Gratzer (14) and stored at 4 "C in 0.3 mM phosphate, 0.1 mM EDTA, 0.1 mM EGTA, 10 p~ sodium azide, pH 8.0. Prior to phosphorylation the solution was dialyzed against a buffer containing 20 mM Tris, 50 mM KC1, 1 mM MgC12, pH 7.4. SDS-gel electrophoresis was routinely used to ensure that therewas no breakdown of the two spectrin subunitsinto smaller fragments. Pyruvate kinase (5-10 mg/ml), spectrin (5-10 mg/ml), and casein (about 10 mg/ml) were phosphorylated by incubating the proteins , pM units of protein with 250-500 pCi of [y3'P]ATP (25-50 p ~ )600 kinase, and 50 p~ CAMPin buffer A for 3-5 h a t room temperature. Excess label was removed by precipitating casein with 10% trichloroacetic acid and pyruvate kinase and spectrin with 50% ammonium sulfate; the proteins were redissolved and dialyzed against buffer B until no radioactivity could be detected in the dialysate. Casein and pyruvate kinase were stored frozen, spectrin was stored at 4 ' C in buffer containing 10 p~ sodium azide. Phosphorylase kinase (15), inhibitor 1 (16), and inhibitor 2 (17) were purified from rabbit skeletalmuscle. An initial sample of inhibitor 2 was kindly donated by Dr. DavidBrautigan (Brown University). Phosphorylase b was purchased from Sigma, 32P-labeledphosphorylase a was prepared from phosphorylase busing Sigma grade phosphorylase kinase and [32P]ATP (18, 19). Proteinphosphatase 1 was purified from rabbit skeletal muscle (20). 3ZP-Labeledphosphorylase kinase was prepared using cyclic AMP-dependent protein kinase purchased from Sigma and [y-32P]ATP (15, 19). The product contained approximately equal 3zPlabel in the CY and @ subunits as determined by densiometric scans of SDS-gel autoradiographs. Phosphatase Assays-p-Nitrophenylphosphatase activity was routinely measured in buffer C by following the increase in absorbance a t 400 nm at 30 "C. The substrate concentration was 10 mM; any variations from these conditions aredetailed in the text. One unit of activity is defined as the enzyme which will hydrolyze 1 nmol of pnitrophenyl phosphate/min. Unless otherwise stated, all phosphoprotein phosphatase activity was measured by following release of [32P]phosphatefrom the proteins. The labeled proteins (50,000-200,000 cpm, about 0.2 mg of protein in 0.5 ml) were incubated with the phosphatase in buffer C (except where stated) a t 30 "C and samples withdrawn and pipetted into 200 pl of 20 mg/ml bovine serum albumin; this mixture was then precipitated by the addition of 750 pl of 10%trichloroacetic acid; 750 pl of the supernatant was then added to 5 ml of ScintiVerse and counted in a Beckman LSlOO scintillation counter. Proteinconcentration was determined by Coomassie Blue dye binding (21). SDS-gel electrophoresis was done according to the method of Laemmli (22). Discontinuous acrylamide gels (5-15% gradient) were run and calibrated with standards: phosphorylase, bovine serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, and lysozyme. Gel isoelectric focusing was done with prepoured Ampholine PAG plates on an LKl3 Multiphor. Nondenaturing polyacrylamide gel electrophoresis was carried out essentially as described by Shuster (23); 5% stacking gels, pH 6.8, and 6% gels, pH 8.8, were used with a running buffer of pH 8.3. Apparent molecular weights (and Stokes radii) were determined by column gel filtration (1.7 X 100 cm) using Ultrogel AcA34 or Sephacryl S200 or by thin layer gel chromatography (Pharmacia PL Biochemicals) using a Sephadex G-200 superfine bed 1-mm thick. Ferritin, pyruvate kinase, y-globulin, hemoglobin, and carbonic anhydrase were used as standard molecular weight and Stokes radius markers. Rabbit Muscle Inhibitor Assay-Activity of holoenzyme and catalytic subunit was assayed in the presence or absence of rabbit muscle inhibitor 1or 2 in buffer C. Assays were against either [32P]phosphorylase a or [32P]phosphocasein(100,000 cpm/l0 pl); incubations were at 30 "C for 30 min. Phosphatase activity was measured by following the increase in acid soluble radioactivity. Phosphorylase Kinase: a and /3 Specificity-Specificity of the holoenzyme and the36,300-Da catalytic subunit for the a and @ subunits of [3ZP]phosphorylasekinase was determined by densitometric scans of SDS gel autoradiographs. The reaction mixture (200 pl) included [32P]phosphorylasekinase, 150 pl of buffer C, and either the holoenzyme or the catalytic subunit. Incubations were at 30 "C for 30 min

2017

and stopped by acid precipitation as previously described. Preparation of Cytosol-For the survey of the phosphatase population in erythrocytecytosol, 200 mlof blood was drawn from human male adults into a Na+:heparin anticoagulant. All subsequent steps were carried out a t 4 'C in thepresence of 100 qM phenylmethylsulfonyl fluoride and 14 mM mercaptoethanol. The wholebloodwas centrifuged at 2,000 X g for 10 min at 4 "C and washed 3 X with isotonic NaCl at pH 7.0. The plasma and buffy layer were carefully removed by aspiration. The cells were then lysed in about 4 times their packed volume of 10 mM Tris buffer at pH7.0. The membranes were immediately sedimented for 45 min at 24,000 X g, resuspended in the same volume of fresh lysis buffer, and resedimented. For purification and characterization of the major Mn-activated protein phosphatase, cytosol was prepared as above except that the starting material was 1500 mlof blood obtained from a local hospital. During the early stages of development of the purification procedure it was found that enzymatic activity was sensitive to both oxidation and proteolysis, especially in the absence of Mn2+,so dithioerythritol or mercaptoethanol was added (to about 10 mM) at all stages of the purification and 100 p~ phenylmethylsulfonyl fluoride was added to the hemolysate. Initial attempts a t purification in the presence of M%+ or without metal, resulted in lower yields of enzymatic activity: Mn2+protected the enzyme and caused no change in its molecular weight properties so MnCll was included in all except the lysis buffers. The purified enzyme in solution tends to spontaneously degrade to the 36.3-kDa active form unless stabilized with glycerol (this will be discussed later), so purified samples of enzyme contained 10% glycerol. CM-Cellulose and Ultragel Chromatography-This procedure was used specifically in the gel filtration survey of the phosphatase activities in the cytosol. The isolated cytosol was adjusted to pH 5.5 and its conductivity lowered to less than 1 mmho via dialysis against a 10-fold excess solution of 20 mM Tris succinate, pH 5.5, containing either 1 mM Mn2+ or 5.7 mM M$+, and passed through a preequilibrated column of carboxymethyl cellulose (CM-cellulose) to remove the hemoglobin. The eluant was concentrated to 2 ml on an Amicon ultrafiltration cell equipped with a YM-10 filter membrane and dialzyed against buffer A or buffer C in preparation for gel filtration chromatography. Details of the gel filtration are given in the legend to Fig. 1. Ammonium Sulfate Fractionation-The lysate was brought to 50% saturation of ammonium sulfate by the addition of the solid salt; the pH was maintained at 7.5. After 60 min at 4 "C the suspension was centrifuged for 30 min at 24,000 X g and thepellet redissolved in 200 ml of buffer C. This solution was made 20% saturated with ammonium sulfate, adjusted by conductivity measurement, left for 1 h a t 4 "C and then centrifuged as before. The supernatant was removed and made 45% saturated by the addition of solid ammonium sulfate; the pH was maintained at 7.4. The suspension was left overnight at 4 "C. Zon-exchange Chromatography-In these stages all the buffers were 20 mM succinate containing 1mM MnC12, adjusted to theappropriate pH by addition of solid Tris base. The 45% ammonium sulfate precipitate was dissolved in succinate buffer, pH 5.5, and dialyzed against this buffer until the conductivity of the solution was less than 1mmho. The suspension was centrifuged for 20 min at 30,000 X g to remove the precipitated protein and the supernatantpassed through a CM Bio-Gel A column (2.5 X 10 cm) equilibrated with succinate buffer, pH 5.5. The percolate from this column together with the first washing of 50 ml of succinate buffer, pH 5.5, was raised to pH 8.0 by the addition of solid Tris, and thissolution passed through a DEAEA50 column (2.5 X 10 cm), previously equilibrated with 20 mM Tris succinate, pH 8.0. The column was washed with 50 mlof Tris succinate, pH 8.0, followed by 500ml of Tris succinate, pH 8.0, containing 0.1 M KC1. The phosphatase was eluted by applying a 500ml salt gradient, 0.1-1.0 M KCl, 3-ml fractions were collected and the pooled active fractions brought to 50% saturation of ammonium sulfate. Nondenaturing Gel Electrophoresis-The ammonium sulfate suspension of enzyme from the DEAE pH 8 column was centrifuged and dissolved in 1 ml of nondenaturing sample buffer (50 mM Tris-C1,20 mM mercaptoethanol, 10% glycerol pH 6.8), centrifuged to remove any undissolved particles, and then loaded onto two 10 X 14-cm nondenaturing polyacrylamide gels (6%). The gelswere run at a constant current of 30 mA/geluntil the bromphenol blue dye reached the bottom; the gels were stained for activity against p-nitrophenyl phosphate; within 1 min a major active band was visible and this was cut out of the gel. The enzyme was electroeluted from the gel into 20

2018

Phosphatase Phosphoprotein Erythrocyte

mM Tris, 50 mM KCl, 20 mM mercaptoethanol, 5% glycerol, 250 p M MnC12,pH 7.4, at 4 "C, at 150 V for 10 h (until no activity could be detected in the compartment containing the sliced gel). The enzyme solution was made 50%saturated in ammonium sulfate by adding the solid salt. Gel Filtration-The ammonium sulfate suspension of protein from the nondenaturing gelwas centrifuged and dissolved in 0.5 ml of buffer D. This solution was applied to an Ultrogel AcA34 column (1.5 X 90 cm) and the protein eluted with buffer D; 1.2-ml samples were collected. Fractions possessing phosphatase activity of greater than 50% of the peak activity were pooled and made 50% saturated in ammonium sulfate. Freezing and Thawing of t k Phosphatase-The ammonium sulfate suspension of the phosphatase from the gel filtration column was centrifuged and the pellet redissolved at a concentration of about 1 mg/ml protein in buffer B containing 15 mM mercaptoethanol. The solution was frozen on dry ice for 5 min and thesample, still frozen, centrifuged at 12,000 X g for 1min at room temperature (Eppendorf Microfuge); during this time the samples melted. The supernatant was removed and assayed for activity and protein concentration and the freeze-thaw cycle repeated. Octyl-SephcrroseChrumutography-The50% ammonium sulfate suspension of the phosphoprotein phosphatase from the gel filtration column was diluted with buffer C to a find concentration of 1 M ammonium sulfate. The solution was passed through an octyl-Sepharose column (1 X 12 cm) previously equilibrated with buffer C containing 1 M ammonium sulfate, pH 7.4, at 4 "C. The column was eluted with an ammonium sulfate gradient (2 X 200 ml; 1-0 M) in 5 mM Hepes, 1 mM Mn2+,pH 7.4; 3-ml fractions were collected and assayed for phosphataseactivity. Sedimentation Velocity Measurements-These measurements were made in linear glycerol gradients with a density range of 1.01-1.07 which had the salt and buffer compositions of buffer A. Mixtures of phosphatase with standards were layered on the gradients which were then centrifuged from between 14 and 16 h at 40,000 or 50,000rpm in Beckman SW56 or SW60.1 rotors at 4 "C. Gradients were tapped by upward displacement and the 20-26 fractions were analyzed by enzyme activity and by absorbance at 420 nm for hemoglobin. Considerations cited by Martin and Ames (24) were taken into account. As internalstandards we used hemoglobin, lactic dehydrogenase, yeast alcohol dehydrogenase, and catalase with s~,,,~values of 4.3,7.3, 10.6, and 11.3, respectively. Three different preparations of phosphatase were examined and on the last occasion an aliquot of the same phosphatase was simultaneously chromatographed on an Ultrogel AcA34 column to establish that its gel permeation behavior was exactly as expected from previous measurements which had given a molecular weight relative to standards of 180,000.

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FRACTION NUMBER

FIG.1. Metal ion activation of cytosolic protein phosphaAn Ultrogel AcA 34 column, 1.6 tases separated by gel filtration. X 100 cm, was eluted at a rate of 8 ml/h and fractions of 1 ml were collected. Protein samples and columns were equilibrated with buffer A or C. Apparent molecular weights were determined by references to standards listed under "Materials and Methods." 0, activated by 1 mM Mn2+;0,activated by 5.7 mM M e .

at all of the states of purification Mn2+-dependentphosphospectrin, phosphocasein, and phosphopyruvate kinase phosphatase activities were coincident with Mn2+-dependentpnitrophenylphosphatase activity. Activity against p-nitrophenyl phosphate and phosphospectrin was proportional to the amount of enzyme added over a 10-fold range of concenRESULTS tration, and theratio of p-nitrophenylphosphataseactivity to Metal Actiuation Profile of Cytosolic Phosphatase-A survey spectrin phosphatase activity remained constant. Activity of the different populations of phosphatase found in the against casein and phosphorylase was not so simple; at higher human red blood cell cytosol is shown in Fig. 1;the forms can concentrations of enzyme, enzymatic activity was not directly be distinguished by apparent molecular weight and differences proportional to enzyme concentration but showed a relative in metal activation. The broken curue represents the activa- increase in activity as theprotein was diluted. tion of the different molecular weight forms by Mn2+and the The overall purification is given in Table I. A purification solid curue represents Mg2+-stimulated activity. The major of about 300,000-foldwith a yield of up to 53% can be achieved Mn2+-activatedpeak is found at anequivalent sphere position routinely. Often during the preparation procedure there was of 180,000 Daand constitutes 60% of all phosphataseactivity: a significant increase in overall phosphatase activity comin the presence of Mg2+ this enzyme constitutes only 20% of pared with that seen in the 20-45% ammonium sulfate fracthe total activity. Overall Mn2+activation is seen to be more tion. This did not consistently occur at a particular point of than 30-fold greater than M$+ activation, The phosphatase the purification procedure and therewas no apparent change we discuss in this paper is the form labeled 180,000 Dain Fig. in thesubunit structureof the enzyme. L2 Molecular Weight-Gel filtration of the 25-45% ammonium Characterization of the Purified Holoenzyme and Its Cata- sulfate fraction of the hemolysate and the purified enzyme lytic Subunit-The purpose of the adopted procedures was to showed one major peak corresponding to a relative molecular purify the major phosphoprotein phosphatase activity without weight of 180,000 based on simple comparison to standard changing the molecular weight of the enzyme. For conven- proteins (Fig. 2, A and B). Sedimentation rates in a glycerol ience, enzymatic activity was routinely followed by assaying gradient, however, indicated a much lower molecular weight. Mn2+-dependenthydrolysis of p-nitrophenyl phosphate but Four samples of three preparationsof the phosphatase showed that thephosphatase sedimented behind lactic dehydrogenase Although the data in this paper demonstratethat the true molec(Mr = 140,000). Based on interpolation on a curve of sediular weight is near 135,000 we have retained the gel permeation mentation distance versus sz0,,,, for the standardproteins, the molecular weight in the figure for comparison with the elution data s20,wfor the phosphatase is 6.1 f 0.2. If we calculate from of others, especially those of Usui et al. (8).

Erythrocyte Phosphoprotein Phosphatase

2019

TABLEI Phosphoproteinphosphatasepurification Sample

Enzymatic activitv

Protein

Specific activitv

units X I@"

mg

nmollminlmg

Purification

0.134 1 132 X 103 Hemolysate 17.6 100 17.7 850 150 20.8 2045% (NHJZSOI 452 60.6 40.3 670 After dialysis 1,090 146 28.0 190 CM percolate 14,230 38.3 20.0 1,907 DEAE pool 68.020 12.0 1.32 9,115 Electroeluted enzyme Gel filtration pooi 0.21 9.4 44,600 332j840 Activity against p-nitrophenyl phosphate assay condition as described under"Materialsand Methods."

I 00 100

1

Yield

100 228 158 216 68 53

6

ao 60 2, c

.-

.? +

40

0

a

20

E 3

.E x

100

0

ao +

c a 0 L

60 10

a

a

20

30

40

FRACTION NUMBER

40

20

40

60

ao

100 120

60

80

100

120

FIG.3. Ion-exchange chromatography of the phosphoprotein phosphatase (prepared as described in the text). DEAEA50 equilibrated with 20 mM Tris succinate, 1 mM MnC12, pH 8.0, eluted with 0.09-1 M KC1 gradient (500 ml). +,p-nitrophenylphosphatase activity; 0, spectrin phosphatase activity; 0, absorbance at 280 nm; 0, KC1 concentrations.

F r o c t i oNnu m b e r

weight forms of the enzyme as detected by gel filtration (Fig. 2C). All show Mn2'-simulated activity against both phosphocasein andp-nitrophenyl phosphate. After three or four cycles of freezing and thawing, the protein concentration of the sample dropped due to precipitation. Gel filtration of the supernatant stillshowed a major peak of activity at anequivalent sphere molecular weight of about 180,000 but a minor band of lower molecular weight was also visible (Fig. 2C). If these freeze-thaw cycleswere continued more protein was removed; gel filtration of the enzyme after these freezing and the gel filtration elution volume compared with the standard thawing cycles showedthat nearly all the activity eluted in a proteins and assume a v of 0.725 we get a molecular weight peak corresponding to an apparentmolecular weight of about estimate of 135,000, substantially lower than the 180,000 36,000 (Fig. 20). The transformation to the lower molecular based on gel filtration alone. The elution of the phosphatase weight form was greatly inhibited by the presence of 10% at theposition of a higher molecular weight equivalent sphere glycerol. The effect of freezing and thawing on the enzyme suggested suggests appreciable asymmetry inthe holoenzyme. The Stokes radius of the phosphatase holoenzyme calculated from that hydrophobic interactions may be involved in the holoenzyme organization so the enzyme was chromatographed on the gel filtration data is 58 A. If the holoenzyme was treated with acetone or ethanol, a octyl-Sepharose. Gel filtration of the enzyme eluted from the procedure frequently used to generate active subunits of pro- octyl-Sepharose column showed that the majority of the entein phosphatases, much activity was lost. This was true both zyme was converted to the 36.3 kDa form. This enzyme is after theammonium sulfate fractionand after the pH8 DEAE identical (by gel filtration) to thelower molecular weight form column (Fig. 3). After ethanol treatment of the ammonium of the frozen and thawed enzyme, which suggests that they sulfate fraction, 20% of the original activity remained; this are thesame small subunit of the enzyme. had a molecular weight of 36,300 by SDS-gel electrophoresis. After several weeks, the pure holoenzyme, kept as an amFreezing and thawing of the purified holoenzyme in thepres- monium sulfate suspension in 10% glycerol at 4 "C, degrades ence of mercaptoethanol produced several different molecular into lower molecular weight forms. This may be due to proFIG. 2. Gel filtration of phosphoprotein phosphatase preparations on Ultrogel AcA 34 (1.6X 100 cm). The samples were applied and eluted with buffer D at 4 "C.The column was calibrated with standards: ferritin, pyruvate kinase, y-globulin, hemoglobin, and carbonic anhydrase; pyruvate kinase and hemoglobin were added to each phosphatase sample as internal standards. 0,activity against pnitrophenyl phosphate; 0,activity against phosphocasein. A , 20-45% ammonium sulfate fraction of red blood cell lysate, B, purified enzyme; C, purified enzyme frozen and thawed three times; D,purified enzyme frozen and thawed seven times.

2020


teolysis from tracecontaminants or to slow spontaneous alteration of the subunit structure. In theabsence of glycerol, the enzyme degrades to the 36.3-kDa form over 24-36 h at 4 “C. Gel Electrophoresis-From the densitometric scans of purified holoenzyme run on nondenaturing gel and stained with Coomassie Blue, it is possible to estimate that the active phosphatase band representsat least 90% of the protein from the Ultrogel column (Fig. 4A). If the phosphatase preparation is either frozen and thawed or chromatographed on an octyl-Sepharose column, the active band of R F value 0.49 disappears and is replaced by one major active band of higher mobility, R F 0.68 (Fig. 4B). Denaturing polyacrylamide electrophoresis of the purified enzyme shows two bands of equal intensity with molecular weights corresponding to 61,700 and 36,300 (Fig. 5A). If the two subunits stain equally with Coomassie Blue, then the

40

30

20

IO

30

20

10

40

30

20

10

MIGRATION

-

FIG. 4. Densitometric scans of purified phosphatase run on a nondenaturing gel (details in text) and stained with Coomassie Blue. A, holoenzyme; B, after freezing and thawing 7 times and centrifuging down insoluble material.

1

MIGRATION

-

FIG. 5. Densiometric scans of purified phosphatase on a SDS denaturinggel (details in text) and stained with Coomassie Blue. A, holoenzyme; B , holoenzyme after freezing and thawing 7times and centrifuging down any insoluble material. Standard positions shown at the top of the figures are: 1, phosphorylase (94 kDa); 2, bovine serum albumin (68 kDa); 3, ovalbumin (43 kDa); 4, carbonic anhydrase (29 kDa); 5, soybean trypsin inhibitor (21.5 kDa); 6, lysozyme (14.3kDa).

IO

20

30

40

Time ( m i n ) FIG. 6. Metaldependence of phosphoproteinphosphatase activity. Hydrolysis of 32P04from: A , pyruvate kinase; B, spectrin; C, casein (prepared as described in the text).A, with no phosphatase added; 0, with phosphatase + 5 mM EDTA; 0, with phosphatase + 5.7 mM MgC12; 0, with phosphatase + 1 mM MnC12. Assayswere performed as described under “Materials and Methods”; about 5 units of phosphatase was Chelex treated and then incubated in the appropriate buffer for 5 min prior to assay.

holoenzyme structure might be two subunits of 36,300 and one of 61,700, for a molecular weight of 134,000, in agreement with calculations from sedimentation velocity and gel filtration chromatography. When the purified enzyme is frozen and thawed five times, then centrifuged to remove the insoluble material prior to SDS electrophoresis, only the 36-kDa form of the enzyme is observed (Fig. 5B). The dissociated 62kDa noncatalytic subunit precipitates out of solution under these conditions. Metal Dependence of Holoenzyme Activity-Enzymatic activity of the holoenzyme againstp-nitrophenyl phosphateand the phosphorylated proteins is verydependent on the presence of divalent metal ions. In the absence of metal ions or in the presence of 5 mM EDTA there is a low but significant level of activity (Fig. 6). Activity is increased in the presence of M e and even more in the presence of Mn2+.With pyruvate kinase and casein as substrates, Mn2+is a far better activator than M e . p-Nitrophenylphosphatase activity shows simple saturation kinetics with increasing concentrations of Mn2+;a K, for Mn2+of 80 PM was obtained (Fig. 7A). With phosphorylated casein as substratethe saturation curve doesnot show simple Michelis-Menten kinetics (Fig. 7B), from the curve a

2021


cium and calmodulin (150 pg/ml) show no activation: millimolar concentrations of zinc are inhibitory, but micromolar concentrations show neither activation nor inhibition. Lack of inhibition by micromolar Zn2+ as well as sensitivity to fluoride (Table 11) is characteristic of phosphoseryl phosphatases in contrastto phosphotyrosyl phosphatase (28). Metal Dependence of the Catalytic Subunit-As in the case of holoenzyme, the 36,300-Da catalytic subunit shows activity in the absence of added metal ion and in the presence of 5 mM EDTA, but is markedly stimulated by divalent cations (Fig. 8). The catalytic subunit differs from the holoenzyme in several respects. Upon conversion of the enzyme to the36,300Da subunit, by freezing and thawing, there is an increase in TABLE I1 Inhibition of erythrocyte phosphoprotein phosphatase Assays were carried out as described under "Materials and Methods,'' in buffer C; 6 units of phosphatase (holoenzyme) were used in each assay. The pH of the inhibitors was adjusted to 7.4 prior to assay. The hydrolysis is expressed as percent of the hydrolysis of the uninhibited substrate in 10 min.

80

% hydrolysis

60

Casein p~~~~~ Spectrin

40

20

0 200

400

3000

CMn2+1 ( p M )

FIG. 7. Saturation curves for Mn2+ activation. A , activity against p-nitrophenylphosphate. The line drawn is a theoretical one for a K,,, of 86 p ~ B., activity against phosphocasein. The rate was released in 10 min at pH7.4.30 "C. In both graphs measured as 32P04 the residual activity which is presentin theabsence of metal has been subtracted from all points. The purified phosphatase was Chelextreated prior to use; assays were done in buffer B with the indicated concentration of Mn2+and 10 mM mercaptoethanol.

No additions 10 mM KF 76 60 10 mM ATP, 10 mM Mn2+ 25 25 10 mM ADP, 10 mM Mn2+ 10 mM Pi 10 mM ATP 10 mM ADP 10 mM 2,3-bisphosphoglycerate 10 mM pyrophosphate, 10 mM Mn2+ 5 mM EDTA

100 78 65 54 22 13 17 35 70 16

100

100 25 25

5 10 46 10

11

h

z

0 Y

Koa ([Mn"] at half of the apparent V,,,) of about 24 PM was observed. After treatment of the holoenzyme with 1 mM Mn2+ and subsequent removal of metal with EDTA, the kinetic properties of the enzyme, against all the substrates, are the same as those of enzyme purified without the addition of Mn2+. The effect of Mn2+was found to be fully reversible. This is in contrast to the behavior reported on phosphatases from the rabbit muscle (13, 27). Although addition of Mn2+ to the lysate of freshly drawn blooddoes not cause a significant stimulation of activity, upon treatment of the lysate with Dowex 2, the phosphatase doesbecome characteristically activated by Mn2+. Presumably Mn2+ activation prior to Dowex 2 treatment of the lysate is obscured by the high concentration of chelating ions found in the red blood cell cytosol. In theabsence of Mn2+the enzymatic activity is very sensitive to oxidized glutathione and air and is also labile to trypsin; in the presence of MnZ+ theenzyme is much more stable (data not shown). The stimulation by M%+does not approach saturation and it is not possible t o obtain a value for K,. Compared with Mn2+, the affinity of the enzyme for M%+ is much lower. Cobalt stimulatesphosphatase activity to about the same extent as Mn2+, but this is only detectable after removal of protective sulfhydryl reagents. Calcium (0.5-3.5 mM) or cal-

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m

a -I

TIME ( m i n ) FIG. 8. The effects of metal-dependent activation of both the 180,000-Da holoenzyme (0, Mn2+; +, M d + ) and the 36,300-Da catalytic subunit (0,Mn2+; 0, M d + ) . Assays were done at 30 "C in either buffer A or buffer C. Six units of the 180,000Da purified enzyme were frozen and thawed 7 times and centrifuged to remove insoluble material, leaving only the 36,300-Da catalytic subunit. After Chelex treatment equal volumes of each were added to the appropriate buffer in the presence of 100,000 cpm/5 pl of 32Pcasein. Final assay volume was 300 p l . Activity was measured as 32P0, released in minutes.

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total Mn2+-stimulatedactivity but there is no marked change in the for Mn2+.In this respect the larger subunit would appear inhibitory. The activity of the catalytic subunit isalso more effectivelystimulated by MgZ+than is the activity of the holoenzyme; activity approaches that achieved upon Mn2+ stimulation (Fig. 8).This change may arise both from a change in the affinity of M e for the enzyme and an increase in maximum velocity upon removal of the 61,700 noncatalytic subunit. Under normal assay conditions, in the presence of MgC1, or 1 mM MnCl,, there is an &fold either 5.7 mM increase in Mg+-stimulated activity and a 2-3-fold increase in Mn2+-stimulated activity when the holoenzyme is converted to thecatalytic subunit. Substrate Profile-The high molecular weight phosphatase shows activity against a broad range of substrates. Those hydrolyzed include: p-nitrophenyl phosphate, phosphospectrin, phosphorylase, phosphorylase kinase (@ subunit), the regulatory subunit of CAMP-dependent protein kinase, phosphocasein, and phosphorylated pyruvate kinase. The enzyme shows very little activity towards simple phosphate esters otherthan p-nitrophenyl phosphate. Eleven low molecular weight phosphate esters were tested as substrates at 30 “C in buffer C at initial substrate concentrations of 1 mM. The phosphate release after 2 h incubation with 100 units of purified phosphatase was measured and compared to a control without phosphatase. The most active substrates were threonine phosphate and phosphoenolpyruvate; 13% of their phosphate was released. That is 0.3%of the rate of hydrolysis of p-nitrophenyl phosphate. Fructose 1,g-bisphosphate and serine phosphate were hydrolyzed at about half that rate. Phosphoethanolamine, phosphoglycolate, and glucose 6-phosphate were hydrolyzed at about 0.08% of the rate of p-nitrophenyl phosphate and 2,3-bisphosphoglycerate,2phosphoglycerate, ATP, and ADP were all hydrolyzed at half that rateor less. Because the degree of hydrolysis is low these rates should approximate initial rates of hydrolysis. The substrate saturation curve for activity against phosphospectrin enzyme appears to follow simple Michelis-Menten kinetics. However, it was not possible to obtain phosphorylated spectrin in sufficient concentration to observe full saturation of the enzyme in this assay system. From data obtained using up to 11 p~ protein-boundphosphate we estimated values of 10 p~ (protein bound Pi) for K,,,. Phosphorylation of spectrin yielded labeled protein containing 1-2 mol of radioactive phosphate/mole of spectrin @ subunit compared with the reported maximum of 4 phosphates/@ subunit.The less than maximal incorporation of 32P may bedue to thefact that thespectrin used was not dephosphorylated prior to phosphorylation. SDS-gel electrophoresis showed that at least 80% of the radioactive label could be attributed to spectrin labeled in the @ subunit. If we assume that the phosphatase does not distinguish among the phosphate groups on the p subunit and assuming 4 mol of Pi/mol of @ subunit, then the K,,, based on spectrin @ subunits would be of the order of 6 pM. In this laboratory pyruvate kinase has been phosphorylated to thelevel of 2-3 mol of Pi/mol of pyruvate kinase tetramer, with a reported maximum of 4 mol of PJmol of enzyme (6). Dephosphorylation of pyruvate kinase shows simple saturation kinetics, similar to those found for spectrin dephosphorylation. Based on a maximum value of 1 mol of Pi/mol of pyruvate kinase subunit, a K,,, of 18 IM (protein-bound phosphate) or 24 p~ (subunit pyruvate kinase), is obtained. Dephosphorylation of casein by the holoenzyme also shows apparent Michaelis-Menten kinetics with an apparent K,,, of 63 p~ casein. However, casein prepared as described by Rei-

mann and co-workers (13) was found to still contain at least 95% of the original phosphate groups leaving a maximum of 5% of the sites available for phosphorylation with radioactive label. Thus dephosphorylation of the labeled phosphate groups may not be representative of overall dephosphorylation. Assuming that all phosphategroups of casein are hydrolyzed at similar rates, the maximum velocity for casein dephosphorylation showed a specific activity between 0.4 and 1.7 pmol/min/mg of phosphatase. Subject to the uncertainties described above, we can summarize the maximum velocities of the 3 protein substrates in buffer C at 30 “C.Spectrin, pyruvate kinase, and casein show maximum velocities within a factor of two of each other at about 1.0-2.0 pmol/min/mg of phosphatase. This is 2-4% of the rate obtained with p-nitrophenyl phosphate which is 44 pmol/min/mg enzyme or 44 X lo3 units/mg of phosphatase. The low molecular weight phosphate esters arehydrolyzed at one-tenth the rate of the protein substances a t similar concentrations. The catalytic subunit shows the same substrate specificity as theholoenzyme. Relative Specificityfor Phosphorylase Kinase a and p Subunits-Assays were carried out asdescribed under “Materials and Methods.” Densitometric scans of the autoradiogram of phosphorylase kinase showed there to be an approximate 1:1 labeling of the a and subunits (Fig. 9A). After 30 min the fraction incubated in the presence of the holoenzyme showed a 10% loss in label in the a subunit and a 79% loss in the @ subunit (Fig. 9B). The catalytic subunit showed similar specificity. Under the same conditions it caused a 6% loss of label in the (Y subunit and a 72% loss in the @ subunit (Fig. 9C). Inhibition of the Phosphatase-Since the enzyme is markedly sensitive to thepresence of Mn2+,any chelating agent is likely to have a pronounced effect on the enzymatic activity. EDTA, ADP, and ATP, all quite strongly inhibit activity against phosphocasein (Table 11). In the presence of higher concentrations of Mn2+,inhibition can still be observed but to lesser extents; ADP and ATP show the most inhibition. It was not possible to measure inhibition by 2,3-bisphosphoglycerate in the presence of higher Mn2+due to precipitation of the metal. Dephosphorylation of both pyruvate kinase and spectrin is markedly inhibited by ADP:Mn2+and ATP:MnZ+; again KF inhibited butless strongly (Table 11).The catalytic subunit showed an identical inhibition profile. Effect of Rabbit Muscle Inhibitors 1 and 2 on Phosphatase Activity-Sensitivity to rabbit muscle inhibitors 1 and 2 is a distinguishing characteristic of type 1 phosphoprotein phosphatases. When measured with phosphorylase a as a substrate, both the holoenzyme and the catalytic subunit were inhibited 40-50% by the same concentrations of inhibitors 1 or 2 that inhibited rabbit muscle protein phosphatase 50% under equivalent circumstances. Similar degrees of inhibition were found with phosphocasein as substrate. pH Profile and Isoelectric Point-The pH dependence of activity against phosphocasein and p-nitrophenyl phosphate shows rather abroad profile with an optimum around pH 7.4. Isoelectric focusing of the purified enzyme proved to be quite difficult because often activity was lost during the focusing, or the protein precipitated out in the gel. However, preliminary data from wide pH range prepoured gels, pH 3.59.5, indicated that the enzyme has a PI of about 5.0. This is in agreement with the characteristics of the enzyme shown during ion-exchange chromatography.


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erythrocyte membrane. We have shown that theenzyme binds reversibly to the membrane and that the membrane-bound form is inactive (34,35). Inside-out red cell membrane vesicles will selectively remove only the holoenzyme form of the enzyme from crude preparations (37) but experiments on binding of purified holoenzyme and catalytic subunit to red cell membranes have given equivocal results (25, 38). Gruppuso et al. (36) have shown that in muscle extracts there is a 60-kDa protein that cross-reacts immunologically with inhibitor 2 and that ina crude fraction of phosphatase, tryptic digestion leads to phosphatase activation in step with destruction of the 60-kDa antigen. Samples of our holoenzyme have been examined by Western blotting in Dr. Brautigan’s laboratory but no evidence of cross-reaction with inhibitor 2 antibody was found. A high molecular weight protein phosphatase purified from human erythrocytes by Usui et al. (8) is particularly interesting for its relationship with the enzyme we have purified. The 104,000-Da enzyme purified by Usui et al. is the major M e activated enzyme of the red cell and is composed of one 32,000Da catalytic subunit and one 69,000-Da subunit. It was not tested for activity toward phosphorylase kinase a and @ subunits butwas judged to be a type 2enzyme because it was not inhibited by rabbit muscle inhibitor 2. That enzyme was also completely inhibited by Mn2+ concentration that fully activates the enzyme we purified. The enzyme that we purified is the enzyme designated phosphatase I in the survey by Usui et al.; the one they purified was their phosphatase IV. Using spectrin phosphate activity as thepoint of comparison of the two papers, we find that the papers are in agreement on the specific activity of the homogenate and on the relative amounts of the enzyme forms. Two points need mention: Usui et al. (8) reported the same Stokes radius for the crude “phosphatase I” that we report for the purified enzyme, butthey found an s20,w for that enzyme of 7.4 while we find 6.1 & 0.2. They therefore calculate MIGRATION FIG.9. Densiometric scans of autoradiograms of phospho- a molecular weight of 180,000 for the crude enzyme in agreerylase kinase subunits separated by SDS denaturing gel elec- ment with the gel permeation estimate. Our sedimentation due to phosphatase. A, phos- results show a much more asymmetric molecule with a molectrophoresis, showing loss of phorylase kinase alone; E , phosphorylase kinase + 18,000-Da phos- ular weight of about 135,000. A second point of apparent phatase; C, phosphorylase kinase + 36,000-Da phosphatase. disagreement is that we find that our enzyme is sensitive to inhibitors 1 and 2 of rabbit muscle, using inhibitors prepared DISCUSSION in this laboratory and also a sample of inhibitor 2 generously Although 30,000-35,000 catalytic subunits of phosphopro- provided by Dr. David Brautigan. Rabbit muscle protein tein phosphatases have been purified from a number of tis- phosphatase was used as a positive control and to assess the sues, purification of the higher molecular weight forms found activity of the inhibitor preparations. Usui et al. reported that in crude extracts has proved difficult. Ingebritsen and Cohen none of the erythrocyte phosphatases were sensitive to inhib(26) have proposed a useful classification of protein phospha- itor 2 but did not report a positive control. The possibility tases based on substrate specificity, metal activation, and exists that thepurified enzyme whichwe studied shows propsensitivity to protein inhibitors 1 and 2. Very recently high erties different from the enzyme in the crude homogenate, molecular weight forms of three type 2 phosphatases have but since substrate specificity, gel permeation behavior, and been purified (22, 30, 31). These enzymes act preferentially metal ion specificity do not change during purification that on the a! subunit of phosphorylase kinase and are not inhibitedpossibility is not high. by proteininhibitors 1 and 2 (32). The enzyme we have The sizeof the catalytic subunitand the noncatalytic purified is, by these same criteria, clearly a type 1 phospha- subunits in the enzyme we have purified are very similar to tase. Purification of a type 1phosphatase from liver glycogen those of the type 2 enzymes isolated from rabbit skeletal particles has been reported by Stralfors et al. (33). That muscle (29), rabbit heart (30), and turkey gizzard (31). In enzyme appears to be a dimer of 1 catalytic subunit (37,000 none of these cases has the function of the large subunit been Da) andone other subunit (103,000 Da) which causes binding understood and despite the type 1-type 2 differences, it may of the enzyme to glycogen particles. The noncatalytic subunit be that these enzymes are much closer in structureand is thus much larger than the one we find but it may have a function than is apparent now. similar function. In the enzyme we have isolated, the 62-kDa The meaning of Mn2+activation is also a problem for future subunit has an inhibitory effect when activities are measured work. Our current expectation is that Mn2+elicits an activity with Mg2+ as activator (presumably the physiological condi- which is cryptic under physiological conditions and that a tion). Italso appears to mediate binding of the enzyme to the physiological activator is to be discovered.

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0

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19. Brautigan, D. (1982) Arch. Bwchem. Bwphys. 219,228-235 20. Stewart, A.A., Hemmings, B.A., Cohen, P., Gorris, J., and Merlevede, W. (1981) Eur. J. Biochem. 116, 197-208 21. Bradford, M. M. (1976) Anal. Bwchem. 72,248-254 22. Laemmli, U. K. (1970) Nature 227, 680-685 23. Shuster, L. (1971) Methods Enzymol. 21,412-433 24. Martin, R. G., and Ames, B. N. (1961)J. Biol. Chem. 236,13721379 25. Yang, T.-T., and Westhead, E. W. (1984) Fed. Proc. 43, 1897 26. Ingebritsen, T. S., and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261 27. Ingebritsen, T. S., Foulke, J. G., and Cohen, P. (1983) Eur. J. Biochem. 132,263-274 28. Brautigan, D. L., Bornstein, P., and Gallis, B. (1981) J. Biol. Chem. 256,6519-6522 29. Paris, H., Ganapathi, M.K., Silberman, S. R., Aylward, J. H., and Lee, E. Y.C. (1984) J. Bwl. Chem. 259, 7510-7518 30. Khandelwal, R. L., and Enno, T. L. (1985) J. Bwl. Chem. 260, 14335-14343 31. Pato, M. D., and Kerc, E. (1985) J. Biol.Chem. 260, 1235912366 32. Ingebritsen, T. S., and Cohen, P. (1983) Science 221, 331-336 33. Stralfors, P., Hiraga, A., and Cohen, P. (1985) J. Biochem. 149, 295-303 34. Carroll, D., Kiener, P. A., vomEigen, P., and Westhead, E. W. (1982) Bwphys. J. 37, 141a 35. Westhead, E. W., Kiener, P. A., Carroll, D., and Gikner, J. (1984) Curr. Top. Cell Regul. 24, 21-34 36. Gmppuso, p. A., Johnson, G. L., Constantinides, M., and Brautigan, D. L. (1985) J. Biol. Chem. 260,4288-4294 37. Carroll, D. (1986) Ph.D thesis, University of Massachusetts Yang, T.-T. (1986) Ph.D thesis, University of Massachusetts