Purification and Characterization of a High Molecular Weight ...

Val. 260, No. 26, Issue of November 15, pp. 14335-14343.1985 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Purification and Characterizationof a High Molecular Weight Phosphoprotein Phosphatase from Rabbit Liver* (Received for publication, April 22, 1985)

Ramji L. Khandelwal and TerryL. Enno From the Department of Biochemistry, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0 WO

A high molecular weight phosphoprotein phospha- protein kinases whose possible control mechanisms are untase was purified from rabbit liver using high speed known. In sharp contrast to our understanding of protein centrifugation, acid precipitation, ammonium sulfate kinases, only limited information is available on the regulaon DEAE-cellulose, tion of phosphoprotein phosphatases, especially in the liver fractionation,chromatography Sepharose-histone, and Bio-Gel A-0.5m. The purified tissue. enzyme showed a single bandon a nondenaturing polGlycogen phosphorylase, which catalyzes the regulatory yacrylamide anionicdisc gel which was associated with reaction of glycogenolysis,exists in two interconvertible active the enzyme activity. Theenzyme was made up of equi- and inactive forms as a result of its phosphorylation and molar concentrations of two subunitswhose molecular dephosphorylation (4-6). Phosphorylase kinase catalyzes the 58,000 (range58,000-62,000)and weightswere 35,000 (range 35,000-38,000). Two other polypep- phosphorylation of phosphorylase b (inactive form) and contides (M, 76,000 and 27,000) were also closely asso- verts it to an active phosphorylase a. The reverse reaction is ciated with ourenzyme preparation, but theirroles, if catalyzed by phosphorylase phosphatase. The initial detailed any, in phosphatase activityare not known. The opti- biochemical investigation on phosphorylase phosphatase (or mum pH for the reaction was 7.5-8.0. K,,, value of phosphoprotein phosphatase) was hampered by the apparent phosphoprotein phosphatase for phosphorylase a was presence of multiple molecular forms of this enzyme in liver 0.10-0.12 mg/ml. Freezing and thawing of the enzyme as well as in other mammalian tissue extracts (7-21). Soon in thepresence of 0.2 M 6-mercaptoethanol caused an after, it became evident that a low molecular weight form (Mr activation (100-140%)of phosphatase activity with a = 30,000-35,000) of this enzyme could be generated from concomitant partial dissociation of the enzyme into a multiple higher molecular forms by either ethanol treatment M, 35,000 catalytic subunit. Divalent cations (Mg2+, ( E ) , freezing and thawing with p-mercaptoethanol (11, 14), Mn2+,and Co2+)and EDTA were inhibitoryat concen- storage at 4 “C for 1or 2 days (161, or by proteolysis (22, 23). trations higher than 1 mM. Spermine and spermidine On the basis of thesedata, it was suggested that higher were also found to be inhibitory at 1 mM concentrations. The enzyme was inhibited by nucleotides (ATP, molecular forms of phosphatase might be associated forms of ADP, AMP),PPi, Pi, and NaF; the degree of inhibition this low molecular weight form (presumably a catalytic subwas different with each compound and was dependent unit) with one or more regulatory subunits (10,14,18,19,24on their concentrations employed in theassay. Among 30). The low molecular weight form of liver phosphorylase phosvarious typesof histones examined, maximum activation of phosphoprotein phosphatase activity was ob- phatase was first purified by Brandt et al. (15) followed by served with typeI11 and type V histone (Sigma). Fur- Khandelwal et al. (16). Both of these preparations exhibited ther studies with type 111 histone indicated that it a broad specificity and dephosphorylated, in additionto phosincreased boththe K , for phosphorylase a and theVmex phorylase a, a number of other phosphorylated protein subof the dephosphorylation reaction. Purified liverphos- strates (15, 16, 31). In 1978,Lee et al. (21) reported the phatase, in addition to the dephosphorylation of phos- purification of a high molecular weight phosphorylase phosphorylase a, also catalyzed the dephosphorylation of phatase from rat liver. The purified enzyme showedtwo 3ZP-labeledphosphorylase kinase, myosin light chain, subunits with apparent molecular weights of M, 65,000 and myosin, histone 111-S, and myelin basic protein. The effects of Mn2+,KCl, and histone 111-S on phosphatase 35,000. In their preparation, a minor band of M, 55,000 and activity were variable depending on the substrate used. several faint bands of M, greater than 70,000 were also observed. In 1980, Tamura et al. (32) and Tamura and Tsuiki (33) also reported the purification of twohigh molecular weight phosphoprotein phosphatases, using phosphorylase a Reversible phosphorylation is one of the major mechanisms as thesubstrate, from rat liver. Analysis of these enzymes by by which hormones exert their effect in theregulation of key sodium dodecyl sulfate-gel electrophoresis indicated that one regulatory enzymes of metabolic pathways (1-3). The phos- form of the enzyme was composed of two polypeptides (M, phorylation reactions arecatalyzed by several protein kinases 69,000 and 35,000) whereas the other one contained three including Ca2+-regulated protein kinases (l),cyclic nucleo- polypeptides (M, 69,000, 58,000, and 35,000). tide-dependent protein kinases (1-3), and a number of other The present study was undertaken with the objectives of (a) purifying a high molecular weight rabbit liver phospho* This study was supported by an operating grant from the Medical protein phosphatase using muscle phosphorylase a as the Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article substrate and ( b ) studying some of its general chemical and must thereforebe hereby marked ‘‘advertisement” in accordance with physical properties. This paper describes a purification pro18 U.S.C. Section 1734 solely to indicate this fact. cedure for a high molecular weight phosphoprotein phospha-

14335

14336

Phosphoprotein Rabbit Phosphatase Liver

tase from rabbit liver and characterization as to its subunit structure,substrate specificity, andthe effect of divalent cations, several metabolites, heat-stable inhibitor 2, and various types of histones on its activity. A portion of our present study has been published in abstract form (34). EXPERIMENTALPROCEDURES

Materials-Crystalline rabbit skeletal muscle phosphorylase b was prepared as described by Fischer and Krebs (35). Rabbit skeletal muscle phosphorylase kinase was prepared according to Hayakawa et al. (36). The heat-stable inhibitor 2 of phosphoprotein phosphatase was prepared from rabbit skeletal muscle by the procedure of Yang et al. (37). One unit of inhibitor was defined as that amount which inhibited 50% of muscle phosphoprotein phosphatase activity under our assay conditions described later in this section. For these assays, muscle phosphoprotein phosphatase used was the enzyme obtained after the chromatography on DEAE-Sephadex step in the above procedure of Yang et al. (37). [y3'P]ATP was obtained from New England Nuclear, and EP-B(or HP-B) solvent for liquid scintillation was a product of Beckman. All reagents for electrophoresis and BioGelA-0.5mwere obtained from Bio-Rad. Sepharose-histone was prepared from CNBr-activated Sepharose 4B (Pharmacia) and histone 11-A (Sigma) as described previously (16). DEAE-cellulose (DE52) was a product of Whatman. Dithioerythritol, various types of histones, ATP, ADP, AMP, and orthovanadate were obtained from Sigma. Molecular weight markers were obtained from Bio-Rad, Pharmacia, and Sigma. All other chemicals were of reagent grade. Preparation of 32P-LabeledSubstrates-[32P]Phosphorylase a was prepared from phosphorylase b using [r-3ZP]ATP,M e , and phosphorylase kinase as described by Krebs et al. (38). All other substrates were phosphorylated using catalytic subunit of cyclic AMP-dependent protein kinase in the presence of [Y-~'P]ATP andM e . Under our conditions, phosphorylase kinase incorporated 2 phosphates (1 in asubunit and 1 in &subunit) per monomeric unit. 32P-labeledmyosin light chain (20,000 daltons) and 32P-labeledmyosin were a kind gift of Dr. Pato, University of Saskatchewan (39). 32P-labeled myelin basic protein was provided by Dr. R. C. Gupta, University of Saskatchewan (40). Purification of Phosphoprotein Phosphatase-New Zealand white rabbits were decapitated, and theblood was drained from the jugular veins. The liver was removed immediately, packed in crushed ice, and cut into small pieces that were homogenized in 4 volumes of 50 mM glycylglycine, pH 7.4, 0.5 M sucrose, and 5 mM EDTA (Buffer A). The homogenate was centrifuged at 10,000 X g for 30 min, and the supernatant solution, collected after filtering throughglass wool and four layers of cheesecloth, was further centrifuged at 105,000 X g for 1 h. The supernatant solution obtained was again filtered through glass wool and cheesecloth and was adjusted to pH5.2 bythe dropwise addition of 1 N acetic acid. The acidified extract was immediately centrifuged at 10,000 X g for 15 min, and the supernatant solution was neutralized to pH7.0 with 2 M Tris base. The neutralized solution was slowly mixed with solid ammonium sulfate (36 g/lOO-ml solution) and let standfor 30 min at 4 "C. The mixture was centrifuged at 10,000 X g for 20 min, and thepellet was dissolved in 0.5 ml of Buffer B (10 mM glycylglycine, pH 7.4, 2% (v/ V) glycerol, and 15 mM 6-mercaptoethanol)/g of tissue with gentle stirring. The suspension was dialyzed for 4-8 h againstBuffer B with two changes. The dialyzed solution was centrifuged at 48,000 X g for 10 min to remove denatured proteins, and the clear solution was adjusted to a conductivity of approximately 1 mS. The dialyzed fraction was applied to a column of DEAE-cellulose (DE52, 0.5 ml of gel/g of tissue) previously equilibrated in Buffer B. The column was then washed with Buffer B, and the enzyme was eluted from the column using a linear gradient (equivalent to 6 gel volumes) from 0-0.5 M NaCl in Buffer B. Phosphorylase phosphatase activity was eluted in two peaks with at least one shoulder. The second peak of the activity was pooled and applied to a second DE52 column (0.25 ml of gel/g of tissue). The conditions for the elution of the enzyme were the same as for the first column. The active fractions were pooled and further chromatographed using Sepharose-histone column (gel volume was equivalent to the second DE52 column). The column was washed with Buffer .B, and then the enzyme was eluted with a linear gradient from 0.1-1.5 M NaCl in Buffer B. The enzyme activity was eluted in two peaks, and only the second major activity peak was pooled and rechromatographed on Sepharose-histone. The column was washed with Buffer B containing 0.2 M NaC1, and the

enzyme was eluted with a linear gradient from 0.2-1.5 M NaC1. The active fractions were pooled. The pooled fraction was concentrated to approximately 2 ml using an Amicon PM-30 membrane. The sample was then applied to a BioGel A-0.5mcolumn (2.5 X 60 cm) previously equilibrated with Buffer B containing50 mM NaC1. Phosphoprotein phosphatase activity was eluted as a single peak. Peak fractions were pooled and concentrated using an Amicon PM-30 membrane. The concentrated enzyme solution was stored at -80 "C in small aliquots either in the absence or presence of 50% glycerol. Phosphoprotein Phosphatase Assay-Phosphoprotein phosphatase activity was determined by the release of 32Pifrom [32P]phosphorylase a a t 30 "C (16). Reaction mixtures contained 25 mM glycylglycine (pH 7.4), 1 mM caffeine, 0.5 mM dithioerythritol, 1 mg/ml [32P] phosphorylase a, and phosphatase preparation in a total volume of 50 pl. Unless otherwise noted, the reactions were started by the addition of ["P]phosphorylase a and terminated after 10 min of incubation by the addition of 0.2 ml of 10% cold trichloroacetic acid. Ten pl of 20 mg/ml bovine serum albumin was then added to each tube and stored at 4 "C for a t least 20 min. All tubes were centrifuged in aclinical centrifuge for 10 min, and 200 pl of the clear supernatants were then counted with 5 ml of Beckman EP-B solvent. In all cases, except some column fractions, the amount of phosphatase added was such that less than 10% of the substrate was dephosphorylated in a 10-min period of incubation. Blank values, i.e. the counts obtained in the absence of enzyme, were subtracted from all the assays. One unit of phosphoprotein phosphatase was defined as thatamount of enzyme which releases 1 nmol of 32Pifrom [32P]phosphorylasea per min. Specific activity was defined as thenumber of units/mg of protein. Polyacrylamide Gel Electrophoresis-Disc gel electrophoresis was carried out by the method of Davis (41). Acrylamide gels (7.5%) were run and stained with 0.25% Coomassie Brilliant Blue for 1 h and were then destained. A duplicate gel wassliced (2-mm segments) and extracted with 100 pl of 20 mM ammonium bicarbonate, 0.5 mM dithioerythritol, pH 7.4, for 3 h. The extracted solutions were either used for the determination of enzyme activity or were further electrophoresed in the presence of SDS' as described below. The purifiedphosphoprotein phosphatase (and extracted slice fractions from gel electrophoresis) were also analyzed by SDS-polyacrylamide slab gels (12.5%) according to the method of Laemmli (42). The gels were stained either with 0.25% Coomassie Brilliant Blue or with silver staining kitobtained from Bio-Rad. For the determination of molecular weights, standard molecular weight proteins obtained from Bio-Rad were used. The proteins were: phosphorylase (M, = 92,500), bovine serum albumin (Mr = 66,200), ovalbumin ( M , = 45,000), carbonic anhydrase ( M , = 31,000), soybean trypsin inhibitor (Mr= 21,500), and lysozyme (Mr= 14,400). Analytical Methods-Protein was determined by the method of either Lowry et al. (43) or Bradford (44). In both cases bovine serum albumin was used as thestandard. RESULTS

Purification of Phosphoprotein Phosphatase-The profiles of phosphoprotein phosphatase activity using phosphorylase a as the substrate, on 1st DE52, 2nd Sepharose-histone, and Bio-Gel A-0.5m columns are shown in Fig. 1. The enzyme from 1st DE52 was eluted in two major peaks (Fig. lA). The second peak of the activity was pooledand rechromatographed on a 2nd DE52 to completely separate phosphoprotein phosphatase activity of the second peak from other contaminating peaks. This was accomplished by this column (results not shown). The decrease in total activity after the 2nd DE52 column was due to the separation of other contaminating enzyme peaks (Table I). The active fractions of the 2nd DE52 column were pooledand furtherchromatographed on a Sepharose-histone column. The enzyme was eluted again in two peaks. The major peak was pooledand rechromatographed on a 2nd Sepharose-histone column (Fig. 1B). Both of these columns achieved a purification of greater than 20-fold with a retention of approximately 40% of enzyme activity (Table 1 The abbreviations used are: SDS, sodium dodecyl sulfate; EGTA, ethylene glycol bis(8-aminoethyl ether)-N,N,N',N'-tetraacetic acid.

Phosphoprotein Rabbit Phosphatase Liver

14337 TABLEI Purification of a high molecular weightphosphoprotein phosphatase from rabbit liver Phosphoprotein phosphatasewas purified as described in the text. The values presentedare the average of four preparations. The average amount of liver used was 208 E. Fraction

0

20

6

2nd

40

60

80

Sepharose Histone

1st DE52 2nd DE52 1st Sepharose-histone 2nd Sepharose-histone Bio-Gel A-0.5 m

Total orotein

Total activitv

Specific activitv

mg

units

units/ mg

%

4926 363574 1665 1467 608

11.8 24.1 378 556 699

loo

419 151 4.40 2.64 0.87

46 30 14

Phosphoproteinphosphatase purified inthis study was separated fromthe majority of other enzymic forms only after the 1st DE52 chromatography. Because of this, Table I summarizes the extent of purification and yields of phosphoprotein phosphatase at different stages of preparation only after this column. The purified enzyme had a specific activity of approximately 700 units/mg of protein. It should be noted that loss in total activity at 2nd DE52 and 1st Sepharosehistone columns was partly due to the separation of other enzymic forms of phosphoprotein phosphatases. In order to obtain a high specific activity enzyme it was essential to avoid (i) freezing dilute fraction(s)at any stage of purification and/ or (ii) leaving dilute column fractions unnecessarily in the cold room for long periods of time. Optimal AssayConditions and Stability-Reaction rates were linear with time andwere proportional to theamount of enzyme under conditions in which no more than 20% of 13‘P] phosphorylase a was dephosphorylated. The optimum pH for the reaction was 7.5-8.0. The reaction was slightly stimulated (10-25%) inthe presence of 1 mM caffeine and 0.5 mM dithioerythritol. K, for [32P]phosphorylasea was 0.10-0.12 mg/ml under our assay conditions. In all assays, therefore, reactions were carried out at pH7.5 in thepresence of caffeine (1 mM), dithioerythritol (0.5 mM), and [32P]phosphorylasea (1mg/ml), under conditions in which less than 10%of [“PI phosphorylase a was dephosphorylated in an incubation pe0 10 20 30 40 50 riod of 10 min. Variations to this assay system have been noted in theappropriate experiments. Purified enzyme prepFRACTION NUMBER arations were stable at -80 “C for at least 6 months. However, FIG. 1. Purification of phosphoprotein phosphatase from each thawing and refreezing resulted in some loss of activity. rabbit liver. A, 1st ion-exchange chromatography on DEAE-celluPurity and Subunit Characterization-The purified phoslose (DE52) at pH 7.4. The dialyzed ammonium sulfate fraction was chromatographed as described under“Experimental Procedures.” phoprotein phosphatase was essentially homogeneous as it Fractions of 225 drops were collected after passing through a hollow migrated as a single band on a native disc gel (Fig. 2). The fiber dialyzed against Buffer B. Fractions 49-60, as shown by the protein band was associated with the enzyme activity. On solid bar, were pooled and further processed as described in the text. sodium dodecyl sulfate-polyacrylamide gel electrophoresis B, chromatography of rabbit liver phosphoprotein phosphatase using (Fig. 3), the enzyme was dissociated into four polypeptides Sepharose-histone (2ndcolumn). Details for this column are given in the text. Fractions of 100 drops were collected after passing through with molecular weights of 76,000 (range 76,000-80,000), and 27,000 a hollow fiber dialyzed against Buffer B. Fractions 40 t o 85, as shown 58,000 (58,000-62,000),35,000(35,000-38,000), by the solid bar, were pooled and further processed. C, gel filtration (27,000-28,000). The ranges given in parenthesis were calcuon Bio-Gel A-0.5m. Pooled fraction from the 2nd Sepharose-histone lated using different molecular weight standard markers supcolumn was concentrated using an Amicon PM-30 membrane and was applied to a column (2.5 X 60 cm) of Bio-Gel A-0.5m. Enzyme plied by scientific companies. Based on these molecular activity was eluted using Buffer B. Fractions of 100 drops were weights, relative molar ratios of these polypeptides calculated collected. Peak activity fractions, 21-27, were pooled and concen- from densitometer tracings were 1.10, 1.00, 1.09, and 0.93, trated using Amicon PM-30 membrane. For all columns: .“--., respectively (average of four preparations). In order to further optical density at 280 nm; O “ - O , phosphoprotein phosphatase establish that these four polypeptides were indeed subunits of activity. a phosphoprotein phosphatase enzyme, proteinextracted from individual slices of a native gel was concentrated and I). The activity profile of phosphoprotein phosphataseon Bio- further analyzed by SDS-polyacrylamide gel electrophoresis. Gel A-0.5m column is shown in Fig. 1C. The enzyme emerged As shown in Fig. 4, only two polypeptides (Mr 58,000 and from the column as a single peak with an apparent molecular 35,000) moved together and represent subunits of the phosweight of approximately 200,000. phoprotein phosphataseenzyme. Although the other two poly-

2

Rabbit Liver Phosphoprotein Phosphatase 10

\

TOP

0

10

DYE FRONT I BOTTOM

30

20

40

50

SLICE NUMBER FIG.2. Polyacrylamide disc gel electrophoresis of the purified phosphoprotein phosphatase from rabbit liver. Twenty micrograms ol'the purified enzvme were suhjected to electrophoresis on polvacrylamide gel ( 7 . 5 s ) in the anionic system as described by Davis (41). Gel was stained with 0.25% Coomassie Brilliant Blue. A duplicate gel, after the run,was sliced (2-mm segments) and extracted as described in the text. Ten pl of extracted solution was used for enzyme assays.

92.5 K 66.2 K 45.0 K

A

B

C 31 .OK 21.5K 14.4 K

D DYE FRONT

24

28

32

36

40

SLICE NUMBER FIG.4. Phosphoprotein phosphatase activity and quantitation of subunits from extracted solutions of polyacrylamide anionic disc gel slices. Fifty micrograms of the purified enzyme were electrophoresed, and the gel wassliced and extracted as described in the legend to Fig. 2. Ten pl of solution was used for the enzyme assays. Fifty pl of extracted solution was lyophilized and was electrophoresed in the presence of SDS as described in the legend to Fig. 3. Recovery of subunits was calculated using a densitometer and represented in arbitrary units.

varying concentrations (0.1-40 mM)ofMe;", Mn2+, Co2+, EDTA, and EGTA on enzyme activity were determined. At concentrations >1 mM, all three divalent cations and EDTA were inhibitory. Cobalt was more inhibitory than Me;" or MnZ+a t equimolar concentrations. For example, a t 10 mM concentration, the per cent inhibition of phosphatase activity by Co" was approximately 45% whereas by M F and Mn'+ it was approximately 20%. At this concentration (10 mM), peptides (M, 76,000 and 27,000) were closely associated with inhibition by EDTA was 10-15%. EGTA, a t all concentrations up to 40 mM, showed no effect on the phosphataseactivity. this enzyme, it is unlikely that they are subunits of this Effect of Polyamines-The effects of polyamines (0.1 and 1 enzyme. Further purification of phosphatase resulted in the mM) on phosphoprotein phosphatase activitywere also deterloss of enzyme activity and, therefore, the preparation obtained from the Bio-Gel A-0.5m column was used for this mined. Spermineandspermidine a t 1 m M concentrations were slightly inhibitory (15-25%). At this concentration, no study. significant effects of cadaverine and putrescine on enzyme Effect of Freezing in the Presenceof &Mercaptoethnol-It has been previously observed that freezing and thawing of activity were observed. At a lower concentration (0.1 mM), high molecular weight protein phosphatases in the presence none of these polyamines affected phosphatase activity. Effect of Nucleotides, PP" Pi,NaF, and Vanadate-The of 0.2 M 8-mercaptoethanol dissociates the catalytic subunit are inFig. 5. Maximum (M, -35,000) from the holoenzyme. Such a treatment of our effects of ATP, ADP, and AMP shown AMP followed by ADP and preparation resulted in a 100-140% activation of enzyme inhibition wasobservedwith of enzyme activitywas obtained activity. No effect of &mercaptoethanol was observed if the ATP. Fifty per cent inhibition with approximately 0.05, 0.2, and 0.3 mM concentrations of preparation was not frozen and thawed. When the thawed respectively. Among other compounds enzyme was chromatographed onBio-Gel A-0.5m column, the AMP, ADP, and ATP, enzyme activity emerged in two peaks, corresponding to the examined, PPi, Pi, and NaFwere inhibitory whereas vanadate holoenzyme and a free M, 35,000 subunit. slightly activated (-30%) the enzyme (Fig. 6). Fifty per cent Effect of Divalent Cations and Chelators-The effects of inhibition of enzyme activity was obtained by approximately FIG.3. Sodium dodecyl sulfate-gel electrophoresis of the purified phosphoprotein phosphatase from rabbit liver. Electrophoresis was carried out using 12.5'6 mini-slab gel according to the method of 1,aemmli (42). Two micrograms of the purified enzyme with the molecular weight standard proteins (Bio-Rad) were used. Molecular weights indicated with standard markers are those published with the Bio-Rad standard kit.

14339

Rabbit Liver Phosphoprotein Phosphatase 100

1

>

L

-I

2 75 I“

2 -I

50

Z

w 0

5a 25 1 0

r 5

I

i

4

3

-Log M NUCLEOTIDES FIG.5. Effect of various concentrations of ATP, ADP, and AMP on the activityof phosphoprotein phosphatase purified from rabbit liver. Assay conditions were the same as described under “Experimental Procedures” except that different concentraATP; W,A D P tions of these nucleotides were added. c”-o, and A-A, AMP. Enzyme activity in the absence of these compounds was taken as100%.

INHIBITOR UNITS FIG.7. Effect of various concentrationsof muscle heat-stable inhibitor 2 on the activity of rabbit liver phosphoprotein phosphatase and rabbit muscle phosphoprotein phosphatase. The activity of phosphatases in the assay was the amount that dephosphorylated 10%of the substrate in incubation time of 10 min. Other conditions of the assay were the same as described under “Experimental Procedures” except that various amounts of inhibitor 2 were first preincubated with phosphatases for 5 min, and then reactions were started with the addition of [32P]phosphorylase a. M, rabbit liver phosphoprotein phosphatase; o ” 0 , rabbit muscle phosphoprotein phosphatase.

0

TABLEI1 Effect of various types of histones (Sigma)o n the activity of phosphoprotein phosphatase purified from rabbit liver Phosphoprotein phosphataseactivity was determined as described under “Experimental Procedures” except that various types of histones, as indicated, were added at a final concentrationof 0.2 mg/ml, and reactions were stopped with the addition of 200 pl of 5% trichloroacetic acid containing 0.25% Na tungstate.

c Z

w

0 CK

Additions

6

5

4

3

2

1

-LOG M FIG.6. Effect of various concentrationsof Pi, PPi, NaF, and orthovanadate on the activityof phosphoprotein phosphatase purified from rabbit liver. Assay conditions were the same as described under“Experimental Procedures” except that different concentrations of indicated compounds were added. Enzyme activity in theabsence of these compounds was taken as100%.

0.06, 8, and 15 mM concentrations of PPi, Pi, and NaF, respectively. Effect of Muscle Heat-stable Inhibitor 2-Heat-stable inhibitor 2 isolated from rabbit skeletal muscle did not inhibit the activity of purified rabbit liver phosphoprotein phosphatase (Fig. 7). Inhibitor 2 also did not inhibit the dissociated liver phosphoprotein phosphatase obtained after the P-mercaptoethanol treatment. Under ourassay conditions, however, the inhibitor was active toward a protein phosphatase isolated from the rabbit skeletal muscle. Per cent inhibition of this enzyme increased with the increase in the amount of inhibitor in the assay. Purified liver phosphoprotein phosphatase con-

Relative activity at phosphorylase a concentrations 0.1 1.0 mdml mdml %

100 None Histone type I11 Histone type V Histone type VI Histone typeVI1 Histone tvoe VI11

100 2 78 231 81 91 61

504 490 180 360 122

tains no heat-stable inhibitor activity. Effect of Histones-It has been recently reported that phosphorylase phosphatase isolated from rabbit’ kidney, rabbit muscle, and bovine vascular smooth muscle are activated by lysine-rich histone H1 (45-47). The effects of various types of histones were, therefore, examined on the activity of phosphoprotein phosphatase purified in this study. In Table 11, the effects of various types of histones (0.2 mg/ml or 10 pg in the incubation reactions) on enzyme activity using two concentrations (approximately equal to K, and 10 times of K,) of the substratephosphorylase a arereported. At lower phosphorylase concentration, lysine-rich histones (Sigma types I11

Rabbit Liver Phosphoprotein Phosphatase

0

a2

0.4

0.6 1

2

HISTONE (mglml) FIG. 8. Effect of various concentrations of histone type I11 (Sigma) on the activity of phosphoprotein phosphatase from rabbit liver. Assays werecarried out using either 0.1 mg/ml (W) or 1 mg/ml (A-A) [32P]phosphorylasea in thereaction. Other assay conditions were the same as described under “Experimental Procedures” except that reactions were stopped with the addition of 200 pl of 5% trichloroacetic acid containing 0.25% Na tungstate.

and V) activated the enzyme whereas other types of histones (Sigma types VI, VII, and VIII) slightly inhibited the enzyme activity. All of these histone types, however, activated phosphorylase phosphatase activity when saturating concentrations (1mg/ml) of [32P]phosphorylasea were employed in the incubation reactions, albeit to varying degrees. The effect of varying concentrations of type I11 histone (lysine rich) using these two [32P]phosphorylasea concentrations is shown in Fig. 8. The response was biphasic with both concentrations of the substrate employed. Initially, the enzyme activity increased with the increase in histone concentrations, maximum activation being observed with 0.1-0.2 mg/ml concentrations. Higher concentrations of histone were either less stimulating or inhibitory. The effect of two concentrations of type I11 histone (0.2 and 1.0 mg/ml) on the K , for phosphorylase a and Vmx for the reaction is shown in Fig. 9. Both concentrationsof histone increased the K , value for [32P]phosphorylasea from a value of0.12 mg/ml to a value of approximately 0.5 mg/ml. As expected, the V, of the reaction was increased approximately 8-fold by the lower concentration (0.2 mg/ml) of histone whereas only a 2-fold increase in V- was observed with the higher concentration of histone. In order to further explore the mechanism by which histone activates the dephosphorylation of [32P]phosphorylasea by purified liver phosphoprotein phosphatase, the product formation (release of 32Pi) was followed as a function of incubation time. Histone was either added at the start of the reaction or 5 min after the start of the reaction. As shown in Fig 10, the initial rate of activation was approximately 50% higher when histone was added after the start of the reaction (5 min). Preincubation of histone with the substrate [32P]phosphorylase a or with the enzyme phosphoprotein phosphatase for 5-10 min before the initiation of the reaction showed

identical rates asobserved when the histone was added at the start of the reaction (0 min). These results indicated that an interaction between phosphorylase a and phosphoprotein phosphatase was essential for the maximum rate of activation of enzyme activity by histone. Substrate Specificity-For the purification and initial characterization of liver phosphoprotein phosphatase, only phosphorylase a was used as thesubstrate. As shown in Table 111, the purified high molecular weight liver phosphoprotein phosphatase was also able to catalyze the dephosphorylation of other 32P-labeledphosphoproteins examined. Basal phosphatase activity (i.e. in the absence of divalent cation or any other effector) was maximum with myelin basic protein followed byphosphorylase a and myosin light chain. Manganese activated the dephosphorylation of myosin, myosin light chain, and histone 111-S but showed no effect on the dephosphorylation of other substrates. The effect ofKC1 on the dephosphorylation of various phosphoproteins was quite complex. It augmented the dephosphorylation of myosin and histone 111-S, had no effect onthe dephosphorylation of myosin light chain and myelin basic protein, and inhibited the dephosphorylation of phosphorylase a and phosphorylase kinase. It was shown previously that the dephosphorylation of phosphorylase a was stimulated by histone 111-S (Table I1 and Figs. 8-10). In addition to this substrate, histone 111-S also activated the dephosphorylation of phosphorylase kinase, myosin light chain, and myosin; maximum-fold activation was observed with myosin and myosin light chain substrates. Histone 1113, on the other hand, inhibited the dephosphorylation of myelin basic protein. Dephosphorylation of phosphorylase kinase by the purified liver phosphatase, under all conditions, was preferentially from its a-subunit. DISCUSSION

In 1975, Kobayashi et al. (14) reported the partial purification and characterization of three high molecular weight forms of phosphoprotein phosphatase in rabbitliver. In 1978, Lee and co-workers (21) reported a successful purification to near homogeneity of a high molecular weight phosphorylase phosphatase, termed phosphatase H, from rat liver. The apparent molecular weight of this native form of the enzyme was estimated to be 260,000 by gel filtration. However, after purification, the value was estimated as 225,000. The purified enzyme contained M , 35,000 and 65,000 subunits in a molar ratio of2:1, together -with other minor components. Tsuiki and co-workers (32,33,48) purified three molecular forms of phosphatase from rat liver, termed IA, IB, and 11, whose molecular weights determined by gel filtration were 40,000, 260,000, and 160,000, respectively. Examination of these enzymic forms by SDS-gel electrophoresis revealed that(i) phosphatase IA contained only one type of subunit with a molecular weight of 48,000, (ii) phosphatase IBwas composed of three subunits (35,000,69,000,and 58,000) in a molar ratio of a&, and (iii) phosphatase I1 contained two types of subunits (35,000 and 69,000) in a molar ratio of a&. In this study, initial steps of purification, up to DEAEcellulose column, were very similar to those of Kobayashi et al. (14) and Tamura et al. (32). The enzyme purified by us was eluted from DEAE-cellulose column at a similar salt concentration as phosphatase I11 of Kobayashi et al. (14) and phosphatase I1 of Tamura et al. (32). A direct comparison of the specific activity of our purified enzyme is difficult to make with those of Tamura et al. (32) since they used a different enzyme assay system. Nonetheless, one can compare the fold increase in thespecific activity of phosphorylase phosphatase from DEAE-cellulose column to thefinal step of purification.


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FIG. 9. Effect of histone type III (Sigma) on dephosphorylation of [SzP]phosphorylasea by purified rabbit liver phosphoproteinphosphatase. Double reciprocal plot of 1/ [32H]phosphorylasea versus l / u with no addition (c".), 0.2 mg/ml histone (A-A), or 1mg/ml histone 1-.( Inset shows the plot of [3ZP]phosphorylase a versus velocity with no addition (W), 0.2 mg/ml histone (A-A), or 1 mg/ml histone (C"D).

- '1

possible that one of the enzyme peaks discarded at theSepharose-histone step might possess a subunit composition similar to those of Tamura et al. (32). Further studies should indicate whether that is true since the purification of that enzyme peak is presently inprogress. 2 Phosphoprotein phosphatase having two subunits has also been purified from other tissues (49-51). Crouch and Safer (49) purified a two-subunit (M, 60,000 and 38,000 eukaryotic initiation factor 2 phosphatase from rabbit reticulocyte lysate. The enzyme consists of equimolar concentrations of these subunits. Werth et al. (50) purified a myosin phosphatase 1 from bovine aortic smoothmuscle, and thisenzyme wasmade up to two subunits (MI = 67,000 and 38,000) in a molar ratio of 1:l.Paris et al. (68) purified a high molecular weight protein phosphatase from rabbit skeletal muscle. It possessed two subunits (M, = 70,000 and 35,000) and catalyzed the dephosphorylation of phosphorylase a,glycogen synthase, phospho0 0 10 20 30 rylase kinase, lysine-rich histone, and p-nitrophenyl phosphate. A major spectrin phosphatase having equimolar conINCUBATION TIME (min.) centrations of two subunits (M, 69,000 and 32,000) had been FIG. 10. Effect of histone on time course of the phosphatase purified from human erythrocyte hemolysate (51). The enreaction. Histone (final concentration of 0.2 mg/ml) was added zyme of Crouch and Safer (49) has similar subunit molecular or 5 min (A-A) of the reaction. A duplicate either at 0 (A-A) set was supplemented with equal volume of H20 and served as weights as of our enzyme but whether there is any relation 5 min). Atappropriate times, between these enzymes needs further study. controls ( O " 0 , 0 min; o"-o, samples were withdrawn, and Pi released was separated and counted It is a well known fact that freezing and thawing of partially as described under "Experimental Procedures" and the legend to purified high molecular weight phosphoprotein phosphatases Fig. 8. with 0.2 M B-mercaptoethanol results in the activation of the enzyme with a concomitant dissociation of a catalytically Tamura et al. (32) needed only %fold purification from active subunit of approximately M, 35,000 (11, 14). Purified DEAE-cellulose column step to homogeneity whereas in our rabbit liver phosphoprotein phosphatase was also activated purification we achieved a fold increase of approximately 60. by a similar treatment, but the enzyme was only partially Our fold purification will even be higher if protein content of dissociated. The reason for this is not clear, but a similar only phosphatase subunits (58,000 and 35,000) are considered observation was made by Tamura et al. (32) with the ratliver in the calculations. As regards subunit composition, our en- enzyme. zyme was composed of equimolar concentrations of two subThe purified rabbit liver phosphoprotein phosphatase was units, and molecular weights of these subunits were 58,000 inhibited by high concentrations of divalent cations. At presand 35,000. Since molecular weight of purified enzyme from ent, the importance of divalent cations in the regulation of gel filtration column was approximately 200,000, this enzyme protein phosphatases is at best controversial. Depending on may have a subunit composition of (58,000)2(35,000)2. Molec- the enzyme preparation and thesubstrate employed in assays, ular weights of these two subunits were always the same even some protein phosphatases are inhibited by divalent cations when composition of homogenizing buffer, including altera- (13, 14,20,52-54) whereas others are unaffected or activated tions in thecomposition of added proteolytic inhibitors, was to varying degrees (7, 50, 52, 55-58). Mn2+or Co2+can also modified. The difference in molecular weight of one of the prevent or reverse the inhibition (or inactivation) by nucleosubunits may, therefore, be a result of different animalspecies side triphosphates, pyrophosphate, and by fluoride of some of used for the purification of this enzyme. Alternatively, it is these phosphatases (30, 56, 58, 59). Some studies have led to 4


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TABLE I11 Substrate specificity of the purified phosphoprotein phosphatase from rabbit liver Phosphatase activity with different substrateswas determined as described under “Experimental Procedures” except that the concentrationsof all substrates, except phosphorylase kinase, used inassays were 1p~ in terms of 32Pbound. Concentration of [32P]phosphorylasekinase was 0.2 pM. Additions of Mn2+,KC1, and histone 111-S were as indicated in the table. In all cases, reactions were started by the phosphatase and were terminated after an incubation of 5 min with the addition of 200 pl of 5% trichloroacetic acid containing0.25% sodium tungstate. Activity in thepresence of Substrate

Phosphorylase a Phosphorylase kinase Myosin light chain (20,000 daltons) Myosin Histone 111-S Myelin basic protein

Mn2+(1 mM)

KC1 (0.1M)

Histone 111-S (0.2 mg/ml)

No addition, specific activity

Specific activity

Ratio, +Mn2+/-MnZ+

Specific activity

Ratio, +KC1/-KC1

Specific activity

475 38 462

587 41 1322

1.23 1.08 2.86

127 25 498

0.27 0.66 1.08

1426 65 3240

3.00 1.71 7.01

128 23 1495

299 35 1655

225 29 1477

950

7.42

1.52 1.11

1.26 0.99

441

0.29

2.33

1.76

the conclusion that phosphatase (especially Mr = 35,000) is a metalloenzyme which requires Mn2+or Co2+for activity and which is inactivated (probably by removal of a metal ion)with pyrophosphoryl-containingcompound or fluoride (54, 56, 58, 60, 61). Other studies, on theother hand, have refuted such a phenomena and have presented alternate plausible mechanisms (62, 63). Data have also been presented to indicate that purified rabbit liver phosphoprotein phosphatase was slightly inhibited by spermine and spermidine and was unaffected by cadaverine and putrescine. In 1978, Killilea et al. (64) also reported that phosphorylase phosphatase (Mr 35,000 form) from rabbit liver or from bovine heart was inhibited by spermine and spermidine. The effect was, however, substrate directed, i.e. due to aninteraction of spermine and spermidine with the rabbit muscle phosphorylase a. Using two molecular forms of the enzyme isolated from pig’heart, Usui et al. (65) reported both activation and inhibition depending on the substrate used in the assay. They also concluded that the effect of polyamines was substrate directed. Our study further indicated that the purified rabbit liver phosphoprotein phosphatase was activated (or inhibited) to varying degrees by various types of histones (Table I1 and Figs. 8-10). Maximum activation was observed by 0.1-0.2 mg/ ml histonetype I11 (Sigma) irrespective of thesubstrate concentration employed in the assay. Further analysis, however, indicated that histone effects were complex and required the presence of both the enzyme and the substratephosphorylase a to achieve the maximum activation (Fig. 10). Kinetic analysis revealed that histone effects were mixed for the dephosphorylation of phosphorylase a, and it increased both the K, for phosphorylase a and Vmaxof the reaction. As indicated before, the activation of phosphorylase phosphatase isolated from rabbit kidney, rabbit muscle, and bovine vascular smooth muscle by histone H1 has also been reported by other investigators (45-47). The optimalconcentration of histone required for maximum activation could not be directly compared since theseotherstudies employed histone H 1 whereas we used type I11 histone obtained from Sigma. All of these studies, however, indicate that the mechanism for the stimulation of phosphorylase (or phosphoprotein) phosphatase by histone isa complex one, and itis possible that histone effects are %fold. First, it acts as an alternate substrate and, therefore, binds to the substrate-binding site and acts as a competitive inhibitor for the dephosphorylation of phosphorylase a. Second, it activates the enzyme activity by binding to some other site on the enzyme or the substrate phospho-

Ratio, +His/-His

rylase a. Because of these 2-fold effects, a mixed type of kinetics is observed. In any case, further studies are required to completely understand their mechanism of action(s) inthe regulation of phosphatase activity. It has been known for several years that substrate specificity of phosphoprotein phosphatases purified from various tissues is nottoo rigid. The relative phosphatase activity toward different substrates is quite often dependent on the assay conditions employed for the measurement of dephosphorylation reactions. It is clear from the data presented in Table I11 that thepurified liver phosphoprotein phosphatase was also able to catalyze the dephosphorylation of all phosphoprotein substrates examined in this study. The effects of Mn2+,KC1, and histone 111-S on phosphatase activity were variable and were dependent on the substrate used. Further detailed studies arerequired to precisely understand the properties of purified liver phosphatase using these substrates. Ingebritsen et al. (52, 66, 67) have classified the phosphoprotein phosphatasesfrom rabbit liver into two major classes. Since the purified rabbit liver phosphatase is not inhibited by muscle heat-stable inhibitor 2 (Fig. 7) and it preferentially dephosphorylates a-subunit of phosphorylase kinase, it would be classified as a type 2 enzyme. However,other properties of the purified liver phosphatase do not conform to their criteria to be classified as a type 2 phosphatase. For example, Ingebritsen et al. (52) have reported that protein phosphatase 2 was inhibited >90% by 0.1 mM ATP whereas purified liver phosphatase was inhibited only approximately 25%. They also reported (67) that protein phosphatase 2A was relatively more active toward substrates like myosin light chain, histones, and phosphorylase kinase as compared to phosphorylase a. In our study, we found that histone and phosphorylase kinase were relatively poor substrates wheras myosin light chain andphosphorylase a were equally better substrates. In view of these discrepancies, classification of purified rabbit liver high molecular weight phosphoprotein phosphatase has to await furtherstudies. The work presented in thisreport describes the purification and some properties of a high molecular weight phosphoprotein phosphatase from rabbit liver. Further studies are, however, required to completely understand the properties of this enzyme. For example, further studies are needed to examine its specificity for the dephosphorylation of small peptide(s) and nonprotein substrates and to known whether the effects of several compounds tested in this study are substrate directed, enzyme directed, or both. In addition, it is widely accepted that theM , 35,000 subunit possesses catalytic activ-


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32. Tamura, S., Kikuchi, H., Kikuchi, K., Hiraga, A., and Tsuiki, S. (1980) Eur. J.Bwchem. 104,347-355 33. Tamura, S., and Tsuiki, S. (1980) Eur. J. Biochem. 111, 217224 REFERENCES 34. Khandelwal, R. L., and Enno, T. L. (1985) Fed. Proc. 44,688 1, Krebs, E. G., and Beavo, J. A. (1979) Annu. Rev. Biochem. 48, 35. Fischer, E. H., and Krebs, E. G. (1958) J. Biol. Chem. 231, 65923-959 71 2. Cohen, P. (1978) Curr. Top. Cell. Regul. 14, 117-196 36. Hayakawa, T., Perkins, J. P., Walsh, D. A., and Krebs, E. G. 3. Flockhart, D. A., and Corbin, J. D. (1982) CRC Crit. Reu. Biochem. (1973) Biochemistry 12,567-573 13,133-186 37. Yang, S. D., Vandenheede, J. R., and Merlevede, W. (1981) FEBS 4. Graves, D. J., and Wang, J. H. (1972) in The Enzymes (Boyer, P. Lett. 132,293-295 D., ed) 3rd Ed., Vol. 7, pp. 435-482, Academic Press, New York 38. Krebs, E. G., Kent, A. B., and Fischer, E. H. (1958)J. Biol. Chem. 5. Hers, H. G. (1976) Annu. Rev. Biochem. 45, 176-189 231,73-83 6. Fletterick, R. J., and Madsen, N. B. (1980) Annu. Rev. Biochem. 39. Pato, M. D., and Adelstein, R. S. (1983) J. Biol.Chem. 258, 49,31-61 7047-7054 7. Kato, K., and Bishop, J. S. (1972) J. Biol. Chem. 247,7420-7429 40. Gupta, R. C., Khandelwal, R. L., and Sulakhe, P. V. (1984) FEBS 8. Kalala, L. R., Goris, J., and Merlevede, W. (1973) FEBS Lett. Lett. 169,251-255 32,284-288 41. Davis, G. J. (1964) Ann. N. Y. Acad. Sci. 121,404-427 9. Zieve, F. J., and Glinsmann, W. H. (1973) Biochem. Biophys. Res. 42. Laemmli, U. K. (1970) Nature 227, 680-685 43. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Commun. 50,872-878 (1951) J. Biol. Chem. 193, 265-275 10. Brandt, H., Killilea, S. D., and Lee, E. Y. C. (1974) Biochem. 44. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 Bwphys. Res. Commun. 61,598-604 45. Wilson, S. E., Mellgren, R. L., and Schlender, K.K. (1983) 11. Kato, K., and Sato, S. (1974) Biochim. Biophys. Acta 358, 299Biochem. Biophys. Res. Commun. 116,581-586 307 46. Mellgren, R. L., and Schlender, K. K. (1983) Bwchem. Biophys. 12. Abe, N., and Tsuiki, S. (1974) Bwchim. Bwphys. Acta 350,383Res. Commun. 117,501-508 391 47. Disalvo, J., Waelkens, E., Gifford, D., Goris, J., and Merlevede, 13. Nakai, C., and Thomas, J. A. (1974) J. Biol. Chem. 249, 6459W. (1983) Biochem. BioDhvs. Res. Commun. 117.493-500 G4G7 14. Kobayashi, M., Kato, K., and Sato, S. (1975) Biochim. Biophys. 48. Hiraga, A.,'Kikuchi, K., Tamura, S., and Tsuiki, S. (1981) Eur. J. Biochem. 119,503-510 Acta 377,343-355 15. Brandt, H., Capulong, Z.L., and Lee, E. Y. C. (1975) J. Biol. 49. Crouch, D., and Safer, B. (1980) J. Biol. Chem. 255,7918-7924 50. Werth, D. K., Haeberle, J. R., and Hathaway, D. R. (1982) J. Chem. 250,8038-8044 Biol. Chem. 257,7306-7309 16. Khandelwal, R. L., Vandenheede, J. R., and Krebs, E. G. (1976) 51. Usui, H., Kinohara, N., Yoshikawa, K., Imam, M., Imaoka, T., J. Biol. Chem. 251,4850-4858 and Takeda, M. (1983) J. Bid. Chem. 258,10455-10463 17. Lee, E. Y. C., Brandt, H., Capulong, Z.L., and Killilea, S. D. 52. Ingebritsen, T. S., Foulkes, J. G., and Cohen, P. (1980) FEBS (1976) Adv. Enzyme Regul. 14,467-490 Lett. 119,9-15 18. Gratecos, D., Detwiler, T. C., Hurd, S., and Fischer, E. H. (1977) 53. Khandelwal, R. L. (1977) Biochim. Biophys.Acta 485,379-390 Biochemistry 16,4812-4817 19. Hsiao, K. J., Chan, W. W. S., and Li, H.-C. (1977) Biochim. 54. Li, H. C. (1982) Curr. Top. Cell. Regul. 21,129-174 55. Imaoka, T., Imam, M., Ishida, N., and Takeda, M. (1978) Biophys. Acta 483,337-347 Biochim. Biophys. Acta 523, 109-120 20. Li, H. C., Hsiao, K. J., and Chan, W. W. S. (1978) Eur. J. 56. Hsiao, K. J., Sandberg, A. R., and Li, H.-C. (1978) J. Biol. Chem. Biochem. 8 4 , 2 1 6 2 2 5 253,6901-6907 21. Lee, E. Y. C., Mellgren, R. L., Killilea, S. D., and Aylward, J. H. 57. Hutson, N. J., Khatra, B. S., and Soderling, T. R. (1978) J. Biol. (1978) in Regulatory Mechanisms of Carbohydrate Metabolism Chem. 253,2540-2545 (Esmann, V., ed) pp. 327-346, Pergamon Press, Oxford 58. Khatra, B. S., and Soderling, T. R. (1978) Biochem. Biophys. Res. 22. Killilea, S. D., Mellgren, R. L., Aylward, J. H:, Metieh, M. E., Commun. 85,647-654 and Lee, E. Y. C. (1979) Arch. Bwchem. Biophys. 193, 130- 59. Khandelwal, R. L., and Kamani, S. A. S. (1980) Biochim. Biophys. 139 Acta 613,95-105 23. Mellgren, R. L. Aylward, J. H., Killilea, S. D., and Lee, E. Y.C. 60. Mackenzie, C.W., Bulbulian, G. J., and Bishop, J. S. (1980) (1979) J. Biol. Chem. 254,648-652 Bwchim. Biophys. Acta 614,413-424 24. Khandelwal, R. L., and Zimman, S. M. (1978) J. Biol.Chem. 61. Goris, J., Pijnenborg-Vercruysse, L., and Merlevede, W. (1972) 253,560-565 Bwchim. Biophys. Acta 268, 158-165 25. Huang, F. L., and Glinsmann, W. H. (1976) FEBS Lett. 62,32662. Shacter-Noiman, E., and Chock, P. B. (1983) J. Bid. Chem. 258, 329 4214-4219 26. Cohen, P., Nimmo, G. A., and Antoniw, J. F. (1977) Biochem. J. 63. Yan, S. C. B., and Graves, D. J. (1982) Mol. Cell. Bwchem. 42, 162,435-444 21-29 27. Khandelwal, R. L., and Zinman, S. M. (1978) Biochem. Biophys. 64. Killilea, S. D., Mellgren, R. L., Aylward, J. H., and Lee, E. Y. C. Res. Commun. 82,1340-1345 (1978) Biochem. Biophys. Res. Commun. 81, 1040-1046 28. Imam, M., Imaoka, T., Usui, H., and Takeda, M. (1978) Biochem. 65. Usui, H., Imam, M., Imaoka, T., and Takeda, M. (1978) Biochim. Bwphys. Res. Commun. 84,777-785 Biophys. Acta 526, 163-173 29. Brandt, H., Lee, E. Y.C., and Killilea, S. D. (1975) Biochem. 66. Ingebritsen, T. S., Foulkes, J. G., and Cohen, P. (1983) Eur. J. Biophys. Res. Commun. 63,950-956 Biochem. 132,263-274 30. Khandelwal, R. L. (1978) Arch. Biochem. Biophys. 191,764-773 67. Ingebritsen, T. S., and Cohen, P. (1983) Science 221, 331-338 31. Killilea, S. D., Brandt, H., Lee, E. Y. C., and Whelan, W. J. 68. Paris, H., Ganapathi, M. K., Silberman, S. R., Aylward, J. H., (1976) J. Biol. Chem. 251,2363-2368 and Lee, E. Y. C. (1984) J. Biol. Chem. 259, 7510-7518

ity, but the role of the other subunit in dephosphorylation reactions has to be examined by future studies.