Discrimination of multiple forms of phosphoprotein phosphatase in bovine thyroid

Discrimination

of Multiple Forms of Phosphoprotein in Bovine Thyroid

Phosphatase

Kikuo Kasai and James B. Field Phosphoprotein with

phosphatases

phosphorylated

(NH&SO,

precipitation,

chromatography, Phosphatase had the

mixed

(phosphoprotein histones.

gel filtration

four fractions

I had an apparent

greatest

Phosphatases

activity

Hl

phosphohydrolase,

histone

of enzyme

with

activity were obtained

histone

Hl

and was

pyrophosphate

(PPi), ATP,

reaction mixture was the most

sensitive

but not by Mg’+. after dialysis,

inactivated

by Mn *’

stimulated

metalloenzyme.

phosphate

to inhibition. extensive

Phosphatases

PPi, ATP

and NaF probably

Ca”,

Zn2’ and Fe”.

its activity was not further in the presence on metal

In bovine thyroid,

Metal ion stimulation

fluoride

of mM

stimulated

concentration

with

phosphohistones

besides

of phosphatase

with a metal ion binding site on the regulatory

(NaF) when

they were

by Mr?.

phosphatase

the inactivated the enzyme

of Mn*+ and had

manner

added

by sodium

directly

to the

The inactivated

of Pi. Moreover,

on substrate

binding

phosphoprotein

The lowest molecular

Ill activity

by removing

Ill an

enzyme could be fully activated by

pretreated

with Pi retained about 60%

(demetallired)

the Mnzt -reactivated

enzyme enzyme

was less was again

These results suggest that Pi may have

and also that phosphatase phosphatases

I and IIA activities may be through subunit.

Ill, a possible catalytic subunit

was generally independent

inactivated

Whereas

there are at least two major

as substrate.

over &fold by Mn*+ and had much higher

them Pi was the most potent inactivator.

ion binding

I, IIA, 56, and Ill.

PPi was the most potent inhibitor and phosphatase

dialysis to remove these inhibitors,

Ba’+, Cu’+. Cd”,

effect

as phosphatases

I, IIA. and Ill were inhibited in a dose-dependent

(Pi) and sodium

chromatography,

and histone-Sepharose

on Mn’+ for maximal activity. The enzyme

by NaCl

molecular weight of 30,000,

mixed histones as substrate.

by Pi, NaF and ATP. Among

inhibitory

properties.

potassium

with phosphorylated

essential metal ion. After

another

and were designated

and was dependent

greatly

DEAE-cellulose

in 0.2 M 2-mercaptoethanol

than with casein in the presence of the cation. Phosphatase

high activities using all three substrates.

reactivated

were partially purified from bovine thyroid Utilizing

IIA and IIB had a molecular weight of about 70,000, were stimulated

of larger molecular weight forms, had an apparent

activity

EC 3.1.3.16)

as substrates.

before and after freeze-thawing

molecular weight of 155,000

activities with phosphohistones

Mn”,

and casein

an interaction

weight

enzyme

Ill might

be a

which may have different with the substrate

(phosphatase

or

Ill) probably

does not exist naturally in the cell.

T

HE COVALENT MODIFICATION of regulatory enzymes and proteins is now established as an important mechanism for the control of a wide variety of cellular functions.‘.’ The recent demonstration of apparent hormonal regulation of phosphoprotein phosphatase and of phosphoprotein phosphatase inhibitors have provided further interest in this enzyme as a control mechanism.-“’ Although some of the data on phosphoprotein phosphatases are apparently contradictory, it is clear that tissue extracts contain more than one form of this enzyme activity. Multiple species of the enzyme have been isolated from a number of mammalian tissues.” I9 While these species show differences in substrate specificity and metal ion requirement, they are thought to have a common active ‘catalytic subunit'.5.?0~?3 Treatment of enzyme fractions with urea or trypsin, or 80% ethanol at room temperature, or freeze-thawing in 0.2 M 2-mercaptoethanol results in dissociation of the higher molecular weight forms to a ‘catalytic subunit’. Moreover, some of the higher molecular weight forms of phosphatase are stimulated by metals, while lack of metal dependence of the catalytic subunit suggests that metals interact with the regulatory subunit as well as with substrates. However, the catalytic subunit itself may contain essential purified or purified meta1s.24 27 Thus the partially catalytic subunit from skeletal muscle, liver or cardiac 296

muscle is inactivated (demetallized) by ATP, PPi or NaF but reactivated by Mn2+ or Co2+.24.26.27 In contrast the inhibitory effect of Pi has been attributed to an action on substrate binding.28.29 However, recently an additional inhibitory effect has been suggested which may be related to removal of an essential metal ion from the enzyme.27,30 While phosphoprotein phosphatase activities have been described in thyroid tissue,3’-34 the enzyme activity has not been well-characterized. In this paper we report the physical characterization and catalytic properties of the enzymes with different substrates before and after treatment by freeze-thawing in 0.2 M 2-mercaptoethanol. In addition, the differential effects of sodium pyrophosphate (PPi), ATP, potassium phosphate (Pi) and NaF on the different enzyme forms are described.

From the Diabetes Research Laboratory: St. Luke’s Episcopal Hospital: P.O. Box 20269: Department of Medicine: Baylor College of Medicine, Houston. Texas. Received for publication May 25. 1982. Address reprint requesis to James 8. Field, M.D.. St. Luke’s Episcopal Hospital, Texas Medical Center, P.O. Box 20269, Houston, TX 77025. 0 I983 by Grune & Stratton, Inc. Supported in part by U.S. Public Health Service Grant AM 26088 from the National Institutes of Health. 00260495/83/3203-00I3$02.00/0 Metabolism, Vol. 32, No. 3

(March),

1983

PHOSPHOPROTEIN

PHOSPHATASE

MATERIALS

297

IN BOVINE THYROID

AND

30 min. An aliquot

METHODS

of 200 ~1 of the acid-supernatant

added to 3 ml of scintillator

Materia1.s Mixed

aliquot

histones

casein

and

kirase

(Type

and HI

partially

histone

purified

I) from rabbit

Co. DEAE-cellulose

Sephadex

(i-200

thymus.

muscle were products

(y-“P)ATP

from

Sepharose-4B

was purchased

protein of Sigma

New

England

of “P-labeled

Histones

and HI)

(mixed

and cyclic

AMP-dependent

by Meisler

and Langan.”

Tris/HCI,

pH 7.0. 5 mM

cpm/p

mole),

2 mM

protein M&I,,

with After

two further

against

acid

was collected

(TCA)

cyclic

precipitations

mM

0.5

and l-2

volume

mg of

of IO ml.

was precipitated 25%).

and redissolved

with TCA,

two changes of 400 volumes

(2OOG400

concentration

by centrifugation

50 mM

AMP,

or HI)

in a total

(final

as described

contained

at 37C for 2-4 hr, the protein

trichloroacetic

precipitate

kinase

(y-“P)ATP

(y-“P)ATP

5 PM

of histones (mixed

protein

incubation

essentially

mixture

0.1 mM

theophylline,

AMP-dependent

Following

kinase

The reaction

with

the solution

The

in water.

was dialyzed

of 5 mM Tris/HCI

buffer,

pH

7.0. Vitamin-free by Reiman essentially tion

dephosphorylated

100 mg/lO

ml and 3-4

with TCA

(final

concentration

protein

400 volumes of 50 mM Tris/HCI,

against

5 mM Tris/HCI.

content

of typical nmole/mg

nmole/mg

pH 8.0. The solution

of mixed

hislones

Phosphoprotein

with

phosphatase

activity

was

from ‘2P-labelled

enzyme

provided

was dephosphorylated.

mixed histones. respectively,

at every

as substrate.

100 mM

NaCl With

I

(“P)-H

pH 7.0. I mM and

was 2.5

mixture

50 mM

I5 FM

activities

with

modified

using (“P)or -HI

50 mM

mixture

(standard

to determine

Tris/

(buffer

or without

the reaction

was omitted

for up

20% of the

(‘2P)-mixed

contained

of substrate

the

were determined.

2-mercaptoethanol

as substrate.

was slightly

than

and (“P)-casein step. With

by

at 30C for 5 or

rate was linear

no more

purification

(“P)-casein

mixture

that

the reaction EDTA,

determined

substrate

Phosphalase

histone

same as above except that NaCl reaction

phosphate

substrates

and approximately

IO min in a final volume of 60 ~1. The reaction to IO min

MnCI,.

was first

ofcasein

release of (“P)phosphate

HCI.

(j*P)

of phosphorylated

or HI

by

pH 7.0 and then

pH 7.0. The alkali-labile

preparations

collected

was washed with water and

in 50 mM Tris/HCI,

against

AMP-dependent

5’X) and the precipitate

The precipitated

then redissolved

histones

was

casein was precipitated

dialyzed

substrate

procedure

mg of cyclic

kinase was used. The phosphorylated

centrifugation.

IO&I6

as described

to use.lh The phosphorylation

the same as for histones except that the casein concentra-

was

protein

casein was partially

et al prior

A),

5 mM was the

assay). The

the phosphatase

in iractions from DEAE-cellulose chromatography, gel filtration or histone-Sepharose chromatography. The substrate activity (mixed MnCl,

phoaphohistones) concentration

Reaction addition

with

concentration

P-mixed

or

~--HI

3% bovine yerum albumin(BSA) P-casein was substrate. of 100 ~1 of I3’X TCA

was stopped

acid (TCA)

or by the addition

acid in 0. I M H$O, the reaction mixture

by the

and 100 MI of of 100 ~1 of 0.1

and IO0 kl of 0.6% BSA. When was terminated

and 100 ~1 of 0.6%’ BSA. After

min on ice, the incubation

to 5 MM, the

was omitted.

histones

of 100 ~1 of 50% trichloroacetic

M silicotungstic

was reduced

to 2 mM and NaCl

was centrifuged

by the addition standing at 2.000

200 ~1 of

was removed

The data obtained

with

(I: I ). 0.8 ml of the organic

One unit of phosphoprotein that

amount

of enzyme substrate

which

phosphatase released

I .O

phase

by both methods were

for 30 x g for

activity I nmol

was defined of

Pi from

Phosphatases

From

Step 1: Preparation of crude thyroid extract. vine thyroid glands obtained from a local abattoir transported

as the

per minute.

Puri$cation of Phosphoprotein Bovine Thyroid

Substrates

were phosphorylated

EDT/\, 2 mM DTT, 50 mg cyclic

mixture

for counting.

studies, an

with

in 4N HCI, and then extracted

ml of insobutanol-benzene

phosphorylated

Nuclear.

Preparation

was mixed

similar.

Whatmann.

were obtained

from

molybdate

was directly

In preliminary

of 200 PI of the acidsupernatant

4.3%, ammonium

vitamin-free

dependent

52 was obtained

and CNBr-activated

Pharmacia.

calf

3’5’ cyclic-AMP skeletal

Chemical from

from

and counted.

to the laboratory

Bowere

in ice and then trimmed

free of fat and connective tissues. The thyroid tissue was further processed at 4C or frozen at -2OC. All steps were carried out at 4C unless stated otherwise. The tissues (140 g) were cut into small pieces and homogenized with a Waring Blendor in 2.5 vol. of cold 50 mM Tris/HCI, pH 7.0, containing 1 mM EDTA and 50 mM 2-mercaptoethanol (buffer A) for I min. The homogenate was centrifuged at 16,000 x g for 30 min and the supernatant was collected after filtration through glasswool. Step 2: DEAE-cellulose chromatograph,l The 16,000 x g supernatant was directly applied onto a DEAE-52 column (2.5 x 28 cm) equilibrated with buffer A. The column was rinsed with 3 bed volumes of the buffer and phosphatase was eluted with buffer A containing 0.4 M NaCI, assayed with P-mixed histones as substrate. Step 3: (NH,),SO, precipitation. The active fractions were pooled and the protein was precipitated by adding (NH,),SO, to 70%) saturation. After stirring for 1 hr. the precipitate was collected by centrifugation (18,000 x g for 20 min), redissolved in a small volume of buffer A and dialyzed against IO0 volumes of buffer A for 3 hr. Step 4a: Sephadex G-200 gel filtration. The dialyzed fraction was applied to a Sephadex G-200 column (2.5 x 90 cm) previously equilibrated with buffer A containing IO?%glycerol). Two major peaks of phosphatase activity were obtained with P-mixed histones as substrate (Fig. IA). The apparent molecular weight of the first peak was 155.000 and the second was 70,000. The active fractions from each peak were pooled as indicated and were designated as fraction I and fraction II in order of elution. Step 46: Freeze-thawing of (NH,)_SO, precipitate. Alternatively, the 70%) (NH,),SO, precipitate of the active fraction from DEAE-cellulose chromatography was extensively dialyzed against buffer A containing 0.2 M 2-mercaptoethanol and then treated twice by freeze-thawing. Denatured proteins were

298

KASAI AND FIELD

oooled

fractions

Fraction I

I

Number 1

I

I -

-

pooledfractions

5 z 6 ,‘

1.0

A 0.8

10 0.6

i % E E 5 E

0.4

which had been equilibrated with buffer A containing 10% glycerol. Elution was performed with 80 ml of a 0.05-0.7 M linear NaCl gradient after rinsing with four bed volumes of the buffer containing 0.05 M NaCl. As shown in Fig. 2, the activity toward P-mixed histones was eluted as a rather broad peak in case of fraction 1 and as a sharp peak in case of fraction III. When fraction II was applied to the column, one major (IIA) and another minor (IIB) activities were resolved. The respective fractions of the enzyme activity from all four fractions were pooled and concentrated by dialyzing against buffer A containing 50% glycerol for 4 hr. These fractions were designated as phosphatase I, IIA, IIB, and III. They were stored in buffer A containing 50% glycerol at -20C and were utilized for further characterization of the phosphoprotein phosphatase activities.

Other Methods Histone-Sepharose was prepared essentially as described by Cobin et al using CNBr-activated Sepharose 4B and mixed histones.” Protein concentration was measured by the method of Lowry using bovine serum albumin as standard.” Apparent molecu-

5

a

0.2

a

0 0

I3

1.0 ooled fraction

0.8 0.6

0

4

Fraction Number

Fig. 1. Fractionation of phosphatases activities on Sephadex G-200. (A) Gel filtration of the redissolved 70% (NH&SO, precipitate on the column. Each fraction was assayed for enzyme activity with P-mixed histones in the presence of 1 mM EDTA end 2 mM MI?+ at 30C for 5 min. (B) Gel filtration of the supernatant after treatment of the redissolved (NH,),SO, precipitate by freezethawing in the presence of 0.2 M 2-mercaptoethanol. Phosphatase activity was assayed in the same condition as in Fig. 1A. Fraction volume was 1.9 ml and flow rate was 12 ml/h. The enzyme activity is expressed as p mole/min/20 ~1fraction (0). Protein concentration was determined by Lowry’s method (0).

removed by centrifugation at 18,000 x g for 20 min and the clear supernatant was loaded onto the Sephadex G-200 column. As shown in Fig. I B, a new, lower molecular weight (approximately 30,000) activity was obtained (fraction III). Higher molecular weight forms of phosphatase activity did not dissociate to the lower one unless the (NH&SO, precipitate was dialyzed against buffer A containing 0.2 M 2-mercaptoethanol prior to freeze-thawing treatment. The active fractions from Sephadex G-200 were pooled and concentrated by dialyzing against buffer A containing 50% glycerol.

Step 5: Histone-Sepharose afinity chromatografraction from steps 4a and phy. Each concentrated 4b was diluted appropriately with buffer A and applied onto a column of histone-Sepharose 4B (0.8 x 14 cm)

c .-0 ‘j 2 *

3 :: \ .:

2 E

0.4

* 0

0.2 0 1.0

10

a

0.8s

6

O.i3E

4

0.45

2 0

1.0

P

0.8 0.6 0.4 0.2 0

0

10

20

30

40

Fraction Number Elution of phosphatase activity from histone-SephaFig. 2. rose by a linear gradient of NaCl from 0.05 to 0.7 M. The three phosphatase fractions (I, II, and Ill) obtained from Sephadex G-200 column (Figs IA and 1Bl were applied to histone-Sepharose. Figs. A, B, and C show the respective elution pattern of fractions I, II and Ill. Fraction volume was 2.0 ml and flow rate was 20 ml/h. Phosphatase activity (0) was assayed as described in Fig. 1A.

PHOSPHOPROTEIN

Table

PHOSPHATASE

1.

Purification

Purification

IN

BOVINE

THYROID

of Phosphoprotein

with

15 /.rM of Three

299

Phosphatases Different

From

Substrates.

Bovine

Assay

Thyroid.

Conditions

Specific are

Activity

Described

Speohc actwq P-mtxed hlstones

1.

16,000

2.

DEAE

3.

70%

4a.

Sephadex

(mg)

x g sup.

(NH,),

so, G-200

EDTA”

+ MnCI,

0.025

0.067

0.076

0.183

0.050

0.102

0.10

0.26

0.33

0.68

0.10

0.16

2,314

0.10

0.38

0.28

0.78

0.09

0.17

chromatography 0.25

0.65

0.58

1.33

0 23

0.46

0.86

0.24

1.39

0.16

0.23

6.45

5.07

of redissolved

(NH&SO,

ppt.

treated

by freeze-thawing

affinity

rn 0.2

M mercaptoethanol

3.42

16.8

III

3.89

7.17

Phosphatase

I

89.8

0.68

1.56

0.64

3.02

0.65

1.31

IIA

27.2

0.51

6.31

0.95

9.05

0.61

0.83

Phosphatase

IIB

7.1

1.44

8.66

Phosphatase

Ill

7.6

6.80

9.50

EDTA

(1 mM)

of enzyme

and MnCI,

activities

(2.5

bovine serum albumin

were determined (Mr

= 68,000),

= 17,000)

by Sephadex (Mr

=

2.

Divalent

Enzyme

Activities

at Least mM.

was

Cation

ovalbumin (Mr

=

as the marker proteins.

20.fold The

Detected

the specific activities at every three different substrates are 1. Chromatography of the fraction on DEAE-cellulose,

Dependency

Using

P-mixed

with

Different the

Buffer Divalent

Designation

of Phosphoprotein

Histones

(P-HI

B to Determine Cations ‘nd’

were

is used.

and

Phosphatases P-casein

the

Added The

Phosphatase

(2.9)

(4.71

100

100

132

1

0.5

Md’

Mg2’

I P-C

% acttvity mM

So.

to the

I. IIA. The

the

Reaction

Substrate

P-H (p moles/min)

(P-C).

Activity.

(Without

Metal

2.10

21.2

2.55

14.5

13.8

followed by precipitation with (NHJ2S0, resulted a 2-3 fold increase in specific activity. Two major peaks of phosphatase activity could be detected after gel filtration of the redissolved (NHJSO, precipitate on Sephadex G-200 and exhibited a molecular weight above _ 60,000 with P-mixed histones as substrate (Fig. 1A). One of these, designated fraction II, was later resolved into two components (phosphatases IIA and IIB) with distinct kinetic properties by affinity

RESULTS

The protein yield and purification step using summarized in Table 16,000 x g supernatant

12.0

2.88 17.4

(5 mM)

x 90 cm) using bovine y-globulin

and myoglobulin (Mr

Control

10.4

chromatography

Phosphatase

gel filtration

Activity

+ MnCI.

0.11

lar weights

0.05

EDTA

293

“Concentration;

than

+ MnCI,

251

Fraction

Diluted

EDTA

EDTA

I

Hlstone-Sepharose

Table

P-C?%S?ln EDTA

II

5.

160,000),

P-HI hlstone

of

Assay

Fraction

Sup.

45.000),

usbng

Stage

Fraction 4b.

G-200

ppt.

(unlf/mg)

at Every as Standard

3,477

25,000

bulk eluate

Measured

Methods

EDTA”

Total Protein FG%XlWl

was

Under

IIB,

EDTA

Divalent

of Various

in Suffer

Concentration

in the

Mixture

and was

Incubated

5 PM.

Results

Divalent

A Containing Respective

at 30C are

for

Assay 10 min.

Expressed

Cations

50%

on the

Glycerol Mixture

Where

as Percent

were was

less

no Enzyme of Control

cation)

Phosphatase P-H

III. Effects

Stored

Concentration any

and

Enzymes

IIA

Phosphatase IIB

Phosphatase Ill

P-C

P-H

P-C

P-H

P-C

(0.7)

(1.31

(0.8)

11.2)

(5.9)

(5.21

100

100

100

100

100

100

234

259

170

139

169

96

111

155

285

594

192

248

203

116

116 89

5

279

247

1190

234

441

272

148

10

335

211

1390

229

591

250

84

67

25

406

186

1460

159

560

152

58

48

50

350

158

703

28

31

0.5

93

112

96

113

84

102

99

97

1

96

118

98

144

95

96

103

98

5

123

147

139

187

103

122

93

92

10

153

148

130

199

88

117

88

88

25

232

140

189

217

105

109

59

73

50

203

120

193

28

50

397

88

CL2’

5

132

109

121

190

66

101

79

87

Ba”

5

93

97

72

94

85

85

85

93

Fe2’

5

2.8

0.3

nd

nd

nd

nd

nd

nd

QJ*+

5

2.2

0.1

nd

nd

0.3

nd

nd

nd

ZnZ

5

11

0.6

18

3.8

nd

Cd2 +

5

2.5

0.2

nd

2.8 nd

3.6

0.3

2.0

nd

0.3

nd

300

KASAI AND FIELD

chromatography on histone-Sepharose (Fig. 2). The redissolved (NH&SO, precipitate containing fractions I and II could alternatively be dissociated by the treatment of step 4b to reveal a single component, as judged by gel filtration and histone-Sepharose affinity chromatography (Figs. 1B and 2). Phosphatase I had an apparent molecular weight of 155,000 and was stimulated 2-5 times by 5 mM Mn’+ with three different substrates. Overall purification of the enzyme activities was IO-30-fold in the absence or presence of Mn”. The enzyme had greatest activity with P-H I histone in the presence of Mn’+. Phosphatases IIA and IIB had molecular weights of approximately 70,000 and were markedly stimulated by 5 mM Mn’+ with P-histones as substrate, but not with P-casein. In the presence of the cation, they had much higher activities with P-mixed or HI histones than with P-casein. The specific activities of phosphatases I IA and IIB increased 50-130 times with P-histones, but only IO25 times with P-casein, compared with those in the crude extract. Phosphatase I I I had a molecular weight of approximately 30,000 and was generally independent of Mn”. In the absence of the cation, the overall purification of the enzyme activity was 200-300-fold. The enzyme had a relatively high activity with P-HI histone. However, it also could catalyze the dephosphorylation of P-casein and P-mixed histones.

cantly dependent on Mn’+. and were stimulated over tenfold and fivefold, respectively, by 5525 mM of the cation with P-mixed histones as substrate. When Pcasein was used as substrate, phosphatase IIA activity was also stimulated about twice by either Mn” or Mg* +. Phosphatase IIB activity was, however, stimulated 2-3 times only by Mn”. It was also possible to distinguish these enzymes forms by the response to Ca’+ with P-casein as substrate. In contrast, phosphatase III was generally independent of Mn’+ or Mg” with both substrates, but like the other forms of the enzyme was strongly inhibited by Fez+, Cu*+, Zn” and Cd’*. NaCl EjSect on the Activities of Phosphatases I. IIA. tlB, and III The effect of NaCl on the various enzyme activities was examined with all three substrates. As shown in Fig. 3A, phosphatase 1 activity was stimulated by lower concentrations of NaCl with P-mixed histones but inhibited with larger amounts of NaCI. The optimal concentration and magnitude of NaCl stimu-

E E

10

(A)

2.0

I

IIA

2.0

IIB

10

Ill

pH Optima The pH optima for phosphatases I, IIA, IIB and III were determined with P-mixed histones and P-casein as substrates. With P-mixed histones, the pH optimum for enzyme activity was between 6.5 and 7.0. With P-casein, the optimal pH values were between 6.5 and 7.0 for phosphatase I and between 6.5 to 7.5 for phosphatases I IA, I IB and I I I, respectively. Eflects of Various Divalent Activity

NaCI(M)

Cations on Phosphatase

The effects of various divalent cations on the activities of phosphatases 1, IIA, IIB, and III were determined with P-mixed histones and P-casein as substrates (Table 2). The anion for each of these was either chloride or sulfate. Phosphatase I was stimulated by a rather broad range (5-50 mM) of either Mn” or Mg”. With P-mixed histones as substrate Mn” stimulated the activity up to fourfold and Mg*+ stimulated it almost twofold. When P-casein was used as substrate, the activity of phosphate I was stimulated 2-3 times by 0.5-25 mM Mn*‘, but less with Mg”. No other ion tested was capable of stimulating activity. In fact, all ions except the alkaline earths (CA*+ and Ba”) strongly inhibited the enzyme activity. The activities of phosphatases IIA and IIB were signifi-

Fig. 3. IIB

and

Effect Ill

using

substrates. different

The

of NaCl

on the activities

P-mixed enzyme

concentrations

histones activities

in

the

incubation

Tris/HCI,

pH

7.0,

50

M NaCI.

were

of phosphatases and

mM

mixture

which

2-mercaptoethanol,

P-casein

measured

(0; 1.25 NM, 0; 2.5 flM,

substrate O-O.4

(A)

A; 5.0

with pM)

contained 1 mM

I, IIA, (B)

as

three of each 50

mM

EDTA

and

PHOSPHOPROTEIN

PHOSPHATASE

IN BOVINE

THYROID

lation was dependent on the substrate concentration. In the absence of NaCI, the enzyme activity was less in the presence of increasing substrate. This substrate inhibition at 2.5 and 5.0 PM could be overcome by the addition of NaCI. The activities of phosphatase IIA and III were slightly stimulated by lower concentrations of NaCl only when 5.0 PM of P-mixed histones was used. Usually the activities of these enzymes as well as phosphatase IIB were inhibited by NaCl in a concentration dependent manner at least with the substrate concentrations examined. Similar effects of NaCl on enzyme activities with P-HI histone as substrate were observed (data not shown). With P-casein ah substrate, the activities of the various fractions were always inhibited by NaCl in a concentration dependent manner (Fig. 3B).

IL . ‘A)

100

-

c c 0 0 :E 100 a 5 75

z

25

8 k

0

3:

Histone

Comparison and P-Casein.

IIB. and III were and P-Casein Mixture

Used,

P-Mixed of 5 mM

as the Standard

Concentrations

were

Reciprocal

I IIA

I

Assay

of Phosphatases

MnCI,

Methods.

10.5-30 from

Seven

NM) were

Double

Plots

P-mlxed Hetones 4.5

I, IIA, Histone

in the Reaction

Under

Calculated

P-HI

P-HI

/.lM

10.5 PM

IIB

8.0

Ill

6.7 /AA

/Al

P-HI Hostone

6.7 pM ll.BpM

P-case,n 3.1 /.&I 2.2 PM

3.2

/.&I

0.9

GM

4.1

@M

3.0

MM

I

I

I

r

Phosphatase

III

75 50 25 0 t

I. [IA,

Histones,

Histones,

of each Substitute

and Km Values

Substrate:Phosphatase

for P-Mixed

Activities

with

in the Presence

Described

Different

Enzyme

Measured

I

a. 100

10-l 10” 10’ Inhibitor (mM)

lo-’

of Km Values

a

25

The apparent Km values of each phosphatase fraction were determined with the three different substrate (Table 3). The apparent Km values for P-mixed histones of phosphatases I, IIA, IIB, and III were 4.5 PM, 10.5 PM, 8.0 PM and 6.7 PM, respectively. The Km values for P-H I histone were 6.7 PM with phosphatase I, 11.X FM with phosphatase IIA, 3.2 yM with phosphatase IIB and 4.1 PM with phosphatase 111. The apparent Km values for P-casein of phosphatases I, IIA. IIB and III were 3.1 PM, 2.2 PM, 0.9 PM and 3.0 PM. respectively.

Table

PO

50

Catalytic. Properties

As shown in Fig. 4, the activities of phosphatases I, IIA, and III with P-mixed histones as substrate were inhibited by pyrophosphate (PPi), ATP, potassium phosphate (Pi) and sodium fluoride (NaF) in a dosedependent manner. The apparent Ki values of these inhibitors are summarized in Table IV. PPi is the most potent inhibitor, while NaF and Pi produce similar inhibition. Since phosphatase III was the most sensitive to these inhibitors and a possible catalytic subunit

I

1

75

50

on Phosphatases

I

I

Phosphatase

;

Efects of Various Inhibitors and 111

I

Fig.

4.

Effects

potassium

of phosphetases The substrate of the and

enzyme buffer

preincubated

then

the

to incubate

3 PM and

pyrophosphate

of the

various

(PPi)

P-mixed at 30C

reaction for

was

5 min

substrate,

histones

started

at 30C. of

(0).

the activities as substrate.

for 2 min in the presence by adding

enzyme

reaction

mixture

The

an appropriate

concentrations

(01, ATP

(0 I on

pH 7.0 (Pi) (A) and NaF

I. IIA and Ill with was

inhibitor,

allowed

contained

of sodium

phosphate,

10’

the

amount

inhibitor

in 60

of the ~1 of

A.

of the larger molecular weight forms, further studies to elucidate the mechanism of the inhibition were done primarily with this enzyme. The results in Fig. 5 demonstrate that the inhibition by PPi, Pi or NaF is consistently observed with a wide range of substrate concentration, but the inhibition by ATP appears to be overcome by increasing the substrate concentration. Double reciprocal plots, however, showed that ATP inhibition was not competitive (data not shown). Reversibility

of Inhibition

ofEn:~~nle

Actil+t!3

In the absence of metal ion, incubation of phosphatase Ill with PPi. ATP and NaF prior to the assay was

302

KASAI AND FIELD

20

I

I

I

I

I

I

5

10

1E

P-mixed

histones

15

10

5

0

1

Fig. 5. Effect of substrate concentration on inhibition of phosphatase III activity by PPi, ATP, Pi or NaF. After preincubation of various concentrations of P-mixed histones with the respective inhibitor at 30C for 2 min. the reaction was performed at 30C for 5 min by adding 0.5 pg of the enzyme. The final concentration of the inhibitor was 0.07 mM (PPi) W.O.2 mM (ATP) (0). 7 mM (Pi) (A) or 8 mM (NaF) (0).

associated with over 80% reduction of enzyme activity after removing the agent (Fig. 6). In contrast, Pipretreated enzyme retained about 80% of its activity after dialysis in spite of using concentration above the Ki. While increasing amounts of Mn” restored the activities of PPi- ATP- and NaF-pretreated enzymes to control activity, the Pi-pretreated enzyme activity was not influenced. The same range of concentrations of Mg” had no effect on enzyme activity. Other divalent cations such as Ca’+, Ba*‘, Cu’+, Cd’*, Zn*+ and Fe” also did not restore the enzyme activity (data not shown). Similarly, pretreatment of phosphatases I and IIA with 2 mM ATP also inhibited enzyme activities after removal of the agent. Thus ATPpretreated phosphatases I and IIA lost about 40% and 50% of their activities, respectively. Basal enzyme activities were progressively augmented by increasing amounts of Mn*’ which were capable of overcoming the inhibition induced by ATP (Fig. 7). Furthermore, the inhibition of phosphatase III activity by ATP (0.05 or I .25 mM), PPi (0.05 or 0.5 mM) or NaF (2.5 or 25 mM) was reversed in a dose-dependent manner by adding Mn’+ (0.25 or 2.5 mM) to the enzyme simultaneously with the inhibitor. In the case of Pi, the inhibition of the enzyme activity by 2.5 or 25 mM of Pi was not reversed by simultaneous addition of Mn” (data not shown). Efects of ATP, Pi and NaF on the Reactivation by Mn” of Inactivated Enzyme (Apoenzyme) As shown in Table 5A, the activity of apophosphatase III obtained by ATP pretreatment, was further inhibited by incubation with Pi, NaF, or ATP. The activities of apophosphatase III incubated with 0.1 or 1 A) PhosphataseI

B) PhosphataseIIA

Control ATP-treated

---

Control

1000

ATP-treated

1 1200

x

I/

Mn’* (mM)

B) F

Mg2+(mtvl)

i

Fig. 6. Effects of Mn’+ and Mg2+ on phosphatase III pretreated with PPi, ATP, Pi or NaF. Phosphatase Ill (25 pg) was preincubated with buffer A (control) or buffer A containing appropriate inhibitor (1 mM PPi, 1 mM ATP, 50 mM Pi or 50 mM NaF) at 30C for 5 min. The

preincubation

taining

0.2%

mixture

BSA,

without

EDTA

dialyzed

mixture

followed

(buffer with

or without

various

diluted

twice

by extensive

8) for 4 hr. After buffer

for 10 min in 60 pl of buffer with

was

8 containing

buffer

against

appropriate

8. the reaction

concentrations

with

dialysis

3 PM of Mnzr

of P-mixed or Mg”.

A

of the at 30C

histones

Inactivation

Fig. 7.

A conbuffer

dilution

was performed

MI? (mM1

by

ATP

and

preincubated 30C

for

mixture

Mn”. with

5 min.

and reactivation Phosphatases

buffer

Following

as described

30C for 5 min with

A or buffer the

same

I

of phosphateses and

various

were

A containing treatment

in Fig. 6, the enzyme or without

IIA

2 mM

of the

activity

concentrations

I and IIA

respectively ATP

at

incubation

was assayed of Mn”.

at

303

PHOSPHOPROTEIN PHOSPHATASE IN BOVINE THYROID

Table 4. Activities

The Apparent

of Phosphatase

Ki Values of the Inhibitors of the I, HA, and Ill. The Enzyme Activity was

Assayed as Described

Effects of ATP, Pi and NaF on Mn” Reactivated Enzyme Activity

in Fig. 6

PhosphataseI

PhosphataseIIA

PPI

0.5f

0.5

0.06

ATP

2.5

1.5

0.25

PhosphataseIll

PI

50

10

7

NaF

20

10

10

lmM concentration I” the reaction mixture.

mM of ATP and 1 mM of NaF were activated manner, respectively by Mn’+ in a dose-dependent while the apoenzyme activity incubated with 1 or IO mM of Pi and 10 mM of NaF were less reactivated. Namely, treatment of apophosphatase III with Pi at or below the Ki (Table 4) effectively prevented reactivation by Mn”‘. Higher concentration of NaF also mimicked the Pi effect. Next, the effects of Pi and NaF on apophosphatase III obtained by ATP or PPipretreatment were compared to those on phosphatase III (Fig. 8). While Mn*+ restored the activity of apophosphatase in a dose-dependent manner up to control activity, the inhibitory effects of Pi and NaF were much greater on apophosphatase III than on phosphatase III when they were added simultaneously to the enzyme with Mn’+. Especially, Pi inhibited almost completely the reactivation of enzyme activity by Mn’+.

As presented in Table 5B, the activities of Mn”reactivated phosphatase III which were obtained by the incubation of apophosphatase III with various were again inactivated by concentrations of Mn”, later addition of Pi, NaF or ATP. Treatment with Pi or NaF at or below the Ki effectively inactivated the Mn” -reactivated enzyme activity. Namely, Pi (1 or IO mM) and NaF (10 mM) inactivated the activity to about or below 10% of control activity, while ATP (0. I or 1 mM) and NaF (1 mM) produced lesser inhibition. Pi was the most potent inactivator among them. DISCUSSION

The present results demonstrate that thyroid tissue contains several different phosphoprotein phosphatase activities. These are clearly different on the basis of molecular weight, substrate specificity, metal ion dependency, response to NaCl and response to various inhibitors. In the starting material ( 16,000 x g supernatant fraction) the majority of phosphatase activity in bovine thyroid exhibited a molecular weight above 60,000. Chromatography on Sephadex and histoneSepharose readily separated three higher molecular weight phosphatases. The dissociation of higher molecular weight forms to a single lower one was obtained by

Table 5. Effect of Inhibitor on Apophosphatase

Ill Activity B

A 1st lncubatlon

2nd lncubatlon 0 (mMI

buffer B

PI (1 mM)

PI (10 mM)

Mn”

Mn“

ATP (0.1 mMI

ATP 11 mMi

2.01

2nd Incubatvx

0 (mM)

13.6

Ml?+ 0.5

5.0

34.8

5.0

0

1.3

0.5

0.7

Mn’+ 0.5

5.0

3.4

5.0

0

0.4

0

2.8 buffer B

18.4 40.5

0

2.6 PI I1 mM)

3.6 5.1 1.9

0

5.0

0

5.0

2.0

0.8

0

2.1

Mr?+ 0.5

9.7

5.0

12.6

MI?- 0.5

Actwlty

Mn’+ 0.5

0 NaF (10 mM)

1st lncubatlon

0.5

0 NaF (1 mM)

Acfw~fv

MI?’

0.5

0

Mn2’ 0.5

5.0

2.7

5.0

0

0.8

0

5.0

32.3

Mn”

Mn”

0.5

0

0.5

3.3

Mr? * 0.5

5.0

23.4

5.0

10.3 1.8

NaF (1OmM)

3.1 3.2 2.7

ATP (0.1 mM)

5.0

0

2.1

10.4

0

0.8

10.2

NaF (1 mM)

5.0

Mn’+ 0.5

Mn*’ 0.5

PI (10 mM)

9.9 35.4

0

2.1 ATP (1 mM)

4.4 17.4

Al Apophosphatase III obtamed as in Fig 6, was first incubated with buffer or inhibitor at 30C for 5 mm. Then, 20 ~1 of buffer or Mn2’ was added to 20 @I of the respectwe rmxture. After 15 min on ice, the enzyme assay was performed by addmg 20 MI of P-mixed hlstones (fInal 6 PM) at 30C for 10 ml”. Bj Alternatwely, apophosphatase III was prewxubated wtth buffer or Mn2+ at 30C for 5 mm. Then, 20 MI of buffer or respectwe tnhlbltor was added to 20 ~1 of the prelncubatlon mtxture. The enzyme actwity was measured by addlng the substrate as in A). l

p moles/ 10 nxn.

304

KASAI AND FIELD

*O”

r

A) (Control) Buffer

I

Pi _

Cl

NaF

5mM

Cl

l-i 1 EI225mM

5mM

q 25mM

in the preincubati

100

III E 2 g .o

0

z 200 L ‘:

6) (ATP-treated) Buffer r-l

0

0.1 0.5

5

Pi

0

0.1

NaF

0.5

5

0

0.1 0.5

5

M’.’ (mM)in the preincubation Fig. 8. The activities of apophosphatase Ill obtained by buffer A, 1 mM of ATP or 1 mM of PPi-treatment as described in Fig. 6, were measured at 30C for 5 min by adding of P-mixed histones (final 3 PM) to 40 ~1 of the mixture which consisted of the appropriate amou# of the respective enzyme, Mn’+ and Pi or NaF.

freeze-thawing treatment in 0.2 M 2-mercaptoethanol. It is possible that this lower molecular weight form was not present in the intact cell, but was formed by the drastic treatment. Phosphatases 1. IIA, IIB, and III respectively had apparent molecular weight of 155,000, 70,000, 70,000, and 30,000. While phosphatase 111 had a broad substrate specificity and was generally independent of divalent cations, the activities of phosphatases IIA and IIB were stimulated by Mn2+ or Mg*+. In the presence of Mn”, they had much higher activities with P-histones than with P-casein. Phosphatases IIA and IIB had several similar properties such as substrate specificity, Mr?+ dependency, molecular weight and response to NaCl, but some differences were also observed in regard to the elution pattern from the affinity column and Km values of the substrate. Phosphatase I activity was also stimulated by by Mn”’ and Mg2+, and was greatly stimulated NaCl with P-histones as substrate but not with Pcasein. Accordingly, treatment of a fraction containing higher molecular weight phosphatases by freeze-thawing in 0.2 M 2-mercaptoethanol resulted in a marked reduction in molecular weight, a loss of metal ion

dependency and changes of substrate specificity and response to NaCl. The present description of multiple forms of thyroid phosphoprotein phosphatase may be compared to earlier preliminary studies.3’,3’ Spaulding and Barrow reported that DEAE-Sephadex chromatography of the 105,000 x g supernatant of thyroid homogenate produced at least three peaks.” Peaks I and II had greater activity with P-protamine, and peak III had relatively more activity with P-histone. Peak III was stimulated
PHOSPHOPROTEIN

PHOSPHATASE

IN BOVINE THYROID

the conformational state of its substrate. The dissociation of the phosphatase I to phosphatase III may be accompanied with loss of this high sensitivity. The discrepancy in divalent cation dependency between higher molecular weight forms and possibly their catalytic subunit suggests interaction of metals with regulatory subunit(s) as well as its substrate. This assumption appears to be further supported by the study of inhibitors on phosphatases I, IIA, and III. It is demonstrated that phosphatases 1, IIA, and III are inhibited by PPi, ATP, Pi and NaF in a dosedependent manner. PPi is the most potent inhibitor and phosphatase III is the most sensitive to these inhibitors. These results are compatible with previous reports using various phosphoprotein phosphatases from other tjssues?“.?x.?9 except the recent one by Khandelwal et al.” The latter authors reported that Pi (0.5-25 mM) and PPi (0.255 IO mM) stimulated the activities of two purified phosphotases from rabbit liver with histone, but not with phosphorylase a and casein as substrate. They did observe slight inhibition when lower concentrations (0.01-0.25 mM), of PPi were used.4’ The reason for this discrepancy is not clear. As shown in Fig. 5, the inhibitory effect of ATP was overcome by 15 PM of the substrate. However, this inhibition by ATP was not competitive. Li and Hsiao reported that the extent of the stimulatory or inhibitory effect of ATP on the enzyme activity may reflect the net result of an interaction of ATP with the enzyme resulting in an inactivation and an interaction of ATP with phosphohistone resulting in a better substrate.” Accordingly. it is suggested that ATP might affect the enzyme activity by interacting with phosphohistones, resulting in a decrease of inhibition at the highest concentration of the substrate. ATP may inactivate the enzyme activity by chelating an essential metal ion as described below. Incubation of phosphatase III with PPi. ATP and NaF followed by extensive dialysis to remove inhibitor inactivated the enzyme activity by converting it to a divalent cation-dependent form (Fig. 6). This inactive form of the enzyme was fully reactivated by Mn”, but not by Mg”, Ca’+, Ba’*, Cu’+, Cd”, Zn” and Fe”. These results are compatible with the previous reports in other tissues.‘4 “.‘Q.‘~Generally, enzymes inactivated in this way are reported to be activated by Mn“ or Co?+ (or Mg’t).‘4 “.‘O Some authors suggest that Mn” is an essential metal ion for catalytic activity.‘h.‘o Moreover, Defreyne et al.4’ using e.p.r. measurement. reported that the phosphorylase phosphatase associated with dog liver particulate glycogen is a manganese metalloenzyme, Mn” is not adsorbed on the protein surface but buried in the structure of the enzyme. Accordingly, it is suggested that thyroid phosphatase 111 may be a metalloenzyme

305

which can undergo interconversion between active (metallized) and inactive (demetallized) forms. Moreover, phosphatases I and IIA may also be interconverted between active and inactive forms by the same treatment. Since Mg’+ cannot substitute for Mn” to activate the apophosphatase III, the metal ion stimulating effects on the activities of phosphatases I and IIA may be through other interactions with regulatory subunit(s) as well as with their substrates. On the other hand, Pi which is a product of the enzyme reaction, has been shown to be a reversible or competitive inhibitor of the enzyme activity.‘h.‘X.29.4’ The activity of phosphatase III treated with Pi above the Ki retains about 80% of control activity after dialysis (Fig. 6). This result suggest that removal of Pi may restore the enzymes activity. However, as shown in Table 5 and Fig. 8, activation of apophosphatase III by Mn’. is effectively prevented by Pi and NaF. Furthermore, the Mn’+-reactivated enzyme is again inactivated by Pi, NaF, and ATP. Especially, inhibition by Pi is much stronger on the once-demetallized phosphatase III than on phosphatase III. These results suggest another inhibitory effect of Pi on metal ion binding besides substrate binding. Khatra et al reported that a phosphoprotein phosphatase from rabbit skeletal muscle was inactivated by a I6 h dialysis against 5 mM potassium phosphate with I mM EDTA. but that the enzyme after appropriate dilution was fully reactivated by preincubation with Mn”.” Burchell et al also reported that phosphorylase phosphatase activity in glycogen complex was inhibited by incubation with 0.5 M potassium phosphate. Preincubation of the treated enzyme with Mn2’ restored only 10% of the activity after extensive dialysis against the buffer without potassium phosphate?” These reports also suggest another inhibitory effect of Pi on the enzyme activity possibly through removal of an essential metal ion for catalytic activity. In these two reports, however, there is a discrepancy concerning the reactivation by Mn” of the inactivated enzyme. Although the reason is not clear, there is one possibility that the concentration of Pi in the tinal reaction mixture may be different. Therefore. in the present study, Pi, at a concentration at or below the Ki was added to the apophosphatase I I I obtained by pretreatment with ATP or PPi, and etfectively prevented the reactivation of the enzyme activity by Mn’. . Accordingly, it is suggested that potassium phosphate may have at least two inhibitory effects on phosphatase activity; one is on substrate binding and the other is on metal ion binding. As suggested by Li et al. the Mn’ ’ -activation of apophosphatase might be associated with loose binding of Mn’. to the enzyme compared to tight binding to its original form.” The

306

KASAI AND FIELD

inhibitory effect of Pi on essential metal ion binding would be manifested on the Mn2+-reactivated enzyme activity. The present study demonstrates that bovine thyroid contains at least two major phosphoprotein phosphatase activities, although it is possible that the enzyme fractions are proteolytic products of the same enzyme. The different properties of the larger molecular weight forms of the enzyme activity in regard to the substrate specificity, metal ion dependency and responses to salt or inhibitors, suggest different regulatory mechanisms which could reflect distinct roles for each enzyme in the control of protein dephosphorylation by the cell. The function of the thyroid is mainly regulated by the action of thyrotropin on the adenylate cyclase-cyclic AMP-protein kinase system, although several other

regulatory mechanisms may also be important.4’m4n In the thyroid, phosphorylation of several proteins was augmented by cyclic AMP.49.50 However, the only endogenous substrate which has been identified with certainty is histone Hl and possibly histone H3.5’.52 Moreover, phosphoprotein phosphatase activity in rat thyroid may also be regulated by thyrotropin.33.34 Accordingly, the presence of multiple forms of phosphoprotein phosphatase activity in bovine thyroid could provide a possible additional mechanism for the regulation of the functional changes induced by thyrotropin. ACKNOWLEDGMENT The authors assistance.

are grateful

to Shelley

Dearing

for her secretarial

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pro-

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Nucleotide

Lamy

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tein kinases:

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IO, 1975

Dumont

histones.

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