BIOCHEMICAL

Vol. 76, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REVERSIBLEINHIBITION BY DIAMIDE OF CYCLICAMP-DEPENDENT PROTEINKINASESFROMBOVINETHYROID

Michael McClung and Joan Miller

Division University

of Endocrinology

of Oregon Health Sciences Center Portland,

Received

April

Oregon 97201

26,1977

SUMMARY: The effects of diamide on protein kinases isolated from bovine thyroid were studied. Cyclic AMP-dependent protein kinase activity was directly, rapidly, and reversibly inhibited by diamide. This inhibition was non-competitive with respect to ATP or histone end could be prevented by thiol-reducing agents. However, a cyclic nucleotide-independent thyroid protein kinase was Our data indicate that diamide specifically inhibits protein not affected. kinases which are cyclic AMP-dependent. INZRODUCTION:The cyclic

AMP-protein kinase cascade has been well studied and

is considered to be sn important step in the regulation Yet the functional

TSH (l-5).

by Von Tersch et al. (6) that protein by diamide was of interest.

inhibitedtboth

protein

function by

role of this cascade has not been defined.

Because of our involvement with thyroid

inhibited

of thyroid

kinase activity

protein kinases, the recent observation kinase activity

from swine kidney was

They subsequently reported that diamide and amino acid and glucose uptake in rat

kidney slices (7) and have suggested that diamide might be useful in studies of the physiological

role of protein

Diamide is an oxidizing thiol

kinases in various systems.

agent which has been used to alter

compoundsin several tissues (8-U).

oxidation teins,

of glutathione,

are also oxidized when intact

low concentrations, addition

but thiol

of thiol

it

is non-toxic

It has a high affinity

intracellular for the

groups of other compounds, including procells

are exposed to diamide (11).

and its effect

In

can be reversed by the

reducing agents.

Copyright 0 1977 by Academic Alt rights of reproduction in any

Press, Inc.

form reserved.

910 ISSN

0006291

X

BIOCHEMICAL

Vol. 76, No. 3, I977

AND BIOPHYSICAL

Our study was deeigned to examine the effects kinaees isolated

f-o?? bovine thyroid

diamide specificslag-

inhibits

glands.

the cyclic

RESEARCH COMMUNICATIONS

of diamide on protein

These data demonstrate that

AMP-dependent protein

kinase activity.

MATERIALSANDMETHODS: Preparation of protein kinases: Fresh bovine thyroid glands were homogenized and fractionated by the method of Wolff and Jones (12). All procedures were performed at k" C. The 37,000 X g supernatant was centrifuged at 105,000 X g for 1 h in sn IEC Model B-60 ultracentrifuge. This post7iicrosomal supernatant was dialyzed overnight vs 10 mMTris*Cl, 1 mMdithiot.hreitol, pH 7.2 (buffer A), added to a DEAE-cellulose column (2.5 x 40 cm) equilibrated with buffer A, end the protein kinases were eluted with a linear gradient of NaCl, O-500 mM, in buffer A. Three peaks of protein kinase activity were found. The first two eluted at 50 and 125 mMNaCl, respectively, phosphorylated mixed histones, were stimulated 5-fold by cyclic AMP, pOSBe55edcyclic AMP-binding activity, and seemedidentical to the two histone kinase peaks previously described in bovine thyroid glands (3,4). The third protein kinase peak eluted at 200 mMNaCl, phosphorylated phosvitin but not hietone, snd was cyclic AMP-independent. In several respects, this phosvitin kinase hes been shownto be distinct from both the cyclic AMPdependent kinase holoenzymes and their free catalytic subunits (M. McClung, J. Miller, manuscript in preparation). The histone kinase peak which eluted at 125 mMNaCl (peak II) was pooled and precipitated by adding solid ammonium sulfate (32.5 g/100 ml) with constant stirring. The precipitate was resuspended in 50 mMpotassium phosphate, pH 7.0, containing 2 mMEDNA(buffer B) and then chromatographed on a 2.5 x 90 cm co1""3 of A M agarose (BioRad) equilibrated in buffer B. Histone kinase and [ H]cyc I ic AMP-binding activity elated as a single peak which was devoid of phosvitin kinase activity. This preparation was used in the following experiments unless otherwise stated. Protein kinase assays: Standard kinase assays were performed in triplicate in an incubation volume of 0.1 ml contt@ing 50 mMpotassium phosphate, pH 7.0, 10 mMmagnesiumchloride, 0.1 mM[y P]-ATP (specific activity 200-400 cpm/pmole), 200 M histone II-A, and enzyme. When added, cyclic AMP concentration was 2 @f. After incubation for 20 min at 37' in a shaking water bath, reactions were terminated by spotting 50 & assay mixture onto 2x2 cm squares of Whatman3!#4 filter paper. The filter squares were processed as described by Corbin snd Reimsnn (13). Using purified enzyme preparations, kinase activity wa8 linear with respect to time and enzyme concentration. Phosvitin kinase activity was assayed in a similar manner except the MgCl concentration was 2 mMand 200 a phosvitin was used as substrate instead of ii istone. Gilman's method of measusing f3H]cyclic AMP-binding to protein kinases was employed using 5 pmoles [ HIcyclic AMP (14). Protein concentration was determined with the method of Lowry (15). Diamide, pr@ein kinase ubstrates, and thiol reducing agents were obtained kom Sigma; [y P]ATP and [ $ ]cyclic AMPwere purchased from NewEngland Nuclear. REWLTS :

Diamide markedly inhibited

dependent manner (Fig. absence of cyclic

Inhibition

was observed in both the presence and

AMP, although the effect

was more pronounced. kinase activity

1).

histone kinase in a concentration-

on cyclic

At &amide concentrations

was less than 5% of initial

911

AMP-stimulated activity

of 10 m&land greater,

total

values, and the response to cyclic

BIOCHEMICAL

Vol. 76, No. 3, 1977

AND BIOPHYSICAL

DIAMIOE

RESEARCH COMMUNICATIONS

(mM)

Mgure 1. Effect of diamide concentration on thyroid histone kinase activity measured in the presence (O-4) and absence (O--O) of 2 @I cyclic expressed as pmoles phosphate trsnsferred/mg protein/min AMP. Kinase activity (mean + S.E. of triplicate determinations).

The half-maximal

AMPwas no longer apparent.

inhibitory

concentration

of

diamide waa 0.8 mM.

Changesin substrate concentration A reciprocal

plot of the data

competitive with histone. inhibition

did not alter the effect

(Pig. 2) revealed that the inhibition

was non-

Similar experiments also demonstrated non-competitive

-5 with respect to ATP. The Km for ATP was 1.9 x 10 M in both the

presence and absence of 20 mMdiamide.

Inhibition

by diamide was also observed

when substrates other than the mixed histone fraction l&ion

of diamide.

were used.

The phosphory-

of protamine, casein, and other histone preparati'ons was decreased

approximately

The effect

90%by adding 20 mMdiamide in the presence of cyclic

ofthiol

to diamide was examined.

reducing agents on kinase activity Addition

and on its

of 10 mM2-mercaptoethanol,

912

AMP.

response

dithiolthreitol,

Vol. 76, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

s Figure 2. LIneweaver-Burke plot of the relationship between histone concentration and diamiae inhibition of thyroid histone kinase activity in the presence ma absence of 2 fl cyclic AMP. l/S = l/histone concentration (mg/ml). l/V = l/pmoles phosphate tranaferred/mg protein/min. (Q-O) NO additions, (-1 20 mMaiamiae, (O--O) cyclic AMP, (O-4 cyclic AMP + 20 mMaiamiae. Km (hiatone) = 2.7 n&ml.

or reduced glutathione

slightly aufricient

(Table I).

increased hiatone phosphorylation

by kinase only

However, when these agents were added in concentrations

to reduce the diamide in the asaay, no significant

kinase activity

decrease in

was observed.

To study the reversibility

of kinase inhibition

by diamide, purified

histone kinase waa incubated on ice for 15 minutes in 0.5 ml 50 mMpotassium phosphate, 2 mMEIYTA, pH 7.0, with and without dialyzed

overnight

10 mMaiamide.

Each sample was

ve 50 mMpotassium phosphate, 2 mMEIYTA, pH 7.0, and then

assayed for kinase activity.

Even after

dialysis,

activity

was decreased in

the sample which had been pre-incubated with diamide (Table II). kinase activity

addition

in the diamide-treated

of 18 mMaithiothreitol

To exsmine the specificity

sample was fully

However,

restored by the

to the assay medium. with which diamiae inhibits

cyclic

AMP-dependent

protein kinases, each of the three kinase peaks which were eluted from the DEAE

913

BIOCHEMICAL

Vol. 76, No. 3, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table I. Effect ofthiol reducing agents on dismide inhibition of protein kinase. Standard histone kinase assay conditions with 2 $4 cyclic AMPwere used. Kinase activity expressed as pmoles phosphate transferred/mg protein/min + SE of triplicate determination.

Protein kinase activity Addition

No dismide

None

534 f 27

208 2 20

641 + 27

614 + 44

655 + 38

626 f 12

680 2 24

626

Dithiothreitol, Glutathione,

10 mM 10 mM

2-mercaptoethanol,

10 mN

5 mhfdiamide

+ 59

Table II. Reversibility of diamide inhibition oC kinase activity following removal of diamide by dialysis. Assays were performed under standard conditions with 2 m cyclic AMPand additions as listed. Results shown are the average f S.E. of triplicate assays expressed as pmoles phosphate transferredfmg proteinrmin.

Kinase activitv ----Additions to kinase assay

Pre-incubation with 10 mMdiamide

Pre-incubation without diamide

None

307 2 21

42 210

Dithiothreitol,

5 mM

351 + 18

283 + 32

Dithiothreitol,

18 mM

317 + l?

322 + 24

column were assayed with and without diamide (Table III). both peaks of cyclic affect

the activity

preferentially [3H]cyclic

AMP-dependent protein kinase. of the cyclic

AMP-binding to either

However, diamide did not

nucleotide-independent

phosphorylates phosvitin

(peak III).

protein kinase which

Dismide did not alter

of the histone kinases.

914

Diamide inhibited

Vol. 76, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table III. Specificity of protein kinase inhibition by diamide. Peaks I, II, and III eluted from IEAE-cellulgse column at 50, 125, and 200 mMNaCl, respecsively, as described under Methods. [ H]cyclic AMP-binding is expressed as pmoles [ H]cyclic AMPbound/mg protein. Histone and phosvitin kinase were assayed under standard conditions, and activity is expressed as pmoles phosphate transferred/mg protein/ min. AU assays were performed in the presence and absence of 20 mMdismide, and the results listed are meansof triplicate determinations.

Activity Fraction

and activity

assayed

Without diamide

$ inhibition by dismide

With diamide

Peak I [3H]cyclic AMPbinding Aistone kinase, no cyclic AMP Ristone kinase, 2 @4cyclic AMP

3::; 162.9

2.0 11.0 14.2

6Ro 91

14.0 42.9 246.1

14.7 17.1 28.3

6: 89

224.5

220.1

0

Peak II [ 3~lcyclic AMPbinding Histone kinase, no cyclic AMP Histone kinase, 2 @4cyclic AMP Peak III Phostitin

kinase

DISCWSION: The data in this report confinn the observation that protein kinase activity

is reversibly

shown that this

inhibited

by diamide (6).

occurs with cyclic

et al. (6) did not convincingly AMP-dependent protein by only lo-16%. presence of cyclic

More importantly,

AMP-dependent protein kinases.

we have Von Tersch

establish that they were studying a cyclic

kinase, since addition of cyclic

As evidenced by the Y-fold

increase in enzyme activity

AMPand the associated [%]cyclic

the kinases which could be inhibited

AMP stimulated activity in the

AMP-binding activities,

in our studies were clearly

cyclic

AMP-

dependent.

The effects

observed with diamide differ

from those seen with other protein investigated bition

the inhibition

in several important respects

kinase inhibitors.

of thyroid

Kariya and Field recently

protein kinase by adenosine (16).

was seen only when adenosine or one of its

915

derivatives

Inhi-

was present in

Vol. 76, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION ol2TPI

was separated

Cl esterase

inhibitor

@l-antitrypsin

inhibitor,

stable

Thus,

inhibited

very

The extent

of the

proteinases was less

it

selectively

inhibitory

sensitive,

Ficin

and fruit

the inhibition

well

known

is

that

other

proteinases

u2TPI

inhibits

inhibition The biological

inhibitor

at inflammatory the prolonged influenced

determine matory

by blood whether

loci

phase

ti2TPI

and by what

this

is

and papain

different

were

most

(2,

proteinases

o(2TPI is not

yet

from known.

be in the control

In fact,

Beloff

and tissue

and Tokaji

which

inhibitors.

thiol

It

is

interacts

with

the proteinases

mechanism

this

occurs.

is

but

(14).

In contrast,

that

the mechanism

of

Oc2-macroglobulin.

also

of

one of the probable proteihases

(16)

depends

It

proteinases

However,

of the

(15)

of inflammation,

that

sensi-

may suggest

combination.

proteinases

different

Bl

less

proteinases

serine

thiol

cathepsin

considerably

indicating

origins.

among several

stoichiometric

13) and acidic

proper-

and plant

sensitive,

were

not only

(ceruloplasmin

a new serum component.

among thiol

inhibits

from another

of animal

however,

and from

physicochemical

inhibitor

was,

and

mobilities.

gTP1

similar

proteinases

substantially

may

sites.

that

due to a simple

thiol

functionof

of this

that

thiol

selectively by @2TPI is

had very

weights,

serum components

and stem bromelains

a*-macroglobulin like

that

inhibitor

electrophoretic

distinguished

of susceptibility not

d-trypsin

molecular

two other

thiol effect

investigated.

that

role

and from

appears

Such a difference

tive.

of their

further

glycoprotein)

to qTP1.

0(2TPI

III,

inter

by their

of antigenicities

antithrombin

and @'*-heat

is

by the difference

and $-antichymotrypsin

The examination

ties

from C$macroglobulin,

have

on thiol

released indicated

proteinases,

of further

interest

to

released

in the

inflam-

REFERENCES 1. Heimburger, N., Haupt, H. and Schwick, H.G. (1971) Proceedings of the International Research Conference on Proteinase Inhibitors, ~~-1-22, Walter de Gruyter, Berlin-New York. 2. Sasaki,M.,Yamamoto, H., Yamamoto. H. and Iida S. (1974) J. Biochem. -'75 171-177. 3. Kimmel, J.R. and Smith, E.L. (1954) J. Biol. Chem. 207, 515-531. 4. Englund, T.R., King, T.P. and Craig, L.C. (1968) Biochemistry 1, 163-175.

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