Influence of Ca++ and Mg++ on the vanadate inhibition of the Ca++-ATPase from pig heart sarcoplasmic reticulum

Vol.

92, No.

February

4, 1980

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27, 1980

INFLUENCEOF Ca++ AND Mg++ ON THE VANADATE INHIBITION OF THE Ca+'- ATPase FROMPIG HEART SARCOPLASMICRETICULUM R. Wierichs,

A. Hagenmeyer and H. Bader

Department of Pharmacology and Toxicology, University of Ulm, D - 7900 Ulm, Oberer Eselsberg, Received

FRG

December 27,1979

SUMMARY:Vanadate inhibits the Ca++- ATPase of sarcoplasmic reticulum from pig heart half maximally at about IO-5 M. Mg++ promotes this inhibition by vanadate whereas increasing Ca++- concentrations protect the enzyme against vanadate inhibition. Keeping the ratio Mg++/ ATP constant there was no influence of ATP on the vanadate inhibition at concentrations up to 5 x IO-3 M ATP. Whenever the ratio Mg++/ ATP was higher than I:? the inhibitory effect of vanadate on the Ca++- ATPase was increased. INTRODUCTION: The stimulus vanadate was identified (1).

At first

vanadate,

in ventricular showed that findings

in ATP derived

like

heart

glycosides,

and papillary several

It

heart

exerts

other enzymes are inhibited the interpretation

half

to find

Mgff-,

maximally

sarcoplasmic

Recent studies, by vanadate

effect

effect however,

(6-9).

function

possible

reticulum

ATP- and Ca++- concentrations

inhibited

inotropic

the different

paper we want to show the inhibitory

of Na++

by the finding

of the biological

now to be of interest

seems

the Ca++- ATPase of pig heart of various

inhibitor

a positive

muscle (3-5)"

mechanisms showing how vanadate influences In this

from equine muscle

This assumption seemed to be confirmed

complicate

vanadate.

as an impurity

in vanadium came when

vanadate was looked at as a specific

K+- ATPase (2). that

to the present interest

These of

common enzymes.

of vanadate

on

and the influences

on the enzyme which was

by vanadate.

MATERIALS & METHODS: Ammonium(meta) vanadate was obtained from Fluka, Switzerland, Adenosine-5-triphosphoric acid disodium salt was from Merck, Darmstadt as well as all other reagents used. 0006-291 Cop.yrrght .A 11 righrs

X/80/041

124-06$01

. OO/@

NC 1980 hv .,I cadenfic Press, oj. reprodk~on in an? Jorrn

1~. reserved.

1124

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ATPase was isolated by differential centrifugation (10) in a medium containing 0,5 M sucrose and IO mM 4-Morpholinopropanesulfonic acid (MOPS). The incubations were performed at 37'C for 15 minutes. c a++- ATPase activity was determined by measuring the liberated inorganic phosphate (11) whereby background ATPase was subtracted. O,25 mM ouabain was added to all incubations to avoid a possible interference of Na++ Kf- ATPase. The standard incubation medium further contained: 4 mMTRIS-ATP, 4 mMMgC12, IO mM&Morpholinopropanesulfonic acid (MOPS), 0,5 mMEthylenglycol-2-(2-aminoethyl)-tetracetic acid (EGTA), IO mM Sodium acide, 140 mMKC1 and 0,4 mM total Ca++. All solutions used were adjusted to pH 7,0 by TRIS, Protein concentrations were determined according to Lowry et al. (12). Vanadate was used as NH4VO3 at the concentrations indicated. Variations of ATP-, Mg"-+- and Ca++- concentrations in the standard incubation medium were made as indicated, The free Ca++- concentrations were calculated according to Anderegg (13). c a++-

RESULTS: As shown in Fig. plasmic reticulum

1

vanadate inhibits

from pig heart.

The half

the Ca++- ATPase of sarcomaximal inhibition

moved

Fig. 1. Effects of vanadate on the Ca+"- ATPase activity of sarcoplastic reticulum from pig heart. Ca++- ATPase activity was measured at a free jja+j of I ,3 x 10-6 M ( 0 ) and 1,O x IO-4 M ( 0 ). Other conditions are described in Materials and Methods. The values indicated are means f S.E. of five experiments.

1125

to

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92, No.

4, 1980

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AND

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t

10-2

[w+j

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u

Fig. 2. Influence of different on the activity of the Ca++[ M$the 3 presence ATPase in the absence ( 0 ) and In of IO-5 M vanadate ( l ). For incubation conditions see Materials and Methods. The relative Ca++- ATPase activity ( + ) is the activity of the enzyme in the presence of vanadate in percent of the enzyme activity without vanadate, The values indicated axe means l S.E. of four experiments.

higher 8 x

vsnadate

10

-6

concentrations

M vanadate

at I,3

IO-4 M Ca+". For all shows the influence activity

at IO

-2

further

10

-6

Ca++-

M Ca++ to I,4

experiments

of different

higher

Mg'+-

than

10

10

M Mg++ whereas after

of 4

protected

10 -5

x

x

10m3

concentrations

at

It

Mgf-t -

was found that effect

was reached without

of

vanadate

of vanadate the maximal Ca++-

vsnadate inhibition

higher (Fig.

than

concen10

-6

M

3). For maximal

more Ca++ was needed in the presence of vanadate

absence (I,3

x

10

-6

M instead

1126

of

4,8

x

2

on the Ca++- ATPase

the inhibitory

M. Free Ca+'- concentrations

the enzyme against

from

M vanadate

decreased by 50 $ and was reached at a MB+-

Ca++- ATPase activity than in its

addition

concentrations

-5 M vanadate was used. Fig,

-3 M increased

Maximal Ca++- ATPase activity

ATPase activity tration

x

increasing

in the presence and absence of vsnadate.

concentrations vanadate.

with

10e7

M free Ca++).

Vol.

92, No.

BIOCHEMICAL

4, 1980

d

1.

BIOPHYSICAL

10-7

10-6

0

10-S

[Cc-i

3

Ii c

lo-&

RESEARCH

COMMUNICATIONS

1

IO

-

10-B

AND

0

0

10-2

lO-L

0

M

4

Influence of different Fig. 3. ATPase in the absence ( 0 ) and ( l ). For incubation conditions Ca++- ATPase activity ( + ) as in means * S.E, of four experiments.

[AT;,

M

of the Ca++cCaf+I on the activity in the presence of IO-5 M vanadate see Materials and Methods, Relative Fig, 2, The values indicated are

Influence of different [AT4 on the activity of the Ca++Fig. 4. ATPase in the absence ( 0 ) and in the presence of IO-5 M vanadate at 4 x lo-3 M. For incubation conditions ( . 1. Q++ was kept constant see Materials and Methods. Relative Cafe- ATPase activity ( + ) as in Fig. 2. The values indicated are means * S.E. of four experiments, When ATPthe In

concentrations

presence

or

contrast

to

inhibition

absence the

at high

was reached

at

of vanadate

whereas

3 x

10

-5 M ATP.

change Mg++

of to

the

ATP.

were

ATP-

in

Mg++-

To clarify

The

concentration

of

absence in

the

Fig.

Mgff-concentrations of Fig, to decrease

maximal

4 x 10q3

maximal

activity

caused

we kept

1127

Ca++-

about

2 and 4 could

concentration this

curves

ATP seems

concentration,

its

constant

the

of MB+,

The results free

at

of vanadate

effects

an ATP-

varied

this

ratio

in

4 were

obtained,,

the

vanadate

ATPase M in

the

presence

was obtained

be influenced

by different constant

activity

ratios (1:l).

at

by a of Fig.

5

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92, No.

4, 1980

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I,

10-L

1o-3 [~~++]and

lO-2

[ATP] I-

M -!

Fig. 5. Influence of different LATFJ on the activity ATPase in the absence ( Q ) and in the presence of IO' ( l )0 The ratio of Mg+ to ATP was kept constant (1:l). conditions are described in Materials and Methods. The ATPase activity ( + ) is defined in Fig. 2. The values means i S.E. of four experiments.

shows

that

M have

under

the

ratio

vanadate

on the

DISCUSSION:

Mg++

culum from the

cardiac

Mg++-

seems that

the

This

Ca++-

presence

the

inhibitory

with

the

isolated results

rabbit

skeletal The

used,

enzyme

from

heart

by St.

sarcoplasmic

reticulum This

pig

Ca ++-

inhibition

can be protected

ATPase

depends

of vsnadate.

not

but

ratio

1128

of Mg++

to

on

against

inhibitory

effect

G.

reti-

whereas

the

-3

of

- concentrations,

promote the

effect

Ca ++

concentrations

10

of vsnadate.

obtained

muscle

muscle.

5 x

4 and 5).

ATPase

from

by increasing

ATP itself

2,

up to

the

(7) on sarcoplasmic

al.

and rabbit conditions

I:1

(Fig.

agrees ATPase

in

than

the Ca++s M vanadate Other +t relative Ca indicated are

concentrations

ATPase

Ca++-

inhibits

Wang et

inhibition

ATP-

was increased

(6) on Ca++-

incubation

vanadate sing

al.

all

/ AT P was higher

reticulum,

and by T. dog

on the

enzyme

Vanadate

sarcoplasmic et

conditions

same effect

When the

O'Neal

these

o

ATP influences

increaIt the

Vol.

92, No.

BIOCHEMICAL

4, 1980

inhibition

by vanadate

since

of Mg++ to ATP increased sary

for

Ca++ is

vanadate necessary

lation

that

the inhibition

above 1:l.

for

This

the phosphorylation ATPase (14)a

ATPase by vanadate

conceivable

BIOPHYSICAL

RESEARCH

increased means that

COMMUNICATIONS

whenever free

the ratio

Mgf+ is neces-

inhibition,

of the Ca++-

the Ca'+-

AND

and. Mg+' for

Since Ca+i prevents

and Mgf+ promotes this

vanadate inhibits

the dephosphorythe inhibition

inhibition

the enzyme by interacting

it

of is

with the

dephosphoform of the Ca++- ATPase, This is in agreement with findings the Na++ K+- ATPase where K+ promotes vanadate inhibition gonizes this

inhibition

on

and Naf mta-

(15-17)p

REFERENCES: 1.

2, ::

5. 6. 7. 80 9” 100 11. 120 13. 140 150 16.

Josephson, I,. and Cantley, L.C., Jr, (1977) Biochem. 16, 4572-4578. Cantley, L.C., Jr., Cantley, L.G. and Josephson, L. (1978) J. Biol. Chem. 253,20, 7361-7368, Balfou~,W.E., Grantham, J.J, end Glynn, I.M. (1978) Nature 275, 768. Hackbarth, I., Schmitz, W., Scholz, H., Erdmann, E,, Krawietz, W. and Phillipp, G, (1978) Nature 275, 67. Grupp, G,, Grupp, I., Johnson, C.L,, Wallick, E,T. and Schwartz, A. (1979) Biochem. Biophys. Res. Commun. 88,2, 440-447* OlNeal, St.G., Rhoads, D,B. and Racker, E. (1979) Biochem. Biophys. Res* Commun. 89,3, 845-850, Wang, T,, Tsai, L., Solaro, R.J., Grassi de Gende, A.O. and Schwartz, A, (1979) Biochem, Biophys. Res, Commun. 91,1, 356-361. Lopez, V,, Stevens, T, and Lindquist, R.N. (1976) Arch. Biochem. Biophys, 175, 31a Choata, G.L. and Mensous,T.E, (1978) Fed. Proc. 37, 1433. Suko, J. and Hasselbach, W. (1976) Eur. J. Bioohem, 64, 123-130. Post, R,L. and Sen, H.H, in Methodes in Enzymology (1967), (Colowick, S.P. and Kaplan, N,O,, eds,), Void IO, ppV 762, Academic Press, New York and London, Lowry, O.H., Rosebrough, N.J,, Farr, A.L, and Randell, R,J, (1951) J. Biol, Chem, 193, 265.

A$~;yg,",

F

17.

Go (1964)

Helv"

47, 1801.

., Maring, E., Murphy, A,J. and MC Farland, B,H, (1970) Arch, Biochem, Biophys. 138, 285-294. Bond, G,H. and Hudgins, P,M, (1979) Biochem. 18,2, 325-331" Grantham, J,J, and Glynn, I.M, (1979) Am, JU Physiol, 236,6, 5WF5350

Karlish,

S,J,D.,

Beauge, L,A.

and Glynn,

333-3350

1129

I,M,

(1979)

Nature 282,