Effect of divalent cations on the structure of human erythrocyte glucose 6-P dehydrogenase

Vol. 42, No. 2, 1971

EFFECT OF DIVALENT

BIOCHEMICAL

CATIONS ON THE STRUCTURE OF HDMAN ERYTHROCYTE GLUCOSE 6-P DEHYDROGENAS

A.Bonsignore,

I.Lorenxoni,

Istituto

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

di Chimica

November

R.Cancedda, Biologica Genova,

M.E.Cosulich

dell'lJniversitl Italy

and A.De Flora di Genova

23, 1970 SUMMARY

The balance between the two and tetramers, is regulated ing effect displayed by high by several divalent cations ions to the protein determines transition.

Glucose catalytically

6-phosphate active

interconvertible previous present

strength (1,

2).

is

tetrsmers

and dimers conditions

weight

and other through

suggesting that 2+ by Mg ions.

largely

at alkaline

divalent

accounting

results cations

association

process

2).

The

the high

ionic

by Cohen and Rosemeyer were

a concentration-dependent the

are

for

ref.

affecting

pH and (or)

with

in two

The two forms

G6PD (see

on factors

in agreement

exists

(1-S).

of active

an investigation and dimers:

prevailed, 2+ Mg ions

toward

induced

with

tetramers

Conversely,

transition

tetramers

to environmental

concerned

dissociation

are reported

namely

from human erythrocytes

on the molecular

between

the balance data

states,

discrepancies

equilibrium

dehydrogenase

according

paper

active oligomeric forms of human G6PD, namely dimers by a number of environmental conditions: the dissociationic strength and alkaline pH values is counteracted resulting in formation of tetramers. Binding of Mg2+ association of two dimers through an allosteric

found

to displace

effect. involves

Moreover,

an allosteric

MATERIALS AND METHODS G6PD was purified

by the procedure

homogeneous

enzyme protein.

same buffer

employed

of 0.05 25'

M Tris-HCl,

C in an Optica

in

Before

et al.

use G6PD was extensively

the experiment.

pH 9.0,

of Bonsignore

containing

The standard 0.05

CF 4 Spectrophotometer.

M NaCl, One unit

159

(5),

yielding

dialyzed

enzyme assay 2 mM G-6-P

against was in

the

1.0 ml

and 0.5 mM NADP at

of G6PD activity

was defined

Vol. 42, No. 2, 1971

as the amount assay

catalyzing

conditions. Sucrose

BIOCHEMICAL

Protein

gradient

(7),

analytical

were

as previously

reduction

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of 1

was evaluated

centrifugation

ultracentrifugation reported

mole of NADP at 340 m under the standard P P by the method of Lowry et al. (6).

was performed and electrophoresis

of pH and ionic

to Martin

on polyacrylamide

and Ames gel

(5).

Table

Effect

according

strength

I

on the dimer-tetramer

equilibrium

of erythrocyte

G6PD

I Phosphate Phosphate Phosphate Phosphate

6.5 6.5 7.5 7.5

0.08 0.12 0.08 0.23

8.46 8.15 7.0 6.42

0.21 0.30 0.63 0.80

Tris-HCl Tris-HCl

8.0 8.0

0.03 0.15

6.65 6.57

0.74 0.76

Tris-HCl

7.5

0.23

6.51

0.78

Tris-HCl

8.6

0.15

6.40

0.81

Glycine-NaOH

7.8

0.18

7.1

0.61

Glycylglyeine-NaOH

7.5

0.08

6.62

0.75

; ;

Sedimentation coefficients are average values of several experiments performed with 1.2 I.U. of dialyzed enzyme at different stages of purification. Centrifugation was at 49,r)OO rpm for 12 hours at 5' C in 5%-20% sucrose gradients containing the actual buffer of each experiment, 0.1 mM EDTA and 5 p NADP (SW 50 rotor). COHb was used as marker protein. Values of $ calculated on the basis of s values, 2o",w as follows (2,5): St - s $=y

‘t

d

where st (9.18 S) and sd (5.75 S) are the sedimentation coefficients of the tetramers and of the dimers, respectively (5), and T is the actual ~~~0,~. 4 defines the extent of dissociation of the dimer-tetramer system: at values of $ approaching 1.0 the active enzyme would be 100% dimers, whereas g-0 would indicate homogeneous tetramers. 160

Vol. 42, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS Effect

of buffers,

parameters,

pH and ionic

including

the

the dimer/tetramer Dissociation variations

buffer,

ratio,

is

favoured

support

ing dependence

strength

on

by actual

at alkaline

-

provided

structure

Several

environrmntal

concentration,

sedimentation

pH and high

evidences

of the quaternary

structure

the pH and the salt

as evaluated

earlier

G6PD

ionic

affect

coefficients

strength

(Table

by Cohen and Rosemeyer of the erythrocyte

of G6PD. I).

(1,

markedly

These 2) concern-

enzyme on solvent

conditions. c

Mgso 4 ‘\.,-. u-7 no

111

I

I

,

$3

l

0.5

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

l-

& 0.5

20

*.L

& 0.25 is

*. I& 5

10

15

20

25

fractions

Figure

1:

Effect

of MgSO

on sedimentation of active G6PD 4 Linear 5%--20X sucrose gradients contained 0.02 M phosphate buffer, pD 7.8, 0.1 m EDTA, 5 p NADP, 0.1% S-mercaptoethanol (vol/vol) and MgS04 where indicated. Centrifugation at 49,000 rpm for 12 hours at 5O C in SW 50 rotor. Arrows indicate theoretical migrations of homogeneous tetramers and dimers along the tube, as relat ed to actual migration of marker COHb (filled circles). The open circles refer to G6PD activity and the dotted line to the sucrose gradient.

161

Vol. 42, No. 2, 1971

Effect resulted control

of Mg

2+

BIOCHEMICAL

ions

-

unexpectedly enzyme

1).

change

the dimer-tetramer extent

system under ra,

toward

conditions

manuscript

behaviour

could

density

the heavier

Moreover

expense model

this

sedimenting

to an increased

of the effect.

at the Spinco these

leading

at the

G6PD species

Although

system

peak occurring

of KC1 by MgS04 to increase

in an active

(Fig.

conformational

marked

Replacement

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

species

MgS04 produced

of the dimers,

more

as shown with

the

by any

(Wrigley,

of

due to the

of the

tetramer

schlieren

optics

tetramerization

microscope

the

a shift

likely

an increase

than

for

of the protein, was more

strength

rapidly

be accounted

E ultracentrifuge(5).Finally,

at the electron

the ionic

was evident

Bonsignore

and De Flo-

in preparation).

Mg SOq(mM 1

Figure

2: Formation

of tetramers

as a function

of Mg

2+

concentration

-

5X-20X

sucrose gradients contained 0.04 M phosphate buffer, pH 7.8, 0.1 mM EDTA, NADP, 0.2% S-mercaptoethanol (vol/vol) and MgS04 at the concentration shown :n?bscissa . Centrifugation at 26,500 rpm for 46 hours at 6' C in a SW 27 rotor (long tubes).

The effect contrary

K2S04,

displayed

Na2S04 and (NH4)2S04

enzyme as compared The extent actual ing

with

sedimentation

0.05

M) result

tetramerfdimer

ratio:

the tetramers

than

the controls,

of association

slightly because

depends

relationship

(Fig.

in an opposite

effect,

this

other

Mg 2+ salts;

dissociation

of the higher

ionic

as expressed

(see below)

162

Higher

active

strength. increase of 2+ of Mg accord-

by a decrease

by a more rapid

and (or)

of the

MgS04 concentrations

as indicated for

on the

by the

on the concentration 2).

may be accounted

of the dimers

with

increased

of the dimers,

coefficients,

to a sigmoid-shaped

(above

by MgS04 was observed

of the

inactivation

by the prevailing

of effect

Vol. 42, No. 2, 1971

of the high according

ionic

strength

to the Hill

significantly cation

BIOCHEMICAL

aggregation by high

leading

equation

above 1.0:

to at least

ionic

to dissociation.

(Fig.

this

is

across

strength

with

and alkaline

that

binding

promotes

of symmetry

centrifuge

data

coefficients

the view

sites

same plane

the

interaction

interacting

the

Plotting

3) yielded

consistent

two positively

of dimers

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a face

which

is

of the to face

inversely

affected

pH.

2-

,“S9.18-S

;;I!

‘n-1.25

3 --

_, 4

-3

-4

-5 In [Mg**]

Figure

3:

Hill

plot

of the Mg

2+

effect

on the dimer-tetramer

equilibrium

-

5%-20X sucrose gradients contained 0.02 M phosphate buffer, pH 7.5, 0.1 mM EDTA, 5 p NADP, 0.2% B-mercaptoethanol (vol/vol) and different concentrations of MgS04. Centrifugation at 26,500 rpm for 41 hours at 6" C in a SW 27 rotor (long tubes).

Effect

of other

divalent

of the dimers

toward

effectiveness

decreases

cations

tetramers;

these

in the

by the individual

produced

an immediate

tetramer

ratio.

are

of divalent

cations

produce

a shift

in Table II, showing that their 2+ 2+ 2+ 2+ 2+ following order: Co > Ca > Ba ) Mn >Mg . 2+ 2+ and show additive effects, a mixture of Ca , Ba

the divalent cations Moreover, 2+ leading to an intermediate Ms determined

A number

-

extent metals.

listed

of tetramerization Among the

making

inactivation,

it

with

cations

tested,

impossible

respect to those 2+ 2+ and Cu Zn

to evaluate

the dimerl

DISCUSSION The divalent erythrocyte

the

cations

producing

concentrations

The on.ly exception of 2-4 mM (8) might

is

of G6PD do not

reach

in the

that

provided shift

tetrameriaation

to

appear to be effective in our experiments. 2+ which at the physiological concentrations by Mg some

extent

the dimer-tetramer

163

equilibrium

toward

Vol. 42, No. 2, 1971

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

Effect

of divalent

cations

II

on the dimer-tetramer

equilibrium

Mgso4

0.05

8.31

(6.34)

0.25

Ml-60 4

0.05

8.37

(6.31)

0.21

&Cl2

0.025

7.93

(6.52)

0.36

CoC12

0.025

9.03

(6.53)

0.04

BaC12

0.025

8.19

(6.48)

0.29

CaC12

0.025

8.9

(6.52)

0.08

0.025

8.30

(6.52)

0.25

CaC12 + BaC12 +

W12

Centrifugations NaOH, pH 7.5, In parentheses

the

latter

were in 5X-20X sucrose gradients containing 0.1 mM EDTA and 10 PM NADP (SW 50 rotor). Sag,, w values of controls are reported. I

form.

In this

values

of pH and ionic

dimers

(2);

the

inactive

species,

monomers Although

escapes

evidence

emerged

as regards The face

aggregation

it

is

for

the binding

to face

of double

could

the dimer

distinct

of NADP (4,

hexagonal

only

the balance

to between

the dissociation

enzyme

cations

still

G6PD which

had already

9).

of "tightened"

164

is

highly E.coli

to

(3).

of divalent

of erythrocyte

of G6PD dimers units

the physiological dissociation

undergoes

of the

M glycylglycine-

the dimer-dimer association involves 2+ Mg -binding sites. This represents

nature

association

slightly

of the effect that

that

in vivo

operation

noteworthy

allosteric

be emphasized

re-equilibrate

mechanism

between

rmst

such as to favour

catalytic

the detailed

interactions

further

are

it

out of which

following

recognition,

cooperative

strength 2+ ions Mg

therefore

two active

connection,

0.04

reminiscent glutamine

of the synthetase

a

Vol. 42, No. 2, 1971

occurring striking

upon

BIOCHEMICAL

addition

since

the

is

obscured

aggregate

of divalent

tendency

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cations

of individual

by high

ionic

(10).

The similarity

molecules

strength

is

of glutamine

evenmore

synthetase

to

(11).

ACKNOWLEDGEMENTS This Ricerche,

investigation

received

We are indebted

Rome.

financial to Mr.

aid

by the Consiglio

F. Giuliano

for

Nazionale

valuable

delle

technical

assistance.

REFERENCES 1.

Cohen,

P.,

and Rosemeyer,

M.A.,

FEBS Letters,

2.

Cohen,

P.,

and Rosemeyer,

M.A.,

European

3.

De 'Flora, A., Cancedda, R., Lorenzoni, I., Nicolini, A., Proceedings 1st International Symposium on "Metabolic of Enzymes", S. Margherita Ligure, p. 35 (1970)

4.

Bonsignore, A., Lorenzoni, De Flora, A., Ital.

I., Cancedda, R., Morelli, J. Biochem., 18, 439 (1969)

5.

Bonsignore, A., Lorenzoni, De Flora, A., Ital.

I., Cancedda, R., Nicolini, J. Biochem., 19, 165 (1970)

6.

Lowry,

7.

Martin,

8.

Greenberg,

9.

Bonsignore, Res.

10.

Valentine,

11.

Shapiro,

O., Rosebrough, 193, 265 (1951) R.G.,

N.J.,

Farr,

and Ames, B.N.,

N.,

J. Biol.

A.L.,

100,

139 (1933)

B.M.,

and Ginsburg,

B.M., A.,

R.,

and Stadtman, Biochemistry,

165

8, 8 (1969)

236,

and Bonsignore, Interconversion

A., A.,

and Randall,

Chem.,

Chem.,

Shapiro,

Biochem.,

J.Biol.

A., Lorenzoni, I., Cancedda, Comms., 39, 142 (1970) R.C.,

J.

1, 147(1968)

Damiani,

R.J.,

J. Biol.

F.,

and

G.,

and

Chem.,

1372 (1961)

and De Flora, E.R.,

Giuliano,

A.,

A.,

Biochemistry,

7, 2153

(1968)

Biochem.

Biophys.

7, 2143

(1968)