Light induced activation of fructose-1,6-bisphosphatase in isolated intact chloroplasts

Vol. 70, No. 1, 1976

LIGHT

BIOCHEMICAL

INDUCED

AND BIOPHYSICAL

ACTIVATION

OF FRUCTOSE-1,6-BISPHOSPHATASE

IN ISOLATED

G.J.

Kelly,

RESEARCH COMMUNICATIONS

INTACT

CHLOROPLASTS

G. Zimmermann

and E. Latzko

Abteilung Chemische Pflanzenphysiologie Technische Universitat Miinchen 8050 Weihenstephan, Germany (BRD) Received

March

lo,1976

SUMMARY: Chloroplasts isolated from spinach leaves previously held in darkness contained no fructosebisphosphatase activity measured at pH 7.5 or 7.9, although hiffh activity at pH 8.8 was observed. Following illumination of these chloroplasts for 7 min, enzyme activity at pH 7.9 was clearly detected, and after 24 min illumination was equal to 36% of the pH 8.8 activity; at this time activity at pH 7.5 also became apparent. The activity at pH 8.8 was not affected by illumination. Similar activation of fructosebisphosphatase in isolated pea chloroplasts was recorded.

INTRODUCTION:

The hydrolysis

fructose&-phosphate step

in

the

The enzyme

Calvin

could

this

has been

could

ensure

(4-7).

sulphydryl

of

however,

reaction,

CO2 fixation

aaent

be analagous

evidence Studies that

is

interest

reductant.

this

any in

is

only activation

(5-7)

intact vivo

Copyright 0 1976 by Academic Press, Inc. ill rights of reproduction in any form reserved.

of the

since

purified 193

have following

have

this

effect

by a photo-

investigations

chloroplasts

(7)

illumby the

mediated

activation

properties

during

Unfortunately,

laboratory

activation

possess

active

dithiothreitol

in

for in

'I-phosphohydrolase,

to an in vi~vo activation

produced

(1-3).

fructosebis-

and shown to

of

replatory

chloroplast

enzyme

One property

of fructosebisphosphatase

ation.

photosynthetic

the

to

to be an important

purified

that

reducing;

synthetically

provide

fructose-1,6-bisphosphate

(D-fructose-1,6-bisphosphate

EC 3.1,3.11),

ination

thought

cycle

catalysing

phosphatase

which

is

of

recently

enzyme

failed illuminshown,

by dithio-

to

Vol. 70, No. 1, 1976

threitol

is

pH of the

BIOCHEMICAL

more

pronounced

stroma

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

at

pH 7.9

illuminated

the

lower

Measurements

of fructosebisphosphatase

chloroplasts

have

is

ing

several

are

presented

in

minutes in

pH 8.8,

now revealed

inactive

the

dark

(3)

optimum

for

at

but

this

remarkably Results

but

the which

enzyme

activity

that,

illumination. this

approximates

chloroplasts

considerably

enzyme

than

which

in lower

activity.

intact pH,

the

activated of these

is

follow-

experiments

communication.

Spinach (Spinacea oleracea L.) and peas MATERIALS AND METHODS: (Pisum sativum L.) were grown undsrvhouse conditions. The plants were left overnight in darkness prior to experiments. Chloroplasts were isolated by homogenising 8 g leaves for 5 set in 40 ml of the isolation medium described by Lilley et al. (8). The homogenate was filtered through Miracloth, centrifuged at 2500 x g for 80 see and the supernatant discarded. Two ml resuspending medium containing 0.33 M sorbitol, 2 mM EDTA, 1 mM MgC12, 1 mM MnC12 and 50 mM HEPES and adjusted to pH 7.6 (8) was added without disturbing the chloroplast pellet and the tube gently agitated to suspend the upper layer of chloroplasts which were discarded. The remaining chloroplasts were suspended in 2 ml of resuspending medium. All operations were at 4OC and in dim light. For illumination, one volume of chloroplast suspension was added to four volumes of resuspending medium in a glass tube (10 cm x 1.3 cm2) held in a water bath at 22OC. Light was supplied from a Paximat-S-500 projector by an Osram 300 W lamp. At the times indicated (see Results) chloroplasts were removed in 0.2 ml samples and broken by direct addition to reaction mixtures containing 100 ,umoles buffer (Tris-HCl, pH 8.8 or 7.9, or imidazole-HCl, pH 7.5), 10 pmoles MgC12, 0.6 pmole fructose1,6-bisphosphate, 1 umole EDTA, 0.3 umole NADP, 2 units phosphoglucose isomerase and 0.6 unit glucose&-phosphate dehydrogenase. The final volume was I ml. Fructosebisphosphatase activity was calculated from the change in absorbance at 340 nm measured with a UNICAM SP1800 spectrophotometer connected to an automatic sample changer and recorder; with this system three reactions could be followed simultaneously (Figure 1). No activity at pH 7.9 was detected with illuminated chloroplasts when fructose-1,6-bisphosphate was omitted or replaced with glycerate-3-phosphate. RESULTS:

Light

bisphosphatase

activation is

released

from

overnight

in

of spinach

shown in

chloroplasts darkness

Figure which

was initially

1.

chloroplast

Fructosebisphosphatase

were isolated inactive

194

fructose-

from at

either

leaves

left

pH 7.5

BIOCHEMICAL

401.70, No. 1, 1976

0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

10

20

30

LO

50

lncubotlon

60 min

1

2 hr

tmte

FIGURE 1: Activation of fructosebisphosphatase following illumination of isolated intact spinach chloroplasts. Incubation conditions and determination of enzyme activity are described under methods. The light intensity was 30 000 lux and the chlorophyll concentration 100 ug per ml. Illumination was begun after 8 min incubation and was removed after 50 min. At the times indicated, enzyme activity was measured at pH 7.5

3r

3.8.

7.9

CM>,

although

7.9,

However,

(+-+I

the

enzyme

followinp

detected

illumination

until

after

the activity

measured

ity

at

this

pH 7.9. returned activity.

When the

light

Control

for

activity 70 min

but

in

off

showed

darkness,

of chloroplasts

broken

at

the

to resuspendinR

medium

containinp;

did

not both

to

or after

start

no sorbitol,

195

of

the

activ-

as that at

alter

at

pH 7.9

the

1iEht

pH 8.8

and no fruc-

in

45 min

of incubation

further

inactive

activation:

developed

at

one-third

light

activity

for

pH 7.9

to

the

that

required at

response than

pH

activity

thereafter

as rapidly

Illumination

were

at

The enz.yme remained

almost

experiments

in

was more

was turned

intactness

tosebisphosphatase

it

illumination,

to a low value.

activity enzyme

and increased

pH 8.8.

pH responded

chloroplast

incubated

15 min

substantial

illumination,

24 min at

(-1.

showed

7 min

oH 7.9 was clearly

3.t pH 7.5 during

8.8

and

chloroplasts illumination by addition

although

in

both

Vol. 70, No. 1, 1976

experiments

BIOCHEMICAL

hiah

activity

tosebisphosphatase in the

presence

was about results of

of

that

activation

in

Activation

for

light

vitro of

of various

activity

at

lux,

but

to be saturated

those

obtained

37 min.

Thus, in

were very

similar.

the

even

with

more

the

fruc-

that after

the

dark

in

at

pH 7.9

at

pH 7.5,

illumination the

viewpoint

of

and dithiothreitol

fructosebisphosphatase is

light

activity

from

vivo

chloroplast

increased

in

and 1.5-times

intensities

pH 7.9

5000

incubated

activation

spinach

by light

below

to

However,

and 7.9 was observed

the

pH 8.8

identical

pH,

pH 7.5

chloroplasts

at

chloroplasts to

was recorded.

mM dithiothreitol;

10

almost

response

at both

of broken

40% of

intact

at pH 8.8

active

a suspension

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

shown in sharply

Figure

at

2. Enzyme

1iEht

activation

process

relatively

high

intensities did

not

light

appear

intensity

of 45 000 lux. Pea chloroplast light.

fructosebisphosphatase

Illumination

activity

of

at pH 7.9

pH 8.8

activity

It

is unlikely

from

after

of chloroplast

evidence

for

light

bisphosphatase Enzyme

the

activity

the

leaf

ination

at

at

pH 8.8

pH 7.5

for

7OC) the at

or 8.8

22 min activity

of

were

in

pH 8.8 took

under

whereas

been

extracts

place. 196

leaves

due entirely (9).

prevailing

After

to illum-

conditions

increased

no change

obtained.

30% and 29% of

probably

pH 7.9

an

fructose-

from

respectively most

were

investigations

chloroplast

has also

and 7.9

at

enzyme

60% of the

here

preliminary

fructosebisphosphatase

of leaves

measured

in

pea leaves

and were

to

described

activation

darkness

cytoplasmic

(sunshine, that

in

darkness

the

by

light.

observations

at pH 7.5

held

of

activated

increased

in

isolation;

intact

activities

previously

the

mediated

in

chloroplasts

no activity 45 min

that

artifact

these

was also

in

the

to 47% of activities

Vol. 70, No. 1,1976

BIOCHEMICAL

AND BIOPHYSICAL

I

1

I

'0

20

xl

Light

intensity

1 lux

RESEARCH COMMUNICATIONS

I

I

LO

50

x 10e3 1

FIGURE 2: Influence of light intensity on the activation of fructosebisphosphatase in isolated intact spinach chloroplasts. Incubation conditions and determination of enzyme activity are described under methods. The chlorophyll concentration was 80 pg per ml and illumination was for 20 min. The pH of reaction mixtures was 7.9.

DISCUSSION: light

These

activation

that

most

only

of the

formation

of

sulphydryl

to

pH of

manuscript

light

chloroplast

when the

enzyme

associated

with

the

activation

does

involve

vivo

reductant

demonstrated

is

the

evidence

from the

assayed

on the

this

number

on the

other,

of

Buchanan

et al.

chloroplast

fructosebisphosphatase,

were

at

have

similar, et al.,

groups than

in

doubled

pH 7.5. the

reported

If

was light

nature

of

the

Reduced to

may be one contender

197

and

and this

question.

(5)

very

(Zimmermann

more

process,

the

one hand,

sulphydryl

of activity

an intriguing

which

available;

dithiothreitol

a reductive

ferredoxin,

mechanism

laboratory

with

appearance

a pH near Reductive

presently

fructosebisphosphatase was incubated

at

activation

enzyme,

enzyme,

becomes

a which

chloroplasts.

activated

preparation)

purified

in

from

experiments in

is

illuminated

activated

other

enzyme

groups

proposed

dithiothreitol and in

of

clearly

fructosebisphosphatase

when the

stroma

logically

responses

have

of chloroplast

can be detected to

experiments

activate for

Vol. 70, No. 1,1976

this

role,

native. that

BIOCHEMICAL

while

natural

Vallejos greater

exposure

not

of natural

with

the

reported

here. hr -1 activity

starch

chlorophyll The enzyme

require

of at

least in

hr -'l

sucrose

(for

(Figure

requirement.

Somewhat

detected

in

the

future,

remain

one Calvin

excess

of the

lihood

that

the

mediated pH (7,

with

of CO2 fixation,

of

substrate

between

these

regulatory

since

both

Figure

I)

over

in

the

the

physiological

per

spinach

this

quite

minimum probably is

activity

not

enzyme

the

activity is

(4,7)

to

properties particularly

be

likely

greatly

increasing

range

mg

cytoplasm).

to

will

be interesting

are

mg

hr"

illuminated

close

and cofactor will

per

mg chlorophyll

or 33 pmoles

of the

It

activation

per

thus

response

closely

fructosebisphos-

fructosebisphosphatase

CO2 fixation,

interaction

of 100 umoles

pH 7.9 in

enzyme

sigmoid

concentrations regulate

cycle

rate

a rate

activities

but

were

fructosebisphosphatase

synthesis

higher

membranes

of

was therefore

I)

follows

dithiols

chloroplasts)

at

evidence

of chloroplast

chloroplast

measured

alter-

dithiols

50 umoles

synthesis

chloroplasts

obtained

similar

at

would

activity

recently

activation

CO2 fixation

chlorophyll

an attractive

vicinyl

that

light

Photosynthetic

offer

changes

inconceivable

involved

(for

(IO)

conformational

is

phatase

dithiols

and Andre0

light-induced and it

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to in

liketo

utilised

to

examine

the

and the

light

sensitive of

7.2

to 8.0

to (3).

ACKNOWLEDGEMENTS: We thank E. Wirth and the Lehrstuhl fiir Gemiisebau for growing the spinach, and the Deutsche Forschungsgemeinschaft for generous support. REFERENCES: 1.

2.

Pedersen, Physiol. Bassham, Biochim.

T.A., Kirk, M., and Bassham, Plant., 19, 219-231. J.A., and Krause, G.H. (1969) Biophys. Acta, 189, 207-221.

198

J.A.

(1966)

Vol. 70, No. 1,1976

3. 4. 5. 6. 7. 8. 9. IO.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Werdan, K., Heldt, H-W., and Milovancev, M. (19'75) Biochim. Biophys. Acts, 396, 276-292. Preiss, J., Biggs, M.L., and Greenberg, E. (1967) J. Biol. Chem., 242, 2292-2294. Buchanan, B.B., Schiirmann, P., and Kalberer, P.P. (1971) J. Biol. Chem., 246, 5952-5959. El-Badry, A.M. (1974) Biochim. Biophys. Acta, 333, 366-377. Baier? D., and Latzko, E. (1975) Biochlm. Biophys. Acta, 396, 741-147. Lilley, R.McC,, Walker, D.A., and Holborow, K. (1974) Biochim. Biophys. Acta, 368, 269-278. Latzko, E., Zimmermann, G., and Feller, U. (1974) Hoppe-Seyler's 2. Physiol. Chem., 355, 321-326. Vallejos, R.H., and Andreo, C-S. (1976) FEBS Lett., 61, 95-99.

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