Download Regulation of Acetyl-Coenzyme A Carboxylase and Acetyl

Regulation of Acetyl-Coenzyme A Carboxylase and
Acetyl-Coenzyme A Synthetase in Spinach Chloroplasts
Andreas Sauer and K laus-P eter H eise
Lehrstuhl für Biochemie der Pflanze der Universität G öttingen. Untere Karspüle 2,
D-3400 Göttingen. Bundesrepublik Deutschland
Z. Naturforsch. 39c, 2 6 8 -2 7 5 (1984); received N ovem ber 24, 1983
Acetyl-CoA Carboxylase, A cetyl-CoA Synthetase, Light D ependence o f Fatty Acid Synthesis
in Chloroplasts
In analogy to chloroplast fatty acid synthesis from acetate the key enzym es o f acetate fixation,
acetyl-CoA synthetase and acetyl-CoA carboxylase, in rapidly Triton X-100 lysed spinach
chloroplasts show an activation by light and deactivation in the dark. The stim ulation o f
acetyl-CoA carboxylase by dithiothreitol in darkened chloroplasts points to an involvem ent o f
reducing equivalents in the light activation o f this enzyme. But more than by alterations o f the
activation state per se, these enzymes appear to be effected by changes in their catalytic activity
due to differences in the proton-, M g2+- and adenine nucleotide levels o f the chloroplast stroma.
Thus the pH dependence o f both enzymes, as im m ediately extracted from Triton X-100 lysed
chloroplasts, resembles that recently found for lipid incorporation o f acetate into intact spinach
chloroplasts in the light with an identical pH optim um o f about pH 8.5 for the acetyl-CoA
carboxylase. Moreover, in the same extracts both enzym e activities show the already postulated
requirement for MgATP and free Mg and are com petitively inhibited by free ATP and A D P with
respect to MgATP. But on account o f the fact, that the extractable acetyl-CoA synthetase as
opposed to the carboxylase activities exceed by far the lipid incorporation rates o f acetate by
illuminated chloroplasts before disruption, acetyl-C oA synthetase will be excluded as rate
limiting step in fatty acid synthesis from acetate. From key enzym es o f acetate fixation only the
carboxylase appears to be involved therefore in the light regulation o f acetate incorporation into
long-chain fatty acids.
Introduction
Materials and Methods
Chloroplast fatty acid synthesis from acetate
appears to be regulated by light [1 -6 ]. L ike the
Calvin cycle enzymes this effect o f light seem s to be
m ediated by light dependent variab le p ara m ete rs in
the chloroplast strom a, e.g. the concentration o f H +,
M g2+ and ATP [6 ] which are know n essential factors
for acetate activation [3,7], F u rth e rm o re lightenhancem ent of fatty acid synthesis from acetate
may be due to an enzym e interconversion m e d iated
by changes in the redox poising [6 , 8 ]. A cetyl-C oA
carboxylase has been exam ined as possible site for
such a light regulation o f fatty acid synthesis [8 ]. In
this study we have exam ined:
Spinach (Spinacia oleracea, U.S. H ybrid 4 2 4 ,
Ferry-M orse Co., M ountain View, CA, USA) was
grown in hydroponic culture [ 1 1 ] and intact ch lo ro ­
plasts were prepared as in [12]. T he chloroplasts
( 1 0 0 (ig Chi • m l-1) w hich show ed an C ^-evolution
rate in the range o f 8 0 - 1 4 0 jim ol • m g - 1 Chi • h - 1
were suspended in a reaction m ixture co n tain in g
0 . 3 3 m sorbitol, 5 0 m M H epes/K O H pH 7.6, 1 m M
M gC b, 1 m M M n C h , 2 m M
ED TA , 0 . 5 m M
K 2 H P 0 4, 10 m M N a H C 0 3 and 4 0 |ig • m f 1 catalase
from bovine liver (Boehringer, M annheim ), unless
otherw ise stated.
. the acvtivation state o f the key enzym es o f
acetate fixation in chloroplasts, e.g. acetyl-C oA
synthetase and acetyl-C oA carboxylase in im ­
m ediately lysed chloroplasts [9] durin g a lightdark transient, and
2 . the kinetic dependence o f these enzym e activities
rapidly extracted from illu m in ated chloroplasts
[ 1 0 ] on various strom al solutes and m etabolites.
Assay o f acetyl-CoA synthetase
1
Reprint requests to Dr. K.-P. Heise.
0341-0382/84/0300-0268
$ 0 1 .3 0 /0
The radioactivity assay of acetyl-C oA synthetase
in chloroplasts by [ l - 14C ]acetate in co rp o ratio n [13]
was carried out with
100 m M
T ricin e /K O H
(pH 8.0), 2 m M M gC l2, 1 m M ATP. 0.5 m M CoA ,
0.5 m M [ l - , 4 C]acetate (specific activity: 57,2 m C i •
m m o r 1) and 0.2% (w /v) T riton X-100 (final
volume: 0.3 ml), if not stated otherw ise. T he
reaction was stopped w ith 0.3 ml o f an activated
charcoal slurry: glacial acetic acid ( 1 : 1 ) m ix tu re
and labelled acetyl-CoA, adsorbed to charcoal, was
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A .Sauer and K.-P. H eise • R egulation o f A cetyl-CoA C arboxylase an d A cetyl-C oA S ynthetase
m easured according to K uhn et al. [13]. C ontrol
sam ples contained neither CoA nor ATP, both of
w hich were essential for high rates o f [ l - 14C ]acetate
adsorption to the charcoal.
Assay o f acetvl-CoA carboxylase
Acetyl CoA carboxylase activity in chloroplasts
was measured as an acetyl-C oA -dependent fixation of
H I4C 0 3 into acid-soluble, h eat-stable products [14].
The standard reaction m ixture contained 100 m M
T ricine/K O H (pH 8,5), 0.5 m M Acetyl-CoA , 1 m M
ATP, 2 m M M gC l2, 30 m M KC1, 7 m M N a H C 0 3,
3 m M N a H l4 C 0 3 (specific activity: 1 m Ci • m m o l-1)
and 0.2% (w /v) T rito n X-100 in a final volum e o f
0.3 ml. The reaction was term in ated by adding
0.3 ml o f 2 n HC1. T he sam ples w ere dried at 70 °C
in a scintillation vial and 0.2 ml 2 n HC1 and 7 ml
scintillator ( 6 g butyl-PBD , 600 ml toluene, 400 ml
methylglycol) were then added and the acid-stable
radioactivity was counted by liquid scintillation.
Control sam ples w ith out acetyl-C oA were included
in each series o f the assays.
Measurement o f enzyme interconversion
F or the study o f the kinetics and the state of
activation and inactivation o f the enzymes ch lo ro ­
plasts in the stan d ard m edium w ere preillu m in ated
or kept dark in the absence and presence o f d iffe r­
ent D TT concentrations at 25 °C for th e tim e
intervals indicated (Figs. 1 and 2) and 50 (il aliquots
were w ithdraw n and im m ediately lysed in 250 |il o f
a reaction m ixture containing 0.2% w /v T riton
X-100 [9, 15] and the essential unlabelled or ra d io ­
active substrates o f the enzym atic reaction to be
assayed. The final chlorophyll conc. was 20 |ig /
sample. This procedure allow s an instantaneous
start of enzym e activity m easurem ents. T he enzym ic
reaction, as initiated by the disru p tio n o f the
chloroplasts, was term inated 30 s later by ad d itio n
o f hydrochloric acid, in o rd er to keep the tim e of
the assay short in relation to the kinetics o f activa­
tion and inactivation [15] and to m easure in the
linear region o f the reaction (up to 90 s).
Preparation o f chloroplast crude extracts
In order to keep the investigated enzym es u nder
approxim ate physiological conditions, m o d u latio n
o f their catalytic activity by ions (H +, M g2+) and
adenine nucleotides (ATP, A D P) has been m ea­
269
sured in substrate free chloroplast crude extracts,
prepared by rapid elution o f T rito n X-100 (final
conc. 0 . 2 % w /v) lysed p reillu m in ated ch lo ro p lasts
over a short Sephadex G 25 colum n acco rd in g to
Laing and R oughan [10] and ad ju sted to 0.1 - 0 .2 m g
Chi m r 1.
In order to d iscrim inate betw een the effect o f free
and adenine nucleotide b o u n d M g2+ on the
activities o f acetyl-C oA synthetase and carboxylase
in above chloroplast extracts we calculated th e free
nucleotide concentration in the in cu b atio n by using
the stability constants o f K räm er [16].
Extraction and detection o f labelled fatty acids
was as described earlier [17],
Results and Discussion
Recent m easurem ents o f the endogenous acyl-acyl
carrier protein pool sizes d u rin g stead y -state fatty
acid synthesis by isolated spinach ch lo ro p lasts [18]
dem onstrated the well know n light d ep e n d en ce o f
fatty acid synthesis from acetate [ 1 -3 ] at th e level
of its thioester interm ediates. T hese results im ply
that the level o f acyl-carrier p ro tein th io esters
reaches a “steady sta te” w ithin 1 m in o f illu m in a ­
tion and declines im m ed iately after d ark e n in g o f
the chloroplasts. Because the latter co m p o u n d s are
direct substrates for specific acyl-thioesterases and
transferases, the decline o f the acy l-carrier p ro tein
pool in the dark leads to an im m ed ia te sto p in the
release of free fatty acids and reduced g ly cero lip id
synthesis [5].
Enzyme interconversion
In order to localize the light d ep e n d en t steps in
the reaction sequence from acetate to lo n g -ch ain
fatty acids we started to investigate to w h at extent
the activities o f th e key enzym es in acetate fixation,
e.g. acetyl-CoA synthetase and acetyl-C oA car­
boxylase, are controlled by light.
Fig. 1 shows a tim e course for th e light activ atio n
and dark inactivation o f acetyl-C oA syn th etase and
acetyl-CoA carboxylase o f chloroplasts in situ. A fter
the onset o f illu m in atio n the activity o f th e acetylCoA synthetase increases ab o u t 2-fold an d th a t of
acetyl-CoA carboxylase increases a b o u t 3-fold.
D arkening leads to a decrease o f b o th enzym e
activities. C om pared to the rap id red u ctio n o f acylacyl carrier protein synthesizing capacity o f chloro-
270
A .Sauer and K.-P. Heise • R egulation o f A cetyl-CoA C arboxylase and A cetyl-C oA S ynthetase
Light
t
E
-
Dark
I
2.0
03
Acetyl - CoA
carboxylase
0 .2
o
01
Acetyl - CoA synthetase
10
Time ( min)
Fig. 1. Changes in the level o f m alonyl-CoA ( • ) and
acetyl-CoA ( a ) formed from [ l - 14C]acetate in spinach
chloroplasts during a light-dark transition. The tim e o f
light activation and dark inactivation o f intact chloroplasts
before their disruption in the Triton X-100 containing
reaction mixture is plotted on the abscissa. In order to
keep the time o f the assay short in relation to the kinetics
o f activation and inactivation and to measure in the linear
region o f the reaction, the test was terminated after 30 s by
addition of acid.
plasts after turning o ff the light [18] the d eactiv atio n
of acetyl-CoA synthetase and acetyl-C oA carb o x ­
ylase appears relatively low (Fig. 1). L atter c o m p a r­
ison suggests, alterations o f the activation state o f
these enzymes, as caused for exam ple by reducing
equivalents, to play a su b o rd in ate role in th e lightenhancem ent o f p lastidary fatty acid synthesis from
acetate. However, recent reinvestigations on the
effect o f the sulfhydryl reagent d ith io th reito l (D T T )
on dark activation o f acetate in c o rp o ratio n into
long-chain fatty acids o f isolated chloroplasts [6 ],
which appeared contradictory in the literatu re
[4, 19], led to the conclusion, th a t beside changes in
stromal solutes (H +, M g2+ and A TP) reducing
equivalents may be in som ehow involved in the
light control o f fatty acid synthesis. L ast notio n
directed our attention to the localization o f the
DTT-sensible step w ithin the reaction sequence o f
the plastidary acetate fixation, particu larly because
o f the recent finding, light to be essential only for
acetyl-CoA carboxylase activity in fatty acid
synthesis o f spinach chloroplasts [8 ],
Therefore, in analogy to the enzym e in terco n ­
version
m easurem ents
(see
m ethods),
intact
darkened chloroplast suspensions w ere in cu b ated
for 10 min with different D T T concentrations in the
dark (Fig. 2), before they were lysed in th e T ri­
ton X-100 containing reaction m ixture. In spite o f a
five-fold dilution o f D T T in the reaction m ix tu re
this method was not fit for a q u an tita tiv e d etectio n
o f the acetyl-CoA synthetase activity by ad so rp tio n
o f l4C-labelled acetyl-CoA (from [ 14 C ]acetate) to
charcoal [13], because D T T undergoes a nonenzymic acetylation with acyl-CoA th io esters [19].
On the other hand carboxylase activities, d e te r­
mined by ,4 C 0 2-fixation to acetyl-C oA , could be
determ ined quantitatively if one allow ed for
adequate acetyl-CoA concentrations (0.5 mM) in the
reaction mixture, to ensure th a t the fo rm a tio n o f
radioactive m alonyl-CoA was not lim ited by D T T binding [19] o f the substrate acetyl-CoA . F u rth e r­
m ore through the acid stop in this test only th e
reaction products were m easured w hile u n reacted
C 0 2 was released. The observed stim u latio n o f the
acetyl-CoA carboxylase by D T T, show n in Fig. 2,
appears to reflect therefore an in volvem ent o f
reducing equivalents in the light activ atio n o f this
enzyme.
0 20
O 0 15
Acetyl- CoA
carboxylase
0
10
0 I
5
10
DTT(mM)
Fig. 2. Effect o f dithiothreitol (D D T ) on the activity o f
acetyl-CoA carboxylase measured as m alonyl-CoA form a­
tion in spinach chloroplasts in the dark. Intact chloroplast
suspensions were preincubated in the dark for 10 m in with
the DDT concentrations indicated and then disrupted in
Triton X-100 containing
reaction-mixture
(further
procedure as in Fig. 1).
271
A .Sauer and K.-P. Heise • R egulation o f A eetyl-C oA C arboxylase and A cetyl-C oA S ynthetase
p H Dependence
As shown in Fig. 3, in chloroplast extracts the
optim al activity o f acetyl-C oA synthetase is o b ­
served at about pH 8.0 and that o f acetyl-C oA
carboxylase at about pH 8.5. T he latter pH -optim um appears identical w ith the strom a pH d eter­
m ined for optim al acetate incorporation into the
lipid fraction o f intact spinach chloroplasts [6 ].
D uring a light-dark transition a decrease o f the pH
from 8.4 to 7.4 was m easured in intact chloroplasts
incubated at pH 7.9 [6 , 12]. But low ering o f the
stroma pH by 1 unit led only to a partial inhibition of
the activities o f acetyl-C oA synthetase and acetylCoA carboxylase (Fig. 3). T he latter observation
suggests that light-induced pH changes in the
strom a may control the activities o f both enzym es, a
suggestion that is consistent w ith recent findings on
the pH dependence o f total fatty acid synthesis from
acetate in intact chloroplasts [6 ].
Effect o f Mg
The absolute req u irem en t for M g2+ in the initial
steps o f fatty acid synthesis from acetate in ch lo ro ­
plasts [14, 20, 21] has been confirm ed in dark
activation experim ents o f fatty acid synthesis in
intact chloroplasts [6 ]. F u rth e rm o re a dual re q u ire ­
m ent o f M g2+ for the form atio n o f M gA TP and for
enzyme activation has been established [14, 2 1 ].
Figures 4 and 5 show the activity o f th e two key
enzymes o f acetate fixation in ch lo ro p last extracts
on the concentration o f free and A T P -b o u n d M gi+.
The prim ary stim ulation o f b oth enzym e activities
in incubations o f a constant A T P content (1 m M ) ,
caused by increasing M g2+ up to eq u im o la r concen­
trations (1 m M ) , w ould be d u e to an accu m u latio n
o f MgATP [22] at the expense o f free A T P (Fig. 4).
The relatively small secondary stim u latio n o f acetylCoA synthetase and carboxylase, found at h ig h er
M g2+ concentrations ( ^ 1 m M ) , w hen th e A T P con­
centration for com plexation w ith Mg is lim ited , can
be ascribed to free M g2+ (Fig. 5). T o find an ex­
planation for the activating influence o f free M g2+,
the dependence o f the K m and Fmax o f b o th enzym es
on the concentration o f free M g2+ (Fig. 6 ) has been
investigated under a constant M gA TP level (1 m M ) ,
calculated according to K räm er [16].
W hile the
o f the carboxylase for M gA TP
(A'm(MgATP) = 0.24 m M ) is significantly reduced
by free Mg2+, its Fmax (Kmax (M gA TP) = 0.36 nm ol •
mg “ ‘ C h l - h -1 ) for the sam e su b strate rem ains
unchanged. In contrast, free M g2+ increases
the Fmax ( Vm (M gA TP) = 1.85 |im ol • mg
Chi • h -1)
Acetyl - CoA carboxylase
MgATP
E Z 0.14
0 °
io >E 010
1 0 O.O6
!§ Ö - 0 02
ifreeATP
01
02
03
0.4
05
ATP, MgATP (mM)
Assay pH
Fig. 3. Effect o f pH on the activities o f acetyl-CoA carbox­
ylase measured as malonyl-CoA formation and acetyl-CoA
synthetase in spinach chloroplast extracts.
Fig. 4. Effect o f free ATP and MgATP on the activities o f
acetyl-CoA synthetase and acetyl-CoA carboxylase in
spinach chloroplast extracts. The free nucleotide concen­
tration during incubation was calculated by using the
stability constants o f Krämer [16].
A .Sauer and K.-P. Heise ■ R egulation o f A cetyl-CoA C arboxylase and A eetyl-C oA S ynthetase
272
Km (mM)
A cetyl - C o A synthetase
free Mg
gZ
Eo
o, cr
<
0 EcO_
1 o
^
0.5
E
0
Acetyl - CoA carboxylase
Vmax ( Hm°l m<3
o
<
a°
1Chi
h
1)
free Mg
° 0.05
CT>
bp
0 01
0
1
2
3
4
5
Mg ATP, free Mg (mM)
free Mg ( mM)
Fig. 5. Effect o f Mg2+ on the activities o f acetyl-CoA
synthetase and acetyl-CoA carboxylase in extracts o f
spinach chloroplasts under a constant MgATP level (1 mM ),
calculated according to Krämer [16].
Fig. 6. Effect o f Mg on
(Fig. A) and Kmax (Fig. B) o f
acetyl-CoA synthetase and acetyl-CoA carboxylase in
spinach chloroplast extracts. The concentration o f M gATP,
calculated according to Krämer [16], was a constant 1 mM.
o f the synthetase but does not change the K m for
M gATP (A^m(M gA TP) = 0.14 m M ;
Fig. 6 ). T he
decrease of the M ichaelis-M enten constant o f the
carboxylase for M gA TP by free M g2+ m ay explain
the difference in
observed here w ith th a t o f the
enzyme (A^mATP = 0.039 m M ) recently found in
crude carboxylase p rep aratio n s from spinach [23].
An evaluation o f the d ata by H ill plots (not show n)
clearly indicated th a t there is no cooperativ ity
between the catalytic binding sites o f b oth enzym es
for the substrates exam ined. T he results o f this
kinetic study support the u n iq u e relatio n sh ip b e ­
tween MgATP as substrate and free M g2+ as an
activator of acetyl CoA carboxylase activity as well
as the notion, th at both ions p ro b ab ly ad d to this
enzyme in an eq u ilib riu m o rd ered m a n n er [14].
F urtherm ore the kinetic constants d eterm in e d for
acetyl CoA carboxylase in the ap p lied cru d e
extracts (Fig. 6 , T able I) resem ble th a t recently
found for partially purified enzym e p rep a ra tio n s
Table I. Kinetic constants o f acetyl-CoA synthetase and acetyl-C oA carboxylase from spinach chloroplast extracts
(mean values).
K m [mM]
^max [nmol • m g 1 Chi • h ' 1]
MgATP
Free Mg
A cetyl-C oA
M gATP
acetyl-CoA
synthetase
0.14
0.14
-
1.85
acetyl-CoA
carboxylase
0.24
0.08
0.06
0.36
Enzymes
Substrate
Free Mg
0.36
K x[mM ]
Acetyl-CoA
Free ATP
ADP
-
1.50
0.71
0.10
0.44
0.10
A .Sauer and K..-P. H eise • R egulation o f A cetyl-C oA C arboxylase and A cetyl-C oA S ynthetase
from spinach chloroplasts [21]. T he data supp o rt
earlier findings [20], th a t free and A T P-bound M g2+
are required for o ptim al activity o f acetyl CoA
synthetase even though the kinetic m echanism for
the activation o f this enzym e by free M g2+ appears
to differ from th at o f the carboxylase (Fig. 6 ). F ree
Mg2+ seems to increase the affinity o f the catalytic
centre for M gA TP in carboxylase. In the synthetase
it accelerates the enzym e catalyzed reaction possibly
by a conform ational change o f eith er the enzym e or
the enzyme substrate com plex. The observed
dependence o f the kinetic constants o f acetyl CoA
synthetase and carboxylase on free M g2+ m ay be o f
regulatory significance for com pensating changes in
the level o f chelating adenine nucleotides, w hich
has been found to influence the activities o f both
enzymes [24], L atter assum ption is sup p o rted by the
observation, that an activation o f fatty acid syn­
thesis in darkened chloroplasts by supplying co­
factors (ATP, N A D P H ) via a dihydroxyacetone
phosphate shuttle [ 1 2 ] was considerably stim ulated
by increasing the strom al Mg concentration beyond
the supposed dark value ( l - 3 m M ; [25]), although
the calculated increase o f the sim ultaneously av ail­
able ATP level (from 0 .2 -0 .4 mM) was com parably
low. Thus, the observed activation by free M g2+
may be ascribed to a com pensation o f the inhib ito ry
effect o f increasing A D P concentrations in the d ark
[23, 25],
Inhibitors
It has been show n th a t free A TP caused a
considerable decrease o f acetyl CoA carboxylase
activity [14, 21]. T his effect has been recently
attributed to co m tam in atio n s o f the carboxylase by
A TPase and adenylate kinase generating significant
am ounts o f A D P and AM P, w hich are inh ib ito rs o f
the carboxylase [23]. T his in terp retatio n cannot be
applied in the present investigation because the
disruption o f chloroplasts in the presence o f T riton
X-100 and subsequent gel filtration [10] elim inate
cofactors and substrates for A TPase and adenylate
kinase. F urther, du rin g the short assay tim es o f only
5 - 1 0 min, A TP hydrolysis in the extracts will play
only a m inor role, as suggested by sim ultaneous
m easurem ents o f the phosphorylation p otential
(data not shown). T herefore, the reduction o f acetylCoA carboxylase and synthetase activities at A TP
concentrations exceeding the adjusted M g2+ content
in the reaction m ixture (Fig. 7), w hich is super-
273
o
E fi
<0 t£
o _
1 o
^ E
A T P (m M )
Fig. 7. Effect o f ATP at a constant M g2+ (1 m M ) in the
assay medium on the activities o f acetyl-CoA synthetase
and acetyl-CoA carboxylase in spinach chloroplast
extracts.
im posed on the stim ulating effect o f M gA T P on
both enzyme activities (Figs. 4 and 5), seem s to be
related m ore to a com petitive in h ib itio n by free
ATP than to an in h ib itio n by A T P hydrolysis [23],
The Kj values for free A TP o b ta in ed for acetyl CoA
carboxylase and acetyl CoA synthetase w ere 0.44
and 1.5 m M respectively (T able I). But th e relatively
high strom al M g2+ concentrations present in intact
chloroplasts (0 .4 -1 |im ol • m g - 1 C hi) [26], suggest
that an inhibition by free A TP is unlikely to be o f
physiological significance. O n th e o th er h an d , the
level o f A D P is know n to increase in d ark e n ed
chloroplasts [25]. T h erefore th e above m en tio n ed
evidence for the inhibitory effect o f A D P [23] has
been extended to b oth key enzym es o f acetate fix a­
tion in the chloroplast extracts. T he
o f both
enzymes for M gA TP increases w ith A D P co n cen tra­
tion (Table II). T his indicates th a t A D P is not only a
com petitive in h ib ito r for acetyl CoA carboxylase
( ^ = 0.1 m M ) but also for acetyl CoA synthetase
(A^ = 0.71 m M ) w ith respect to M gA TP (T ab le I). In
Table II. Effect o f A D P on the K m o f acetyl-C oA syn­
thetase and acetyl-CoA carboxylase in spinach chloro­
plast extracts for MgATP.
[mM]
ADP
Concentration
Acetyl-CoA
carboxylase
Acetyl-CoAsynthetase
0.14
0.18
0.24
0.32
0.02
0.12
0.26
0.52
[mM]
0
0.2
0.5
1.0
274
c
o_
O SI
E _
A .Sauer and K.-P. Heise • R egulation o f A cetyl-C oA C arboxylase and A cetyl-C oA S ynthetase
030
0.24
£ 6
< 7 0.18
O oi
^ E
>* *5 0.12
g
o i.
2 0 06
Fig. 8. Effect o f the A T P /A D P quotient in the assay
medium on the activities o f acetyl-CoA carboxylase and
acetyl-CoA synthetase in spinach chloroplast extracts. The
samples were incubated for 10 min in the standard
medium with 2 m M M gCl2 and 1 m M ATP at different
ADP-concentrations. The ÄTP used for the A T P /A D P
quotient is total ATP.
order to determ ine the regulatory significance o f th e
stromal adenine nucleotide levels on the activities o f
both enzymes during a light-dark tran sitio n , the
dependence o f th eir activities on the A T P /A D P
quotient in incubations o f chloroplast extracts has
been com pared w ith corresponding values d e te r­
mined in the strom a o f illum inated (A T P /A D P
= 2 - 3 ) and darkened (A T P /A D P = 0 .4 -0 .6 ) iso ­
lated chloroplast [25, 27], T he results (Fig. 8 )
suggest that the calculated decrease o f the strom al
A T P/A D P quotient during a light-dark transient, at
least in isolated chloroplasts, led to a strong red u c­
tion o f the activities o f acetyl CoA synthetase and
carboxylase and could th erefore exert a regulato ry
function in fatty acid synthesis [6 , 23]. T he relatively
higher stromal A T P /A D P q u o tie n t ( ^ 1) found in
chloroplasts from w heat le af protoplasts [27] in the
dark, which appears to be m ain tain ed by A TP
im port from the cytosol, m ay fu rth er explain, why
leaf discs [5] and w heat le a f protoplasts (u n p u b ­
lished results) as opposed to isolated chloroplasts
[5, 18] were still capable o f fatty acid synthesis
(about 2 0 % o f the corresponding light rates) in the
dark.
[1] B. P. Smirnov, Biokhimia 2 5 ,4 1 9 -4 2 2 (1960).
[2] J. B. Mudd and T. T. McManus, J. Biol. Chem. 237,
2056-2063 (1962).
[3] P. K. Stumpf and A. T. James, Biochim. Biophys.
Acta 70, 2 0 -3 2 (1963).
The above evidence suggests th a t a co o p e ra tio n o f
enzyme interconversions and light d ep e n d e n t
changes in the H +- and M g2+-co n cen tratio n and
A TP/A D P quotient affect the catalytic activ ities o f
acetyl-CoA synthetase and carboxylase in the
chloroplast strom a. Evidently these play an im ­
portant role in the light activation o f acetate in ­
corporation into long-chain fatty acids [ 1 -6 ] . T he
rates of lipid incorporation o f acetate by illu m i­
nated
chloroplasts
before
d isru p tio n
w ere
100-360 nmol • m g “ 1 Chi • h _1. In ch lo ro p last ex­
tracts under optim ized conditions, m alo n y l-C o A
was synthesized w ith sim ilar rates (up to 360 nm ol •
mg - 1 Chi • h_l), w hile acetyl-C oA fo rm a tio n show ed
significantly higher values (3 (.tmol • m g - 1 C hi • h -1).
Even allowing for the effect o f stro m al p a ra m ­
eters (e.g. H +, M g2+ and A T P /A D P q u o tie n ts) on
enzyme activity in illum inated ch lo ro p lasts th e h igh
activity o f acetyl-CoA synthetase m easu red suggests
that it is unlikely a rate lim itin g step in th e lig h t
regulation o f fatty acid synthesis from acetate. T his
conclusion is consistent w ith the findings th a t no
substantial difference in acetyl-C oA fo rm a tio n was
observed between illum inated and d ark e n ed c h lo ro ­
plasts [28], although fatty acid synthesis in th e d ark
was nearly totally in h ib ited [ 1 -6 ]. In co n trast, the
activity o f acetyl-CoA carboxylase in ch lo ro p last
extracts, calculated w ith respect at th e know n levels
o f above strom al solutes (H +, M g2+ and A T P /A D P
quotient) in illum inated chloroplasts [27], was
sim ilar to the rate o f lipid in co rp o ratio n o f acetate
in intact chloroplasts in the light. T his suggests th at,
as proposed [8 , 23], the carboxylase is the key enzym e
o f acetate fixation that w ould a p p e a r to be involved
in the light control o f acetate in c o rp o ratio n into
long-chain fatty acids in chloroplasts.
Acknowledgements
This work was supported by the D eu tsch e F o r­
schungsgemeinschaft. The au th o rs are g ratefu l to
Prof. H. W. H eldt and Dr. M. S titt for stim u la tin g
discussions and Dr. K. C. W oo for corrections.
[4] Y. Nakamura and M. Yamada, Plant Cell Physiol. 16,
139-149 (1975).
[5] J. Browse, P. G. Roushan, and C. R. Slack. Biochem .
J. 1 9 6 ,3 4 7 -3 5 4 (1981).
[6] A Sauer and K.-P. Heise, Plant Physiol. 73, 1 1 -1 5
(1983).
A.Sauer and K.-P. Heise • R egulation o f A cetyl-C oA C arb o x y lase and A cetyl-C oA Synthetase
[7] P. K. Stumpf, J. Brooks, T. G alliard, J. C. Hawke, and
R. Simoni, Biochemistry o f Chloroplasts, Vol. II
pp. 2 1 3 -2 3 9 (T. W. Goodwin, ed.), A cadem ic Press,
New York 1967.
[8] Y. Nakamura and M. Yamada, Plant Sei. Lett. 14,
2 9 1 -2 9 5 (1979).
[9] H. W. Heidt, C. J. Chon, and G. H. Lorimer, FEBS
Lett. 9 2 ,2 3 4 -2 4 0 (1 9 7 8 ).
[10] W. A Laing and P. G. Roughan, FEBS Lett. 144,
3 4 1 -3 4 4 (1 9 8 2 ).
[11] R. McC. Lilley and D. A. Walker, Biochim. Biophys.
Acta 3 6 8 ,2 6 9 -2 7 8 (1974).
[12] K. Werdan, H. W. Heidt, and M. M ilovancev, B io­
chim. Biophys. Acta 396, 2 7 6 -2 9 2 (1975).
[13] D. N. Kuhn, H. Knauf, and P. K Stumpf, Arch.
Biochem. Biophys. 2 0 9 ,4 4 1 -4 5 0 (1981).
[14] N. C. Nielsen, A. Adee, and P. K. Stum pf, Arch.
Biochem. Biophys. 1 9 2 ,4 4 6 -4 5 6 (1979).
[15] W. A. Laing, M. Stitt, and H. W. Heidt, Biochim .
Biophys. Acta 637,348 - 359 (1981).
[16] R. Krämer, Biochim. Biophys. Acta 592, 6 1 5 - 6 2 0
(1980).
[17] A Sauer and K -P. Heise, Z. Naturforsch. 37 c,
2 1 8 -2 2 5 (1982).
275
[18] J. Soll and P. G. Roughan, FEBS Lett. 146, 1 8 9 - 1 9 2
(1982).
[19] G. B. Stokes and P. K Stumpf, Arch. Biochem.
Biophys. 162,638 - 648 (1974).
[20] P. K Stumpf, The Biochemistry o f Plants, Vol. 4
(P. K Stumpf, ed.), pp. 177 —204 (1980).
[21] S. B. Mohan and R. G. O. Kekwick, Biochem . J. 187,
6 6 7 -6 7 6 (1980).
[22] A C. Storer and A J. C om ish-Bow den, Biochem.
J. 1 5 9 ,1 -1 4 (1 9 7 6 ).
[23] K C. Eastwell and P. K. Stumpf, Plant Physiol. 72,
5 0 - 5 5 (1983).
[24] P. K Stumpf, T. Shimakata, K. Eastwell, D. J.
Murphy, B. Liedvogel, J. B. Ohlrogge, and D. N.
Kuhn, Biochemistry and M etabolis, o f Plant Lipids
(P. C. J. Kuiper and J. F. G. M. W intermans, ed.),
pp. 3 —11, Biochemical Press, Amsterdam 1982.
[25] U. I. Flügge, M. Stitt, M. Freisl, and H. W. Heldt,
Plant Physiol. 69,263 - 267 (1982).
[26] A R. Portis, Plant Physiol. 6 7 ,9 8 5 - 9 8 9 (1981).
[27] M. Stitt, R. McC. Lilley, and H. W. Heldt, Plant
Physiol. 7 0 ,9 7 1 -9 7 7 (1982).
[28] P. G. Roughan, T. Kagawa, and H. Beevers, Plant
Sei. Lett. 1 8 ,2 2 1 -2 2 8 (1980).