ANALYTICAL BIOCHEMISTRY
216, 77-82 (1994)
On the Assay of Acetyl-CoA Synthetase Activity in
Chloroplasts and Leaf Extracts
P. Grattan Roughan* and John B. Ohlrogget
*Horticulture and Food Research Institute of New Zealand, Mt. Albert Research Centre, Private Bag 92 169, Auckland New
Zealand; and tDepartment of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824
Received April 19, 1993
Acetyl-CoA synthetase activity in vitro is assayed
quickly and conveniently by incubating whole chloro­
plasts, chloroplast extracts, or leaf extracts with la­
beled acetate, CoA, ATP, and Mg and transferring ali­
quots
of
the
reaction
mixture
to
pieces
of
either
Whatman No. 1 or DE81 filter paper. Unreacted ace­
tate is quantitatively washed from the papers while the
acetyl-CoA, which binds quantitatively, is determined
by scintillation counting. Enzyme activity is absolutely
dependent upon the presence of CoA, ATP, and Mg in
reaction mixtures. The reaction has a broad pH opti­
mum around pH 8.5. Potassium is required for maxi­
mum activity, and lithium strongly inhibits the reac­
tion.
The
product
retained
on
the
papers
was
characterized as acetyJ-CoA by several methods. On a
chlorophyll
basis,
acetyJ-CoA
synthetase
activities
were about 25% higher in leaf homogenates than in in­
�
ta
chloroplasts isolated from similar leaves. Enzyme
activities in the optimized assay were three- to fourfold
greater than previously reported.
,c
1994
Academic Press, Inc.
The synthesis of acetyl-CoA, catalyzed by acetyl-CoA
synthease (ACS,1 EC 6.2.1.1), is the first step in the syn­
thesis of long-chain fatty acids from exogenous acetate
by isolated, illuminated chloroplasts. Although ACS is
unlikely to be rate limiting in de nova fatty acid synthe­
sis (FAS), since its activity is 5-20 times that of FAS in
the same preparation (1), there is considerable interest
in the physiological role of the enzyme. For instance,
chloroplasts have been shown to be the sole repository
of ACS in spinach leaf cells (2), an extrastromal ACS
activity has been suggested for spinach chloroplasts (3),
1 Abbreviations used: ACS, acetyl-CoA synthease; Taps, 3-[tris(hy­
droxymethyl)methylamino-1-propanesulfonic acid; FAS, fatty acid
synthesis; 3-PGA, 3-phosphoglycerate; PDH, pyruvate dehydroge­
nase; OTT, dithiothreitol.
ooo:l-2697 /94 $5.oo
Copyright ct• 1994 by
and the enzyme from spinach and maize chloroplasts
may be light-activated (4,5). However, the role of ACS
in generating acetyl-CoA for fatty acid synthesis in vivo
has been disputed because of conflicting reports on con­
centrations of free acetate within leaves (2, 7), a lack of a
convincing system for generating the required acetate
(7), and a conviction that carbon for fatty acid synthesis
should be derived directly from products of photosyn­
thesis (7). In the latter scenario, 3-phosphoglycerate (3PGA) would give rise to pyruvate which would be con­
verted to acetyl-CoA by pyruvate dehydrogenase
(PDH). Although PDH activity was not initially de­
tected in isolated spinach chloroplasts (1,7) more recent
work appears to establish its presence in chloroplasts
isolated from pea ( 9), spinach ( 6,10), and maize (10).
Nevertheless, ACS activity has been detected in all
chloroplast preparations so far examined and, where
comparisons can be made, its activity normally exceeds
that of PDH by at least two- to fivefold (1,8,11; see,
however, Ref. 12). In addition, in almost every case
where comparisons have been made, acetate has proved
to be the preferred substrate for chloroplast FAS and
the presence of acetate in incubations has strongly in­
hibited the incorporation of other precursors into fatty
acids (1,13-15). There is also evidence that some plas­
tids may lack 3-PGA mutase, an essential enzyme for
converting 3-PGA, the immediate product of C02 fixa­
tion, to pyruvate (16,17).
Some perceived shortcomings of the assay for ACS
activity in plants originally proposed by Huang (18)
have led to the development of two alternative proce­
dures (2,19) which are, however, considerably more
complicated than the original without apparently im­
proving sensitivity or specificity. In one case, the [l-14C]­
acetyl-CoA formed in the reaction is trapped on char­
coal and subsequently transformed overnight into
acetyl hydroxamate to permit elution and measurement
of the radioactivity (2). In the other case, (l-14C]acetyl­
CoA is converted nonenzymatically to O-acetyl77
Academic Press, Inc.
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78
ROUGHAN AND OHLROGGE
dithioerythritol within the reaction mixture, and the
latter is extracted into chloroform for radioactivity mea­
surement (19). Neither the conversion of acetyl-CoA to
the corresponding hydroxamate nor the extraction of
0-acetyl-dithioerythritol into chloroform is strictly
quantitative so that standards must be included with
the assays to compensate for incomplete and variable
recoveries (2,19,20). Compared to the Huang procedure,
both alternative procedures are more laborious and
time consuming.
We previously ( 1) measured ACS activity in chloro­
plasts using a slight modification of an assay for long­
chain fatty acyl-CoA synthetase (21). The principle is
similar to that of the Huang assay. Aliquots of the reac­
tion mixture are transferred to discs of Whatman No. 1
filter papers which are then washed in diethylether/eth­
anol/acetic acid to remove unreacted [l-14C]acetate but
not [l-14C]acetyl-CoA. The latter is determined by di­
rectly measuring the radioactivity on the papers. This
procedure is simple, sensitive, and rapid, permitting
time-courses to be conducted routinely on multiple as­
says. However, in this paper we describe improvements
to this basic assay and reaffirm its simplicity and utility
by assessing concentration requirements for the various
reactants in the reaction. Acetyl-CoA is quantitatively
retained on the papers under a variety of conditions
likely to be encountered during enzyme purification,
e.g., assaying column fractions or fractions from density
gradient centrifugations. In addition, the product of the
reaction may be recovered intact for further verification
or utilization. Acetyl-CoA synthetase activities in chlo­
roplasts and leaves as measured in the optimized assay
are three- to fourfold greater than previously reported.
MATERIALS AND METHODS
[l-14C]Acetate and [l-14C]acetyl-CoA were from
Amersham. The latter was also prepared from [l-14C]­
acetate and CoA using acetyl-CoA synthetase (Sigma),
and was purified by TLC and HPLC. CoA, ATP, and
Percoll were from Sigma. Filter paper squares (1.5 X 1.5
cm) were cut from sheets of Whatman No. 1 (No. 1) or
Whatman DE81 (DE81) chromatography papers.
Amaranthus, pea, and spinach plants were grown in
controlled environments, and chloroplasts prepared as
previously described (22) were suspended in isotonic
buffer at 1 mg chlorophyll/ml. Protein (23) to chloro­
phyll (24) ratios in these preparations were 6-8 for
amaranthus, 12-16 for spinach, and 18-22 for pea
chloroplasts. For maximum ACS activities, 0.25-0.5 ml
of chloroplast suspensions was frozen and thawed, di­
luted to 1 ml with 25 mM Hepes/KOH, pH 7.8, 4 mM
MgC12, and 2 mM dithiothreitol (DTT), and centrifuged
at 13,000g for 2 min. Most (750 µl) of the supernatant
was passed through a 5-ml column of Sephadex G-25
equilibrated with 10 mM Hepes/KOH, pH 7.8, 2 mM
MgC12, and 1 mM DTT. Intact chloroplasts were also
added directly to incubation media containing 0.1% Tri­
ton X-100. Expanding leaves were homogenized in a
mortar with a little sand and 3-4 vol of 50 mM Hepes/K,
pH 7.8, 4 mM MgC12, 1 mM EDTA, and 10 mM mercap­
toethanol. The homogenate was filtered through two
layers of Miracloth, the filtrate was centrifuged 2 min at
13,000g, and 750 µl of supernatant was immediately
passed through a 5-ml column of Sephadex G-25 as
above. Equivalent chlorophyll concentrations of Sepha­
dex eluates were calculated from protein concentrations
of eluates and soluble protein to chlorophyll ratios of
the preparations loaded onto the columns. The final rou­
tine assay for ACS activity contained 50 mM Hepes/
KOH, pH 8.5, 8 mM MgCl2, 2 mM ATP(2Na), 0.25 mM
CoA, 0.4 mM [l-14C)acetate (8 Ci/mol), and 10 µl chloro­
plasts, stromal proteins, or leaf extract equivalent to
1-4 µg of chlorophyll in a final volume of 50 µI. This
volume was scaled up for carrying out longer time­
courses or when accumulating semipreparative amounts
of reaction product. Reactions were carried out at 25°C
and were started by adding enzyme, and 20-µl aliquots
of the reactions were transferred to filter papers at 2.5
and 5 min. One minute following the transfer, the
loaded papers were dropped into either 2% acetic acid in
acetone (for No. 1 papers) or 2% acetic acid in water (for
DE81 papers). Up to 10 papers were washed simulta­
neously in 25 ml of solvent in a 250-ml beaker with gen­
tle swirling on an orbital shaker. After 5 min the wash­
ing solution was replaced and shaking continued for
another 5 min. Finally the papers were washed in ace­
tone alone and then air dried. Initially, fixed radioactiv­
ity was determined by counting the papers in 3 ml of
nonaqueous scintillant in 15-ml vials, but it was subse­
quently found that the same counting efficiencies (6570%) were achieved by counting the papers in 1 ml of
scintillation fluid in a 1.5-ml microfuge tube. The orien­
tation of the papers within the scintillant was not criti­
cal. Papers could be recovered from the scintillation
cocktail and washed in acetone so that the radioactive
material could be eluted and characterized. Thin-layer
chromatographic separation of acetyl-CoA (R10.5) was
performed on 0.25-mm layers of Sigmcell Type 20 using
butanol-1-ol/acetic acid/water (5/2/3) for development,
and the labeled product was detected by autoradiog­
raphy.
RESULTS AND DISCUSSION
Removal of [14C]acetate from the filter papers. The
first requirement of this assay is that unreacted acetate
should be completely washed from the papers while ace­
tyl-CoA is quantitatively retained. When 25-µl aliquots
of the full reaction mixture, without enzyme, were
transferred to the No. 1 or DE81 papers, which were
then washed as described, >99.9% of the applied acetate
ASSAY FOR ACETYL-CoA SYNTHETASE ACTIVITY IN PLANTS
was eluted. This elution of acetate was unaffected by the
presence of 20 mM MgCl2, 0.33 M sorbitol, 0.5 M sucrose,
or 0.2 M LiCl in the respective reaction mixtures. By
'
contrast, authentic (l-14C]acetyl-CoA was quantita­
tively retained on the papers during the entire washing
procedure. Loaded papers could be transferred to the
washing solution without loss of label as soon as the 20to 25-µl sample had been completely absorbed into the
paper ( <5 s). However, as there was no difference in
apparent reaction rates when the papers were allowed
to stand for 10 s or 5 min before washing, a time of 1 min
was adopted as a convenient standard time between
loading the papers and initiating washing. The labeled
product formed when chloroplasts were incubated in
the presence of acetate, CoA, and ATP was identified as
acetyl-CoA primarily by TLC and HPLC (25) and also
by its reaction with DTT and neutral hydroxylamine
(below).
Dependence of acetyl-CoA formation upon added cofac­
tors. In the presence of chloroplasts or chloroplast ex­
tracts, acetate was transformed into material which
bound to the papers only when CoA, ATP, and Mg were
also present in reaction mixtures. Maximum rates of
acetyl-CoA synthesis were achieved at 0.2-0.5 mM CoA,
1-2 mM ATP, 4-8 mM MgCl2 and 0.4 mM acetate (Fig. 1
and results not shown). Higher concentrations of CoA
inhibited the reaction in spinach (see also Ref. 26), pea,
and amaranthus chloroplast extracts (Fig. 1) but not in
corn chloroplasts (result not shown). Approximate Kms,
derived from substrate concentration at one-half maxi­
mum velocity, for the various substrates in the reaction
were 30-75 µM for acetate, 25-50 µM for CoA, and 100200 µM for ATP. Whereas the reaction catalyzed by the
enzyme from pea and amaranthus was maximal at 2 mM
MgCl2 (apparent Km < 1 mM; kinetic results not shown),
maximum rates using the spinach enzyme were only
achieved with Mg in excess of that required for stiochio­
metric association with ATP (apparent Km 2 mM). This
suggests either that free Mg is a cofactor for spinach
ACS (27) or that it stabilizes the enzyme. Magnesium
concentrations up to 10 mM did not adversely affect the
assay and 8 mM was chosen for routine use. This depen­
dence upon added substrates and cofactors was demon­
strated equally well by whole chloroplasts, chloroplast
extracts, and leaf homogenates when chlorophyll con­
centrations were > 100 µg/ml in the reactions. Acetyl­
CoA formation was linear with respect to chlorophyll up
to at least 120 µg/ml in a 2.5-min assay and was linear
with time for at least 10 min when chlorophyll did not
exceed 50 µglml. The reaction exhibited a broad pH op­
timum in Hepes/Taps/KOH buffers, pH 7.5-9, being
most active around pH 8.5, and reaction rates were
equal in Tris, Tricine, Taps, and Hepes buffers. A re­
quirement for potassium was demonstrated by a 55100% stimulation of the reaction by 20 mM KCl in Tris-
79
HCl buffer and this stimulation was greater at pH 7.5
than at pH 8.5. By contrast, 20 mM NaCl was slightly
inhibitory. Maximum rates of ACS activity measured
here for spinach and pea chloroplasts were at least
three- to fourfold higher than those reported previously
(2,4,18,26) when expressed relative to chlorophyll.
Interference in the assay. Substances which might be
present in preparations to be assayed for ACS activity
were checked for possible interference. The reaction
was mildly inhibited by sorbitol and glycerol and more
strongly inhibited by sucrose, LiCl, and (NH )2S0 (Ta­
4
4
ble 1) but not by 0.1% (w/v) Triton X-100. Pea ACS was
50-60% inhibited by 10 mM LiCl when the reaction was
run in Tris, pH 8. Hence, it is not the recovery of prod­
uct as suggested in (2) but rather the activity of the
enzyme which must be considered when interpreting the
influence of these compounds on the measured activity
(see also 28). Also, Triton X-100 may be used to ensure
chloroplast lysis without affecting the reaction. The for­
mation of acetyl-CoA was not affected by the presence
of 0.5 mM DTT in the reactions but was 60 and 80%
inhibited in the presence of 5 mM and 10 mM DTT,
respectively. The 0-acetyl-dithiothreitol that was
formed by acetyl transfer from acetyl-CoA did not bind
to DE81 papers.
Separate advantages of No. 1 and DEBI papers. Be­
cause the acetyl-CoA bound more strongly, and washing
those papers with 2% acetic acid is probably more conve­
nient and economical than washing in acetone, the
DE81 papers were preferred for routine assays. How­
ever, the DE81 papers became slightly fragile when wet
and the No. 1 papers retained their integrity better dur­
ing the acetone washing. Other advantages of using the
No. 1 papers are a slightly higher counting efficiency
(70% compared to 65%) and the greater ease with which
the fixed acetyl-CoA may be eluted (compared to
DE81). In fact, it is important that anhydrous acetone
be used for washing the No. 1 papers since only 15% of
the reaction product was retained when No. 1 papers
were washed in 80% compared with 100% acetone.
Characterization of the product as acyl-CoA. When
the same reaction was analyzed using both the filter
paper method and TLC on cellulose, there was excellent
correspondence between the values obtained (results
not shown). The reaction product eluted from washed
Whatman No. 1 papers was transformed, either in 10
mM dithioerythritol in 50 mM Bicine, pH 8.6, or in 0.5 M
hydroxylamine, pH 7, to compounds which no longer
bound to the papers. The product bound to DE81 papers
was 50% eluted in 0.1 M K-P0 , pH 5, and quantatively
4
eluted in 0.25 M LiCl. It was also quantitatively solubi­
lized from these papers within 5 min by 40 mM Tris, pH
9, containing 20 mM DTT and by 0.2 M NH20H, pH 7
(Fig. 2), presumably due to the formation of acetyl-DTT
and acetylhydroxamate, respectively. These reactions
80
ROUGHAN AND OHLROGGE
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SUBSTRATE CONCENTRATION (mM)
FIG. 1.
Dependence of spinach (A), pea (B), and amaranthus (C) chloroplast acetyl-CoA synthetase activities on concentrations of acetate,
CoA, and ATP in reactions. Each point represents a mean of four different preparations of stromal proteins recovered from Sephadex G-25
columns.Reaction media (50 µl) were as described in the methods but with varying concentrations of either acetate (0), CoA (L::.) , or ATP (0) as
shown. Incubation was at 25°C and 25 µl was transferred at 5 min to filter papers for determination of acetyl-CoA formation.
are characteristic of acyl thioesters. When DE81 papers
loaded with reaction product were moistened and ex­
posed to ammonia vapor at room temperature for 15-30
min, > 90% of the radioactivity was eluted in a subse-
TABLE I
Inhibition of Spinach and Pea Chloroplast ACS by Sorbitol,
Sucrose, Glycerol, LiCl, and (NH4)2S04
% Inhibition
Additions to assay
(final concentrations)
0.33 M sorbitol
0.33 M sucrose
0.33 M glycerol
0.1 M LiCl
0.1 M (NH,)SO,
Spinach
Pea
12
14
26
15
70
32
30
13
59
45
Note. Additions were to the routine assay of stromal ACS as de­
scribed under Materials and Methods.
quent wash in 2% acetic acid. The 5-min reaction prod­
uct trapped on No. 1 papers was eluted into water and
analyzed by radio-HPLC. In incubations containing de­
tergent-lysed chloroplasts, both acetyl-CoA and ma­
lonyl-CoA became labeled (the latter presumably
through acetyl-CoA carboxylase activity), and the pro­
portion of the malonyl-CoA was usually enhanced when
10 mM KHC03 was included in the reaction.
Comparison of ACS in chloroplasts and chloroplast ex­
tracts. The dependence of the reaction upon added
CoA, ATP, and acetate was determined (Fig. 1) using
chloroplast extracts free of endogenous low-molecular­
weight compounds. However, virtually the same result
was achieved by lysing intact chloroplasts directly into
the reaction medium. The very low blank values, vir­
tually zero as in Fig. 1, measured when CoA was omitted
from reactions initiated with whole chloroplasts con­
firm the low concentrations of endogenous CoA in
chloroplasts (25). On the other hand, rates of [l-14C]­
acetyl-CoA formation increased by 12-40% when lysed
ASSAY FOR ACETYL-CoA SYNTHETASE ACTIVITY IN PLANTS
chloroplasts were passed through Sephadex, suggesting
that intact chloroplasts may contain either endogenous
substrate which dilutes the supplied [1-14CJacetate or
low-molecular-weight compounds that inhibit the reac­
tion. Judging from enzyme activities when intact chloro­
plasts were used to initiate reactions, both spinach and
pea chloroplasts were completely lysed in the medium
containing 33 mM sorbitol but amaranthus and corn
chloroplasts were not. However, maximum rates of ACS
activity using intact chloroplasts from amaranthus and
corn were obtained by including 0.1 % (w/v) Triton
X-100 in reactions (see also Ref. 29). Therefore, it is
probably advisable to include Triton X-100 in all assays
of ACS where intact chloroplasts are used to initiate
reactions.
81
TABLE 2
Activities of Acetyl-CoA Synthetase in Isolated Chloroplasts
and in Leaf Homogenates
µmol Acetyl-CoA/h/mg chlorophyll
Plant species
Chloroplast
Whole leaf
Spinach
Pea
Amaranthus
Corn
15.1±2.4
14.4±1.6
17.6±2.l
2-5
18.0±2.2
19.6 ± 3.6
20.8±2.5
3-6
Note. Chloroplast stromal proteins and centrifuged lead homoge­
nates were passed through Sephadex G-25 columns prior to assaying
for enzyme activity as described under Materials and Methods. n = 6
for spinach and pea and 7 for amaranthus.
Levels of ACS activity in chloroplasts and leaf homoge­
nates. Typical activities of ACS in chloroplasts from
different sources were 12-20 µmol/h/mg chlorophyll
(Table 2) where rates of acetate incorporation into
long-chain fatty acid are typically 1-2 µmol/h/mg chl
(22). On a chlorophyll basis, enzyme activities were
about 25% higher in Sephadex-treated leaf homoge­
nates than in isolated chloroplasts (Table 2). Activities
of crude leaf homogenates were always increased,
whether on the basis of corrected chlorophyll or total
protein, following the Sephadex treatment. The higher
activity in cell-free extracts compared with isolated
chloroplasts may mean that ACS is not strictly confined
to chloroplasts as has been suggested (2) or that chloro­
plasts lose some of their activity during isolation. The
ACS activity in crude homogenates of amaranthus, pea,
and spinach leaves was stable for at least 60 min at 0°C,
yet some leaves (barley, oats) yielded no ACS activity
even when the usual protein protectants were added to
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TIME (min)
FIG. 2. Reactions of acetyl-CoA on the filter papers with dithiothre­
itol and with neutral hydroxylamine. DE81 papers loaded with known
amounts of reaction product (approx 150 X 10' dpm) were immersed
in 0.5 ml of 40 mM Tris/HCl, pH 9 (0), 40 mM Tris/HCl containing 20
mM DTT (6), or 0.2 M NH20H, pH 7 (0), and 25 µl of solution was
removed for counting at the times shown.
the homogenization buffer. However, exogenous acetate
is an excellent precursor for FAS in those barley and oat
leaves.
Advantages of the filter paper assay. Convenience, ra­
pidity, and accuracy are the principle merits of the pres­
ent assay compared with the alternatives for measuring
ACS activity in plants. Because repeated extractions
(2,19) are not involved, large numbers of assays, as for
example in measuring [SJ vs product formation, may be
performed with relative ease and in a relatively short
time. Quantitative recovery of the product on the papers
is virtually assured. Since the washed papers are color­
less, determining radioactivity by scintillation counting
does not present the problems of color quenching and
chemiluminescence associated with counting materials
containing plant pigments (19). Counting efficiency on
the papers is determined by recounting samples under
conditions where the product is completely eluted into
the scintillation cocktail. In addition, following count­
ing in a nonaqueous cocktail, the labeled product may
be recovered intact for further characterization and uti­
lization. The main disadvantage of the method is the
limited sample size (25 µl) that may be applied to the
2.25-cm2 papers, although the size of the papers could
presumably be at least doubled without causing much
inconvenience.
Both carnitine acetyltransferase and PDH activities
can be measured sensitively by using filter papers to
trap the acetyl-CoA synthesized (1,30). The [1-14C]­
acetylcarnitine and r2-14C]pyruvate used as substrates
in the reactions are readily washed from the papers and
the labeled acetyl-CoA retained for counting in the sys­
tem described.
ACKNOWLEDGMENT
This work was supported in part by Research Grant DCB 9005290
from the National Science Foundation to J.B.0.
82
ROUGHAN AND OHLROGGE
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