J. Plant Physicl. Vol. 141. pp. 276-280 (1993)

Isolation and Characterization of Acetyl-CoA Synthetase from Etiolated Radish Seedlings

ANDREA GOLZ and HARTMUT K. LICHTENTHALER

Botanical Institute (Plant Physiology and Plant Biochemistry), University of Karlsruhe, Kaiserstr. 12, D-7500 Karlsruhe, Germany

Received October 5,1992 . Accepted October 12, 1992

Summary

Acetyl-CoA synthetase (ACS; E.C. 6.2.1.1.) is a key enzyme in plastids of higher plants which provides the starting substrate acetyl-CoA for de novo fatty acid biosynthesis. The ACS enzyme activities, meas­ured in leaves and chloroplasts of different plants, are described. The enzyme was purified 183-fold from etiolated radish cotyledons to a final specific activity of 193 nmol min -I mg- I protein. The properties, the substrate specifity and the Michaelis-Menten constants (Km for ATP, acetate and coenzyme A) of the pu­rified enzyme were determined. The ACS exhibits a high substrate specifity for acetate as compared to other organic acids (e.g. propionic, acrylic or butyric acid).

Key words: Acetyl-CoA Synthetase; Properties; Purification; Raphanus sativus.

Abbreviations: ACS = acetyl-CoA synthetase; DTE = dithioerythritol; Tris = Tris(hydroxymethyl)­amino methane; Tricine = N-Tris(hydroxymethyl}methyl glycine.

Introduction

In the plastids of higher plants acetyl-CoA plays a central role in several biosynthetic pathways. Besides de novo fatty acid biosynthesis, acetyl-CoA is needed for the synthesis of mevalonic acid and isoprenoid lipids as well as branched­chain amino acids. It is well known for a long time that iso­lated chloroplasts are able to incorporate HC-acetate into fatty acids (Smirnov, 1960; Mudd and McManus, 1962; Stumpf and James, 1963). Therefore, isolated chloroplasts and etioplasts have been established as a suitable test system for the de novo biosynthesis of fatty acids from HC-acetate which includes the enzyme systems acetyl-CoA synthetase (ACS), acetyl-CoA carboxylase (ACC) and the fatty acid synthetase complex (FAS) (Kobek et al., 1988; Kobek and Lichtenthaler, 1989; Lichtenthaler and Focke, 1992). In higher plants it was first shown by Millerd and Bonner (1954) that ACS activates acetate to acetyl-CoA. Free acetate can permeate through the chloroplast envelope, but the membrane is not permeable to acetyl-CoA (Brooks and Stumpf, 1966; Jacobson and Stumpf, 1970). In higher plants the enzyme ACS is exclusively located in the plastids (Kuhn

© 1993 by Gustav Fischer Verlag, Stuttgart

et al., 1981). ACS forms acetyl-CoA from acetate, ATP and CoA by releasing acetyl-CoA, AMP and pyrophosphate:

acetate + ATP + CoA ~ acetyl-CoA + AMP + PP j

Another important acetyl-CoA-producing enzyme system is present in plastids, the pyruvate dehydrogenase complex (Reid et al., 1977; Williams and Randall, 1979), which can provide acetyl-CoA from pyruvate. In the case of intact spin­ach chloroplasts the incorporation of HC-acetate is, however, preferred to HC-pyruvate (Mudd and McManus, 1962; Stumpf and James, 1963; Roughan and Slack, 1977). At pre­sent there is still some discussion which enzyme supplies the major part of acetyl-CoA for the different acetyl-CoA con­suming pathways of the chloroplast. There are several ob­servations that the acetyl-CoA source can depend on the plant species (Liedvogel and Bauerle, 1986; Springer and Heise, 1989), on the development of plastids (Heintze et al., 1990) and on the plastidic acetate and pyruvate levals (Kuhn dt al., 1981; Liedvogel, 1985 a; Dreede et al., 1986). Only in the case of isolated chloroplasts from the endosperm of Ri· cinus communis it was shown that pyruvate was incor-

porated into fatty acids four times faster than acetate (Miernyk and Dennis, 1982; 1983), but the Ricinus endo­sperm rapresents a particular case which seems not be rdpre­sentative for the leaf and other tissues of higher plants.

The ACS enzyme has been characterized from a number of animal tissues (Londesborough et aI., 1973) and from bak­er's yeast (Frenkel and Kitchens, 1977). Several attempts had been made topurify the ACS from plant tissue, but the en­zyme had not yet been purified to homogeneity. From non­green plant tissue (potato tubers) the enzyme has been only partially purified (Huang and Stumpf, 1970). A purer ACS preparation was obtained only recently from spinach leaves (Zeiher and Randall, 1991). In order to obtain more informa­tion on the plant ACS we determined its activity in leaf and shoot tissues and also in chloroplast preparations of different plants. Furthermore, the isolation of the ACS enzyme from etiolated radish seedlings and its substrate specifity are de­scribed here as well.

Materials and Methods

Radish seedlings (Raphanus sativus L. var. Saxa Treib) were cul­tivated on water for 6 d in darkness. The other plants were cul­tivated on a mineral-containing peat (TKSll, Floratorf) in a 14/10 h day/night cycle.

Direct assay 0/ A CS in the leaf tissue: The leaves (shoots) of differ­ent plants were homogenized at 4 °C in a hypo-osmotic buffer system consisting of 10 mM Tricine pH 8, 2 mM DTE and protease inhibitors. After filtration through 10 layers of cheesecloth and cen­trifugation for 30 min at 16,000 x g, the supernatant was used for ammonium sulfate sedimentation of the plant ACS (see below).

ACS from isolated plastids: For the determination of the ACS ac­tivity in the plastid fractions, the leaf material was homogenized at 4°C in an iso-osmotic isolation medium which contained 330 mM sorbitol, 100 mM Tris pH 9, 2 mM MgCI 2, 2 mM DTE and as pro­tease inhibitors, 2 mM benzamidine, 2 mM foaminG-caproic acid and 0.2 mM phenylmethylsulfonyl fluoride. Homogenisation was carried out within 10 s in a mixer with replacable razor blades (Kan· nangara, 1977). After filtration through 10 layers of cheesecloth and centrifugation for 10 min at 4000 x g, the supernatant was discarded. The plastids in the sediment were osmotically broken in a medium with 10 mM Tricine pH 8, 2 mM DTE and protease inhibitors by using a cell mill for 2 min (VI 4, Buhler) and then centrifuged for 30 min at 16,000 x g.

Ammonium sulfate fractionation 0/ ACS: The supernatants from the direct leaf homogenization or from the broken chloroplasts were brought to 40 % saturation by slow addition of saturated ammonium sulfate solution and stirring was continued for 20 min. After centrifugation (20 min, 16,000 x g), the sediment was dis­carded and the supernatant was brought to a 70 % ammonium sulfate saturation. The 40-70 % pellet was resuspended in buffer pH 8 (100 mM Tricine pH 8, 2 mM MgCI2, 2 mM DTE and protease inhibitors) and desalted by passing through a small gel-filtration col­umn (PD 10, Sephadex G·25M, Pharmacia).

Further purification 0/ the A CS enzyme {from etioplasts 0/ radish cotyledons}: The desalted pellet was applied to a FPLC anion ex­change column (Fractogel EMD TMAE-650(M), Merck) in SO mM potassium phosphate buffer pH 7 which contained 2 mM DTE and protease inhibitors. The protein, including ACS, bound to the col­umn was eluted with a linear KCI gradient (0 to 1 M KCI in 60 min). The active ACS fractions were pooled and applied to a dye-ligand af­finity column (TSK AF-orange, Merck) in a buffer which contained 330 mM sorbitol, 150 mM potassium phosphate pH 7.5, 2 mM

Plant acetyl-CoA synthetase 277

DTE, 2 mM MgCh and protease inhibitors. The column-bound en­zyme was eluted with 1 M KCl in the same buffer. The active frac­tions were applied directly to a gel-filtration column (Merck HW­TSK SO). After addition of 2 mM DTE the active fractions were stable at - 20 °C for a few weeks. Protein concentrations were de­termined after the method of Lowry et al. (1951) as modified by Bach et al. (1986).

A CS enzyme assay: The assay of the ACS is based on the forma­tion of 14C-acetyl-CoA in the first step and the non-enzymic acyla­tion of dithioerythritol (DTE) from acetyl-CoA in a second step (Stokes and Stumpf, 1984). The assay was performed as described (Liedvogel, 1985 b) with some modifications (Focke et al., 1990). The standard assay contained 0.1 M tricine buffer pH 8, 5 mM MgCb, 0.25 mM acetate (0.027IlCi 14C), 0.5 mM CoA, 2 mM ATP in a final volume of SO ilL. After a 10 min incubation at 30°C the enzymic reaction was stopped with an equal volume of a solution which contained 3 M NaCl, 0.2 M Tricine pH 8, 1 M sodium acetate and 20 mM dithioerythritol. The acylation of DTE proceeded for 1 h at 30°C. Thereafter the resulting acylation product was ex­tracted twice with diethylether and the radioactivity counted in a li­quid scintillation counter after evaporation of the ether.

All values given represent means of at least eight determinations from three independent experiments, maximum standard deviations being ± 5%.

Results and Discussion

Several green and etiolated plant tissues were examined in order to find an appropriate starting material for the isola­tion of the acetyl-CoA synthetase. It appeared that in most plants it was necessary to isolate the chloroplasts in a first step (sedimentation at 4000 x g for 10 min) before other methods such as hypo-osmotic rupture of plastids,

Table 1: Specific activities of acetyl-CoA synthetase preparations from different plants (in nmol· mg- l

. min-I). The specific activities were determined for the desalted 40 -70 % ammonium sulfate pellet fractions. The values obtained for different preparations of the same plants varied up to 30 %.

Plant species Age of Specific activity seedlings plastid fraction without isolation

a) dicotyledonous plants

Spinacia oleracea L. (var . Matador) Pisum sativum L. (var. kl. Rheinlanderin) Raphanus sativus L. (var. Saxa Treib) Vicia [aba L. Phaseolus vulgaris L.

b) monocotyledonous plants

Zea mays L. (var. P rotador) A vena sativa L. (var. Flammingsnova) Hordeum vulgare L. (var. Alexis)

c) greening of etiolated tissue

Raphanus sativus L.

40d 6.4

10d 4.5

6d 8.1

lOd 0.9

10d 0.7

10d 2.1

7 d 1.2

7d 1.7

etiolated cotyledons 6 d 7.2 etiolated + 12 h light 6 d 8.0 green cotyledons (3d light) 6d 7.1

of plastids

5.9

4.3

0.0

0.3

0.1

0.0

0.0

0.2

278 ANDREA GOLz and HARTMUT K. LICHTENTHALER

Table 2: Purification and isolation scheme of the ACS from cotyle-dons of 6 d old etiolated radish seedlings.

Protein Specific activity Yield Purification [mg] [nmol mg- I min- I] [%] factor

Homogenate of 2541 1.06 100 1.0 cotyledons

Plastid sediment 117 0.53 2.3 0.5 (tricine·buffer pH 8)

40-70% NH.(SO' )2 32 13.3 15.5 12.5 pellet (desalted)

Fractogel fraction 7 47 12.6 45.0 (anion exchange column)

Dye-ligand fraction 0.8 99 3.0 93.0 (orange column)

Gel-filtration fraction 0.2 193 1.7 181 (TSK-HW 150)

ammonium sulfate precipitation, etc. could be applied. In several plants tested in this way the ACS activity was com­pletely lost when the isolation of chloroplasts did not pre­cede the enzyme isolation (Table 1). For spinach and pea the isolation of intact plastids was, however, not necessary, since the specific activities with or without plastid isolation were about the same.

The cotyledons of radish seedlings, as a starting material and source for ACS, resulted in the highest specific ACS ac­tivity after the 40 -70 % ammonium sulfate precipitation step (Table 1). Since there were no differences in specific acti­vities of ACS between green and etiolated radish seedlings, we selected etiolated radish cotyledons as source for the ACS purification. The complete purification protocol developed is shown in Table 2.

After isolation of etioplasts and ammonium sulfate precip­itation of the soluble stroma proteins, we obtained a 12-fold enrichment of the ACS activity in the desalted 40 to 70 % ammonium sulfate pellet. This fraction was applied to an FPLC anion exchange column from which the active ACS fractions were eluted at 250 mM KCl. This step led to an en­richment of about 45-fold. The ACS fractions were directly applied to an AF-orange dye-ligand column. Other dye-li­gand materials successfully bound ACS, but the elution of the enzyme in an active form was not possible except from the AF-orange dye. Though the application of the AF­orange dye-ligand material proved to be very useful under optimized conditions, this step of purification, however, re­duced the yield of enzyme drastically, while increasing the purification factor to 93. Without further concentration, the active fractions of the dye-ligand column were further pu­rified and desalted by gel-filtration and resulted in a final 181-fold enrichment with a specific activity of 193 nmol mg- I

min-I. The ACS enzyme, while fairly pure, has not been pu­rified to homogeneity so far; further steps with specific affin­ity materials are, however, in preparation.

Concerning the stability of the purified ACS preparation, we observed large differences depending on the purification stage. The desalted 40 -70 % ammonium sulfate fractions were stable for more than 6 months at -20 °C in Tris-buffer pH 8. After the anion exchange step, the samples could be stored at - 20 °C for about 2 weeks without significant loss of activity. After the AF-orange dye-column the stability of

the ACS was drastically reduced; the presence of sorbitol al­lowed, however, the storage at - 20 °C over night. In con­trast to observations made by Zeiher and Randall (1991) with an ACS preparation from spinach, the presence of 20 % glycerol did not increase the stability of the radish ACS. After gel-filtration, a storage of the ACS enzyme fraction at - 20 °C was possible for about 2 weeks without significant loss of activity.

ACS is salt-tolerant, which allows the purification of the enzyme by application of salt gradients for its elution from column materials. Even 1 M KCl or NaCl in the enzyme assay reduced the activity only by 30 % relative to a control without salt. Thiol protection of the enzyme with DTE or DTT increased significantly the stability of the enzymic preparation between the different purification steps. It has to be mentioned that small amounts of DTE (about 1 mM) were already sufficient, since there was no difference in ACS activity with 1 mM DTE or 20 mM DTE in the assay system. Also preincubation of the enzyme with 1 mM DTE without further addition of DTE did not reduce the ACS activity. Therefore we added 20 mM DTE together with the stop­solution which is needed for the non-enzymic acylation of DTE as described by Stokes and Stumpf (1974). The purified radish ACS was stable over a broad pH range and showed a broad pH optimum between 7.5 and 9 (Fig. 1). The enzyme assay had to be modified in the case of pH variation because the non-enzymic acylation of DTE is pH-dependent (Lied­vogel, 1985 b). In these experiments the stop-solution con­tained 1 M Tris buffer pH 8 in order to have reliable pH con­ditions during the acylation. The ACS activity increased with an increase of the assay temperature up to 50°Cj at higher temperatures the enzyme was, however, rapidly inac­tivated (Fig. 2 a). The corresponding Arrhenius plot allowed the determination of the apparent activation energy of 48.9kJ/mol (Fig.2b).

Using an ACS fraction purified by gel-filtration we de­termined from Lineweaver-Burk plots (Fig. 3) the Km values of the enzyme for the three substrates. The Km(acetate) was 55 11M, the Km(ATP) was 161 11M and the Km(CoA) was 93 11M. For ATP and acetate the Km-values obtained here for Raphanus are in good agreement with the values given by Zeiher and Randall (1991) for the purified spinach enzyme: 57 11M (acetate) and 150 11M (ATP). In the case of CoA, how-

>. +' .> :;:::; 100

u 0 80 E :l

E 60

·x 0 40 E '0 20

~

a 3 4 5 6 7 8 9 10 11 12 13

pH value

Fig. 1: pH-optimum of a purified ACS preparation isolated from radish cotyledons (post gel-filtration). 100 % activity corresponds to the maximal ACS activity at pH 8.5.

.r:--' 500 Ie a E 400

* '0'> E 300

* 0 200 E e

L-.J 100

>

280 290 300 310 320 330

T [K} 6.0

> b Arrhenius Plot e

5.0

4.0

3.0

2.0 .j----_~~~I---<~~__I~~-___1 3.30 3.40 3.50 3.60 3.70

1 IT [K -1]

Fig. 2: a) ACS activity with increasing temperature and b) Arrhenius plot for the determination of the activation energy. An activation energy of 48.9 kJ/mol was determined from the slope of the Arrhenius plot, applied were the values from 273 to 298 K. (Par­tially purified radish ACS, specific activity at 273 K: 185 nmol minot mg-!).

ever, the Km(CoA) of the spinach enzyme was 5 J.1M and a substrate inhibition was described with a Ki value for CoA of 700 J.1M. In contrast, with the radish ACS preparations, we never observed such a CoA substrate inhibition even at the very high CoA concentration of 2 mM. This difference be­tween the radish and spinach ACS preparations cannot be explained; it may indicate that the ACS enzymes of different plants possess different characteristics. This topic needs de­tailed investigation using different plants and the same assay system.

The incorporation of HC-acetate into acetyl-CoA can be diluted with unlabelled acetate as expected (Table 3). Besides acetate, we tested some other carboxylic acids as potential competitive inhibitors of HC-acetate incorporation in order to elucidate the substrate specifity of the ACS enzyme. Fluoracetate, formate and butyrate had only little influence on !4C-acetyl-CoA formation. Only in the case of propionate and acrylate was significant competition and dilution observ­ed. Other tested carboxylic acids and acetate-related com­pounds such as methyl-butyrate, malonate, oxalate, acet­ylene-dicarboxylic acid and glycine, had no effect on the incorporation rate of HC-acetate. This demonstrates that the radish ACS has a very high substrate specifity for acetate. Its affinity towards the substrate analogues, propionate and acrylate, is much lower than towards acetate. A similar high substrate specifity for acetate has been shown for the spinach ACS (Zeiher and Randall, 1991), the ACS from mammals (Londesborough et aI., 1973) and that from baker's yeast (Frenkel and Kitchens, 1977).

Plant acetyl-CoA synthetase 279

., 0.05,-----,-----------,;r--,

....., c 'E "'-01 E "'­(5 E

O.O~

0.03

0.02

oS 0.Q1

.~ 0.00+--....".."--+-------------l

~ ~ -0.Q1 +-''--_-+--__ -+-__ --+---'

-0.05 0.00 0.05 0.10

1/[acetate] in Ji.M -1

., O.O~

~ c ·E 0.03

"-0> E 0.02 "-0 E

0.01 oS .!: > 0.00 ~

-0.01 -0.01 0.00 0.Q1 0.02

T-, 0.09 C

~ 0.07 01 -t 0.05 (5

E 0.03 oS .~ 0.Q1

~

1/[ATP] in JLM -1

~ -0.Q1 :l"-::---:+:---:+:--+----,--:-+---II---+--' -0.02 0.00 0.02 O.O~ 0.06 O.OB 0.10

1/[CoA] in Ji.M-1

Fig. 3: Determination of the Km-values with respect to the substrates ATP, acetate and CoA using purified ACS preparations from radish seedlings. Concentrations of the non-variable substrates were 5 mM MgClz, 2 mM ATP, 1 mM CoA and 1 mM acetate.

Table 3: Substrate specifity of purified acetyl-CoA synthetase for different carboxylic acids as compared to acetate. The applied acids were unlabelled and their dilution effect on the incorporation rate of HC-acetate (given as percent of control without unlabelled sub­strate) was determined; this is a measure of the specifity of the ACS for acetate (mean of 6 determinations from 2 isolations).

Concentration [mM] 2 5

Acetate 18.9±O.4 9.6±O.5 4.5±O.6 Propionate 81.1 ± 1.5 73.5± 1.7 57.6±1.9 Acrylate 89.5±2.8 81.9±2.3 65.1± 1.2 Fluoracetate 95.9±2.9 92.3± 1.8 88.6±2.1 Butyrate 97.2±1.7 93.0±2.3 90.0±1.9 Formate 99.5±2.3 98.5± 1.2 99.7± 1.8

Acknowledgements

We wish to thank the Deutsche Forschungsgemeinschaft, Bonn for financial support, Ms. Sabine Zeiler and Stefan Herzog for excel­lent technical assistance and Theodore Williams, Tallahassee for checking the English text.

280 ANDREA GOLZ and HARTMUT K. LICHTENTHALER

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