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Time-dependent inactivation of acetyl CoA carboxylase by adenosine triphosphate
Int. J. Biochem., 1978, Vol. 9, pp. 227 to 234. Pergamon
Press.
TIME-DEPENDENT CARBOXYLASE
Printed in Greor Brirain
INACTIVATION OF ACETYL CoA BY ADENOSINE TRIPHOSPHATE
P. R. DESJARDINS and K. DAKSHINAMURTI* Department
of Biochemistry,
Faculty of Medicine, Winnipeg, Canada
(Received 30 September
University
of Manitoba,
1977)
Abstract-l. ATP and ADP but not AMP, cyclic AMP, GTP or phosphate caused a time-dependent inactivation of acetyl CoA carboxylase. 2. The protein phosphorylated during inactivation of partially purified acetyl CoA carboxylase in presence of y-32P-ATP was separated from acetyl CoA carboxylase on DEAE-cellulose. 3. The inactivation of acetyl CoA carboxylase by ATP has been shown to be directly dependent on the concentration of citrate in the medium. 4. These observations suggest that acetvl CoA carboxylase is not regulated by a phosphorylationdephosphorylation mechan&.
concentration of 30 mg/ml as previously described (Desjardins et al., 1972). Y-~*P-ATP was either purchased from Amersham/Searle, or synthesized by the method of Butcher (1971). Sepharose 4B was purchased from Pharmacia (Canada), Montreal. NaH“‘C0, was purchased from New England Nuclear Corporation, Montreal and acetyl CoA was purchased from Boehringer Mannheim Co., New York. Calf thymus histone (type II) and all other chemicals were purchased from Sigma Chemical Co., St Louis, Missouri.
INTRODUCTION
Acetyl CoA carboxylase (Acetyl CoA: CO* ligase; EC 6.4.1.2) catalyzes the first step in the series of reactions leading to the synthesis of long chain fatty acids from acetyl CoA. This enzyme has been recognized as the key regulatory enzyme of fatty acid synthesis because of its rate limiting nature (Ganguly, 1960; Numa et al., 1967, 1965; Matsuhashi et al., 1964; Korchak & Masaro, 1964). Although Lane and coworkers (Ryder et al., 1967; Stoll et al., 1968) established that citrate is an allosteric activator of acetyl CoA carboxylase, the in uiuo role of citrate as a regulator of acetyl CoA carboxylase and fatty synthesis has not yet been conclusively established (Lane et al., 1974). However, the role of palmityl CoA as a physiological inhibitor of acetyl CoA carboxylase seems to be well established (Goodridge, 1973; Goodridge et al., 1974). Inoue and Lowenstein (1972) have reported that pure rat liver acetyl CoA carboxylase contains phosphate. Following this, Carlson and Kim (Carlson & Kim, 1973; Lee et al., 1973; Carlson & Kim, 1974a, b; Lee & Kim, 1977) have presented evidence which would indicate that both rat liver and adipose tissue acetyl CoA carboxylase are regulated through phosphorylation and dephosphorylation of the enzyme to an inactive and active form respectively. These findings have been disputed by Lane et al. (1974) and Halestrap and Denton (1974) who claim that the results could be due either to disaggregation of the enzyme by ATP to its inactive form or carboxylation of the enzyme by endogenous bicarbonate. We have investigated that possible role of protein phosphorylation as a mechanism by which acetyl CoA carboxylase activity can be regulated.
Preparation of extracts Male Holtzmann rats (25G-300 g) fed ad libitum on Purina lab chow were sacrificed by decapitation and the livers removed and placed in an ice-cold solution containing 0.05 M Tris (HCl), 0.15 M KCI, and 0.1 mM EDTA, pH 7.2 at 25” (buffer A). The livers were minced with scissors and homogenized in a Potter-Elvehjem homogenizer with a Teflon pestle in 3 vol of buffer A. The homogenate was centrifuged at 10,000 g for 15 min. The pellet was discarded and the supernatant was further centrifuged at 105,oOOg for 1 hr. The 105,000 g supernatant was gelfiltered on a Sephadex G-25 column (2.5 x 45 cm). The fractions containing the protein fraction of the 105,OOOg supernatant protein were pooled. Assay of acetyl CoA carboxylase Enzyme activity was measured by the method previously described (Dakshinamurti & Desjardins, 1969). In all of these experiments, unless otherwise specified, the gel filtered enzyme was preincubated in the presence of citrate, bovine serum albumin and magnesium in order to activate it. Preincubation The gel filtered cytosol was incubated at 37” for 30 min in a mehium containing 60 mM Tris (HCl), pH 7.0, 3 mM elutathione (reduced form). a 8 mM M&I,, 0.1 mM EDTA, 5 rnh potassium &ate, 0.6 mg/m< bovine serum albumin and 0.5-1.0 mg protein from the gel-filtered cytosol per ml.
MATERIALS AND METHODS
Assay
Chemicals Vitamin-free casein (Fisher Canada) was dephosphorylated * To whom
all correspondence
Acetyl CoA carboxylase activity was measured at pH 7.0 and 37” in 0.5 ml of a reaction mixture containing the following: 60 mM Tris (HCl), 0.1 mM EDTA, 8 mM MgC12, 3 mM glutathione (reduced form), 2 mM ATP, 0.2 mM acetyl CoA, 5 mM potassium citrate, 0.6 mg/ml
Scientific Co., Toronto, and adjusted to a protein should
be addressed. 227
‘2X
P. R. DESJARDINS and K. DAKSHINAMUKTI
bovine serum albumin, 10 mM NaH14C0, (0.2 pc/flmole) and the enzyme. Blank tubes contained all of the components of the reaction mixture except acetyl CoA. The reaction mixture was incubated for either 2 or 5 min and the reaction was stopped by the addition of 0.1 ml 6N HCI. Aliquots (0.2 ml) of the reaction mixture were placed in a liquid scintillation vial and heated at 85” for 30 min. To this were added 0.2 ml water followed by 15 ml of a toluene-ethanol scintillator (Dakshinamurti & Desjardins, 1969) and the radioactivity was measured. Assq
of protein
kinase
Enzyme activity was assayed using the method previously described for cyclic AMP-dependent histone kinase (Desjardins et al.. 1975). Enzyme extracts were incubated at pH 7.0 and 306 in 0.1 ml of a reaction mixture containing the following: 50 mM Tris (HCl), 0.2 mM EDTA, 0.3 mM ethylene glycol his@ aminoethyl ether)-NJV’tetraacetic acid (EGTA), 6 mg/ml casein or 1.2 mg/ml calf thymus histone (type II), 0.1 mM Y-~‘P-ATP (20,~90,000 cpm/ mpmole), 1 mM magnesium acetate and enzyme. All reactions were started by the addition of enzyme, and initial velocities were determined over a 20 min period. All assays were done in duplicate. The reaction was terminated and “P determined by method B of Reithe protein-bound mann et al. (1971). This consisted of pipetting 0.05 ml of the incubation mixture on to filter paper squares (2cm x 2cm) and immediately placing them in ice-cold 10% trichloroacetic acid. The filter paper squares were washed in the following manner: 30 min in ice-cold 10% trichloroacetic acid, 20 min in ice-cold 5% trichloroacetic acid, 15 min in room temperature 5% trichloroacetic acid (repeated once), 10 min in 957; ethanol and 5 min in diethyl ether. The paper squares were dried, placed in liquid scintillation vials and the radioactivity was measured in a Beckmann liquid scintillation counter using as scintillator toluene containing 5.9 g/l 2,5-diphenyloxazole. Inactioation
qf acetyl
CoA
In order carboxylase
to investigate by ATP and
carboxylasr
the inactivation of acetyl CoA other nucleotides, the partially
$1 Q
, 10
Ttme,
, 20
min
purified acetyl CoA carboxylase or the gel-filtered htghspeed supernatant was first preincubated as previously dcscribed to activate the enzyme. The activated enzyme (1.0 ml) was then incubated (pH 7.0 and 37’) in 2.0 ml of a reaction mixture containing the following: 50 mM morpholino ethane sulfonic acid. 30 mM Tris (HCI). 1.5 mM glutathione (reduced). 14 mM magnesium acetate, 0.25 mM EDTA, 0.3 mM EGTA, 20 mM NaF. 1 mM aminophylline, 0.3 mg/ml bovine serum albumin, 5.0 mM potassium citrate, 2 mM ATP (when added) and the activated enzyme. The control had no ATP. At various time intervals a 0.2 ml aliquot was removed and the acetyl CoA carboxylase activity was measured in a 2 min incubation.
Rat liver acetyl CoA carboxylase was purified by the method of Inoue and Lowenstein (1972). Step I. Ten rats were sacrificed and their livers were removed and homogenized in 2 vol of buffer B (50mM Tris HCl, 20 mM potassium citrate, 1 mM EDTA, 10 mM 2-mercaptoethanol, pH 7.0) in a Potter-Elvehjem Teflon homogenizer. The resulting homogenate was centrifuged at 15,OOOg for 45 min. The pellet was discarded and the supernatant was centrifuged at 105,OOOg for I hr. Step 2. Ammonium sulfate (194 g/1000 ml) was added with stirring to the supernatant of Step 1. The pH was kept at 7.3 by cautious addition of 5N KOH. The resulting precipitate was collected by centrifugation at 14,OOOg for 60 min, and homogenized in 40 ml buffer C (10 mM potassium phosphate, 10 mM potassium citrate. 0.5 mM EDTA, 5 mM 2-mercaptoethanol, pH 7.5) and dialysed enzyme was then centrifuged at 105,OOOg for 30 min and the precipitate was homogenized in 20ml buffer C. The resulting suspension was centrifuged at 105,000 g for 30 min. The precipitate was washed once more and the supernatants were combined. Step 3. The combined supernatants from Step 2 were applied to a DEAE-cellulose column which has been equilibrated in 10 mM potassium phosphate, pH 7.4. After the enzyme solution has soaked into the DEAE-cellulose, the column was washed with 50ml buffer C. Elution of the
,“I
,
,
,
30
IO
20
30
Time,
mm
Fig. 1. ATP-dependent inactivation of acetyl CoA carboxylase activity. A. The acetyl CoA carboxylase activity of the gel-filtered high-speed supernatant was preincubated as described in Methods to activate the enzyme. The activated enzyme was incubated under the conditions described for Inactivation of acetyl CoA carboxylase, with no additions, W-0; 2 mM ATP, U; 2 mM ATP and 2.4 x 10m6 M cyclic AMP, W---M; 2.4 x 10e6 M cyclic AMP, O---O. Acetyl CoA carboxylase activity was measured in a 5 min incubation at various times (0, 10, 20 and 30min). B. The rate of endogenous protein phosphorylation was measured in the complete system. Conditions were identical to part A (U) except that 2mM cold ATP was replaced by 1 mM y-32P-ATP (2.0 x lo6 cpm/pmole ATP). The incubation was started by the addition of ATP and aliquots (0.1 ml) were removed at 0, 5, 10, 20, 25, 30 min and the protein-bound 32P was determined as described in the protein kinase assay.
Inactivation of acetyl CoA carboxylase Table 1. Effect of ATP concentration
229
on acetyl CoA carboxylase activity Acetyl CoA carboxylase activity* o/0original activity
Time ATP concn (mM)
0.01 118 f 9 41 f I
Zero time 5 min
0.1 84 _+ 5 25 + 6
0.05 94 _+ 3 30 f 4
* Conditions are identical to those described in Fig. lA, except that the ATP concentration was varied. The activity of the control without ATP was taken as 100%. Results are mean + standard deviation of 6 experiments. material absorbed on the column was conducted with a linear gradient from 350ml of buffer C and 350ml of a solution containing 150 mM potassium phosphate, 150 mM potassium citrate, 0.5 mM EDTA, and 5 mM 2-mercaptoethanol, pH 7.0 (buffer D). The resulting preparation had a specific activity of 1.1-1.3 units/mg protein. Purification of chicken liver acetyl CoA carboxylase
Chicken liver acetyl CoA carboxylase was purified according to the method of Hashimoto and Numa (1971). The resulting enzyme had a specific activity of 4 units/mg protein. Protein determination
Protein determinations were by the method of Lowry et al. (1951), using crystalline bovine serum albumin as standard. RESULTS ATP produced a time-dependent inactivation of the activated acetyl CoA carboxylase of the gel-filtered high-speed supernatant (Fig. 1A). Only 32% of the acetyl CoA carboxylase activity remained after 10 min when compared to the control with no additions. The inactivation was essentially complete by 10 min. Cyclic AMP at a concentration of 2.4 x 10m6 M had no effect on the time-dependent inactivation by ATP or on the enzyme alone. Phosphorylation of endogenous protein occurred concurrently with the time-dependent inactivation by ATP (Fig. 1B). Endogenous pro-
tein phosphorylation was essentially complete by 10 min. Although inactivation has been shown to occur at ATP concentrations of 2.0 mM (Fig. 1) ATP causes significant inactivation at concentrations as low as 0.01 mM. (Table 1). The greater the ATP concentration the greater the degree of inactivation. In Fig. 1, ATP inactivation was complete in 10 min. It can be seen (Fig. 2) that the rate of inactivation of acetyl CoA carboxylase is very rapid and almost complete after 2 min. Since ATP can be converted to ADP and AMP in crude enzyme preparations, the effects of equivalent concentrations of these nucleotides on the activity of acetyl CoA carboxylase were studied (Fig. 3). Surprisingly, 2 mM ADP inactivated acetyl CoA carboxylase in an identical time-dependent manner. AMP did not cause a time-dependent inactivation of acetyl CoA carboxylase, but rather inhibited the enzyme at all times, 0, 5 and 10 min. This inhibition was likely due to the carryover of the AMP into the assay. GTP and inorganic phosphate had essentially no effect on acetyl CoA carboxylase activity at these concentrations. The acetyl CoA carboxylase activity of the gel-filtered high-speed supernatant was inactivated in the
$1 a
, Time,
0
2
Time,
3
4
5
min
Fig. 2. Initial rate of ATP-dependent inactivation of acetyl CoA carboxylase. Conditions are identical to those described in Fig, lA, except that the ATP concentration was 0.05 mM. Aliquots were removed at various times from the inactivation incubation and acetyl CoA carboxylase activity was measured immediately.
, IO
5
min
Fig. 3. Effect of other nucleotides on acetyl CoA carboxylase. Conditions were identical to those described for Fig. 1A. The activated enzyme was then incubated as follows: O---O, no additions; M, 2mM ATP; 0-U; 2 mM ADP; m-m, 2 mM AMP; x-x, 2 mM GTP or A--A, 2 mM potassium phosphate. Acetyl CoA carboxylase activity was measured in a 2 min incubation at various times (0, 5, 10 min).
P.
R.
DESJAKIMN
and
K.
DAKSHINAMUI~I
Fractions
Fig. 4. DEAE-cellulose chromatography of phosphorylated high-speed supernatant. The gel-filtered high-speed supernatant (12 ml) was activated by preincubating at 37 for 30 min. The preincubated enzyme (12ml) was then incubated with 1 mM .!-“P-ATP i as described in Fig. I B for 15 min. The incubation was stopped by placing the reaction mixture in ice. The reaction mixture was made 65”i saturated with respect to ammonium sulphate by the addition of solid ammonium sulphate. The precipitate was resuspended and dialyzed against buffer C. The dialysate was then applied to a DEAE~cellulose column (1.5 x 30cm) under the conditions described in Methods for the purification of acetyl CoA carboxylase, except that a linear gradient of 400 ml was used. The molarities of the elution gradient are marked. Each fraction contained 5 ml. Aliquots (0.2 ml) were used for 3ZP counting and determination of acetyl CoA carboxylase.
of 1 mM Y-~‘P-ATP for 15 min. The reaction mixture was concentrated by ammonium sulfate precipitation and dialyzed. This removed excess 32Pi and Y-~‘P-ATP. The mixture was subjected to DEAEcellulose chromatography. (Fig. 4). All of the phosphoprotein was eluted ahead of the peak of acetyl CoA carboxylase activity. It is possible that the peak of phosphoprotein represents inactive acetyl CoA carboxylase. This is unlikely since phosphorylated acetyl CoA carboxylase would have additional negative charges and would require an eluting buffer of greater ionic strength to remove it from the column. Fractions 12-30 were pooled, concentrated. and mixed presence
with an equal amount of fresh gel-filtered high-speed supernatant. The mixture was then activated by preincubating in the presence of magnesium, citrate, and bovine serum albumin. The phosphoprotein peak itself contained no acetyl CoA carboxylase activity. Although phosphate was released during the activation, no additional acetyl CoA carboxylase activity could be detected over and above what was already present (results not shown). One possibility which cannot be excluded is that the phosphorylated enzyme was labile and irreversibly denatured. When the crude acetyl CoA carboxylase was inactivated in the presence of Y-~*P-ATP and chromato-
IO
1 -109
Fractions,
5ml
Fig. 5. Sepharose 4B chromatography of phosphorylated high-speed supernatant. The gel-filtered highspeed supernatant (6 ml) was activated by preincubation and then incubated with y-32P-ATP as described in Fig. 3. The phosphorylated protein was concentrated by ammonium sulfate precipitation, resuspended and dialyzed against buffer B. The dialysate was applied to a Sepharose 4B column (2.5 x 74cm) equilibrated with buffer B. The column was eluted with buffer B at room temperature. M, optical density 280 nm; O-O, “P cpm; m---m, acetyl CoA carboxylase activity.
Inactivation of acetyl CoA carboxylase
Fractions,
IO ml
Fig. 6. Separation of acetyi CoA carboxylase from protein kinase I and II on DEAE-cellulose. The partially purified acetyl CoA carboxylase preparation (40 ml) obtained from Step II in the purification was applied to a DEAE-cellulose column as described in Step III of the purification. m----m, acetyl CoA carboxylase activity; M, protein kinase activity without added substrate; w, protein kinase activity with added substrate (casein). graphed on Sepharose 4B rather than DEAE-celluIose, 2 peaks of radioactive phosphate were found (Fig. 5). The first peak co-chromatographed with the acetyl CoA carboxylase activity and the second peak co-chromatographed with standard ATP. The major portion of the protein in the gel-filtered high-speed supernatant was not phosphorylated. During the purification of rat liver acetyl CoA carboxylase by the method of Inoue and Lowenstein (1972), it was found that the DEAE-cellulose step separated the major portion of the acetyl CoA carboxylase activity from 2 protein kinases (Fig. 6). Using casein as substrate 2 peaks of protein kinase were detected, which we have designated as I and II. Both protein kinases were dependent on the addition of substrate for maximal activity. Without added substrate, protein kinase activity was still detectable, suggesting the presence of endogenous substrate within these fractions. A third peak of protein kinase activity chromatographed with the acetyl CoA carboxylase activity. It is not known whether this represents a distinct protein kinase or if it is a non-
231
specific interaction of protein kinase I or II with either acetyl CoA carboxyiase or another protein eluting in this region. The substrate specificity and cyclic AMP dependency of protein kinase I and II were investigated (Table 2). Protein kinase I phosphorylated casein more extensively than histone when compared to protein kinase II. Cyclic AMP did not stimulate the activity of protein kinase I when histone was the substrate and showed only a slight stimulation with casein as substrate. Protein kinase II was equally effective with either protein as substrate in the absence of cyclic AMP. Cyclic AMP stimulated the activity of protein kinase II when either casein or histone was the substrate. The stimulation by cyclic AMP was greater when histone was the substrate. It is possible that protein kinase I is the free catalytic subunit which has dissociated from protein kinase II, the holoenzyme (Reiman et nl., 1971). The effect of ATP and ATP in the presence of added protein kinase I or II on the purified acetyl CoA carboxylase were investigated (Fig. 7). ATP alone, at a concentration of 2 mM, inactivated the purified acetyl CoA carboxylase. The degree of inactivation by ATP was moderate when compared to the results in Fig. 1. The purified acetyl CoA carboxylase retained 65% of its activity after 10 min incubation with 2 mM ATP, compared to the control with no additions. The crude enzyme retained only 32% of its activity after 10 min under similar conditions. The addition of ATP and either protein kinase I or II enhanced neither the rate nor the degree of inactivation. The addition kinase I alone had no effect although the addition of protein kinase II alone caused inhibition of acetyl CoA carboxyiase at all three incubation times (0, 5 and 10 min). Since the purified enzyme contained citrate in the buffer, ATP-inactivation studies were done at a citrate concentration of 8.8 mM. Due to this we could not conclude that the purified enzyme was, inactivated to a lesser extent than the crude enzyme. The effect of citrate concentration on ATP-dependent inactivation of rat liver and chicken liver acetyl CoA carboxylase was investigated. Representative results of 3 separate experiments are given in Fig. 8. Both the crude gel-filtered enzyme and the DEAE-purified enzyme from rat liver were inactivated to a greater extent at low citrate (2.5 mM) than at high citrate concentration (20 mM). The crude enzyme was inactivated to a
Table 2. Cyclic AMP dependency and substrate specificity of protein kinase I and II
Substrate Casein Histone
Protein kinase activity (cpm,/assay) Protein kinase I Protein kinase II plus minus minus plus cyclic AMP cyclic AMP cyclic AMP cyclic AMP 328 234
510 214
1418 1302
2378 3146
The fractions from the DEAE-Sephadex column (Fig. 3) which contained protein kinase I and Ii were concentrated by ammonium sulfate precipitation (65%) as described in Fig. 6. Protein kinase activity was measured as described in Methods with casein or histone as substrate. The activity is expressed as cpm “P incorporated per assay in a 20 min incubation at 30”. Blank tubes contained all of the constituents except the enzyme.
Protein
I
klnase
I 5
0
I
I 10
Time ,
I 0
P. R.
DESJARUINS
Protein
klnase
I 5
and K. II
I IO
min
Fig. 7. Inactivation of purified acetyl CoA carboxylase by ATP with and without added protein kinase. The fractions containing protein kinase I, II and acetyl CoA carboxylase from the DEAE-cellulose chromatography were individually pooled and concentrated by ammonium sulfate precipitation. Protein kinase I and II were resuspended and dialyzed against a buffer containing 10 mM Tris (HCl), and 10 mM 2-mercaptoethanol, pH 7.0. Acetyl CoA carboxylase was resuspended and dialyzed against buffer D. The partially purified acetyl CoA carboxylase was incubated without preincubation under the conditions described for the inactivation of acetyl CoA carboxylase in the Methods. O-O, acetyl CoA carboxylase alone (2.4 mg/ml); O-O, acetyl CoA carboxylase with protein kinase I or II at a concentration of 0.26 and 0.59mg/ml respectively; m---m, acetyl CoA carboxylase with protein kinase I or II and 2 mM ATP; O--U, acetyl CoA carboxylase with 2 mM ATP without protein kinase. Incubation conditions were identical to those of Fig. 1 except that the citrate concentration was 8.75 mM. Acetyl CoA carboxylase was measured in a 2 min incubation.
The pure chicken liver enzyme behaved in the same manner as the rat liver enzyme except that the crude and purified enzyme were inactivated to the same extent at all citrate concentrations. Again greater inactivation was observed at low citrate concentration (2.5 mM) than at high citrate concentration (20 mM). If the cold ATP present during the inactivation in Fig. 8 was replaced with radioactive 1;-3ZP-ATP no significant protein phosphorylation occurred in the presence of pure chicken liver enzyme at any citrate concentration. Thus significant enzyme inactivation occurred at low citrate concentration, and yet no protein phosphorylation was observed. greater
extent
at all concentrations.
DISCUSSION
It is well established
that insulin stimulates the rate of fatty acid synthesis from glucose in rat epididymal fat-pads, whereas adrenaline inhibits fatty acid synthesis (Cahill et nl., 1960; Denton & Halperin, 1968; Saggerson & Greenbaum, 1970). Recently, Halestrap & Denton (1974) have provided evidence that insulin and adrenaline control fatty acid synthesis by regulating the concentration of fatty acyl CoA’s, citrate and ATP within the tissue. They suggest that these compounds regulate the activity of acetyl CoA carboxylase through polymer-protomer transitions.
DAKSHINAMURTI
Other investigators (Heimberg et al., 1969; Bricker & Levy, 1972; Allard & Ruehrig, 1973) suggest that insulin and adrenaline mediate their effect on fatty acid synthesis, through cyclic AMP. Kim and coworkers (Carlson & Kim, 1973; 1974u, h; 1977) have suggested that insulin and adrenaline control fatty acid synthesis through the interconversion of acetyl CoA carboxylase from a dephosphorylated active form to be phosphorylated inactive form. They have suggested that the phosphorylation-dephosphorylation system is analogous to that which occurs with pyruvate dehydrogenase (Jungas, 1970; Coore Ed rrl., 1971; Weiss ef al., 1971), since the phosphorylation of acetyl CoA carboxylase appears to be insensitive to cyclic AMP. Carlson & Kim (1973, 19740, b) have based their hypothesis primarily on their observations that (1) inactivation of crude rat liver acetyl CoA carboxylase in the presence of y-“P-ATP resulted in (a) the parallel incorporation of 32P into protein which copurified with the carboxylase and (b) 32P incorporation into the carboxylase-antibody precipitate using antibody prepared in rabbits against the rat liver enzyme, (2) inactivation of the enzyme was dependent on the presence of ATP and a crude protein kinase fraction (fraction K). We have shown that ATP produced a time-dependent inactivation of the enzyme, which was paralleled 1 30 -
A-Rat
SO-
.
liver
\
to-
‘L.
!O-
i I
B-
iO-
I
Chlcken
I
I
lwer
. L.
. IO-
!O -
I 5
I 10
Cltrote.
I 15
20
J
mM
Fig. 8. Effect of citrate concentration on ATP inactivation of rat and chicken liver acetyl CoA carboxylase. A. Acetyl CoA carboxylase from rat liver at 2 stages of purification. (04, crude gel-filtered high-speed supernatant: -0, DEAE-cellulose enzyme) were incubated with 0.2 mM ATP for 10 min as described in Methods. The citrate concentration during the inactivation was varied from 2.5 mM to 20 mM. Acetyl CoA carboxylase activity was measured al the end of each inactivation incubation. B. Same as A except that pure chicken liver enzyme was used. (ed, Crude, gel-filtered high-speed supernatant; O-0, pure enzyme preparation.
Inactivation of acetyl CoA carboxylase by endogenous protein phosphorylation. Surprisingly, the same concentration of ADP produced an identical time-dependent inactivation of the enzyme. It is possible that the ADP was converted to ATP by an ATP generating system. Adenylate kinase catalyzes such a reaction and would be present in the high-speed supernatant. Similar concentrations of AMP, phosphate and GTP did not produce a time-dependent inactivation of the enzyme. Cyclic AMP did not inactivate the enzyme nor did it enhance the time-dependent inactivation by ATP. This latter result may be misleading since conditions may not have been favorable for detecting an effect of cyclic AMP. It is also possible that the protein kinase responsible for the phosphorylation of acetyl CoA carboxylase is not stimulated by cyclic AMP. It is also to be noted that under conditions of maximal phosphorylation the crude enzyme preparation still retained 25-30x of the original carboxylase activity. In case of enzymes like pyruvate dehydrogenase where a phosphorylationdephosphorylation mechanism has been established, the maximally phosphorylated enzyme had no enzyme activity (Wieland L Siess, 1970). We have also shown that ATP can cause very significant inactivation of acetyl CoA carboxylase at a concentration as low as 0.01 mM. In earlier work Greenspan and Lowenstein (1967) showed that when 4 mM ATP was included in the pre-incubation medium the activation of the enzyme was reduced considerably. However, at this concentration ATP inhibits the carboxylation reaction itself (Desjardins & Dakshinamurti, 1970). Lane and coworkers (1966) studied the sedimentation rate of the chicken liver enzyme in sucrose density gradient medium containing 2 mM ATP, 8 mM Mg2+ and 10 mM KHCO,. The enzyme sedimented in the protomeric form. Similar results were reported by Numa et at. (1967). These authors have concluded that the ATP-Mg complex is responsible for dissociation of the enzyme which is present in the carboxylated state. This is supported by the observation that malonyl CoA which also carboxylates the enzyme produces a similar disaggregation of the enzyme. In our experiments, the incubation medium contained only ATP and Mg2+ and no added HCO;. Inactivation, reversible with citrate, occurred at a very low concentration of ATP in the preincubation medium. Although Carlson and Kim (1974) stated that the 32P incorporated from Y-~~P-ATP during inactivation of acetyl CoA carboxylase copurified with this enzyme, we were able to separate the 32P-labelled protein from the acetyl CoA carboxylase on DEAEcellulose. The 32P-labelled protein and acetyl CoA carboxylase were found to co-chromatograph on Sepharose 43. These latter results suggest that the labelled protein(s) is (are) of a high molecular weight although not necessarily acetyl CoA carboxylase. DEAE-cellulose chromatography of the partially purified rat liver acetyl CoA carboxylase separated this enzyme from the bulk of the protein kinase activity. Two major protein kinases were identified. Protein kinase I which phosphorylated casein rather than histone and which was basically unaffected by a cyclic AMP, and protein kinase II which was stimulated by cyclic AMP and showed some preference to histone as substrate over casein in the presence of cyclic
233
AMP. A small peak of protein kinase activity was associated with the peak of acetyl CoA carboxylase activity. Neither protein kinase I or II could enhance the ATP inactivation of the purified acetyl CoA carboxylase. Although Carlson and Kim (1974) showed that increasing amounts of protein kinase (fraction K) caused increased inactivation of acetyl CoA carboxylase in the presence of ATP they did not show what effect the protein kinase fraction alone (minus ATP) would have on a&y1 CoA carboxylase. We have shown that inactivation by ATP of acetyl CoA carboxylase in both purified and crude acetyl CoA carboxylase from rat and chicken liver is directly dependent upon the citrate concentration. This result would appear to indicate that citrate prevents the ATP induced disaggregation (and thus inactivation) of the enzyme, a fact which has long been established. We have offered evidence which suggests that the time-dependent ATP inactivation of acetyl CoA carboxylase may not be due to phosphorylation of the enzyme to an inactive form, but that this may be entirely due to the disaggregating effect of ATP on the enzyme. Our evidence suggesting the latter is basically the following: (1) ATP inactivation occurs at a very low concentration and is dependent on the citrate concentration. (2) The protein phosphorylated during the time-dependent inactivation can be separated from acetyl CoA carboxylast on DEAE-cellulose. (3) Addition of protein kinase I or IT to purified acetyl CoA carboxylase does not enhance the inactivation of the enzyme by ATP. The evidence presented here strengthens the suggestion of previous investigators (Lane et al., 1974; Halestrap & Denton, 1974), that the time-dependent ATP inactivation is due to the disaggregation of the enzymes by ATP, to its inactive protomeric
form.
Acknowledgements-This investigation was supported by a grant from the Medical Research Council of Canada. P.R.D. acknowledges a grant from the University of Manitoba. REFERENCES
ALLARDJ. B. & RUEHRIGK. L. (1973) Inhibition of rat liver acetyl CoA carboxylase by N6,02’-dibutyrl cyclic adenosine ~:S-monophosphate in vitro. J. bid. Chem. 248,4131-4133. BRICKERL. A. & LEVEY G. S. (1972) Evidence for regula-
tion of cholesterol and fatty acid synthesis in liver by cyclic adenosine 3’,5’-monophosphate. J. bid. Chem. 247, 49144915. BUTCHERF. R. (1971) A rapid filter paper disk assay for picomole amounts of cyclic AMP using a cyclic AMP
dependent protein kinase. Hormones Metub. Res. 3, 336340.
CAHILLG. F., LEBOEFB. & FLINN R. B. (1960) Studies on rat adipose tissue in vitro. VI Effect of epinephrine on glucose metabolism. J. biol. Chem. 235, 1246-1250. CARLSONC. A. & KIM K.-H. (1973) Regulation of hepatic acetyl CoA carboxylase by phosphorylation and dephosphorylation. J. bid. Chem. 248, 378-380. CARLSONC. A. & KIM K.-H. (1974a) Regulation of hepatic acetyl CoA carboxylase by phosphorylation and dephosphorylation. Archs. Biochem. B&phys. 164, 478-489. CARLSONC. A. & KIM K.-H. (1974b) . , Differential effects of metabolites on the active and inactive forms of hepatic acetyl CoA carboxylase. Archs Biochem. Biop~ys. 164, 49g-501.
234
P. R. DESJARD~NSand K. DAKSH~NAMURT~
CHANG H.-C., SE~DAN I., TEEBOR G. & LANE M. D. (1967) Liver acetyl CoA carboxylase and fatty acid synthetase: relative activities in the normal state and in hereditary obesity. Biochem. hiophys. Res. Commun. 28, 682-686. COORE H. G., DENTON R. M., MARTIN B. R. & RANDLE P. J. (1971) Regulation of adipose tissue pyruvate dehvdroeenase bv insulin and other hormones. Biochem. J: 125: l&12?. DAKSHINAMUR~~K. & DESJARD~NSP. R. (1969) Acetyl CoA carboxylase from rat adipose tissue. Biochem. hiophys. Actu. 176, 221-229. DENTON R. M. & HALPER~N M. L. (1968) The control of fatty acid and triglyceride synthesis in rat epididymal adioose tissue. Biochem. J. 110, 27.-38. DESJARD~NS P. R. & DAKSH~NAMURT~K. (1970) Rat epididymal adipose tissue acetyl CoA carboxylase. Can. J. Biochem. 48. 915-92 I. DESIARD~NS P. R., L~EW C. C. & GORNALL A. G. (1975) Rat liver nuclear protein kinases. Can. J. Biochem. 53, 354-363. DESJARDINS P. R., LUE P. F., L~EW C. C. & GORNAL A. G. (1972) Purification and properties of rat liver nuclear protein kinases. Can. J. Biochem. 50, 1249-1259. GANGULY J. (1960) Studies on the mechanism of fatty acid synthesis. VII Biosynthesis of fatty acids from malonyl CoA. Biochem. hiophys. Actu 40, 1 lO_ 118. GOODRIDGE A. G. (1973) Regulation of fatty acid synthesis in the liver of prenatal and early post natal chicks. Hepatic concentration of individual free fatty acids and other metabolites. J. biol. Chew. 248, 1939-1945. GOODRIDGE A. G., GARAY A. & S~LPANANTA P. (1974) Regulation of lipogenesis and the total activities of lipoge
phosphate and concentration of free fatty acid on lipid metabolism. J. hid. Chem. 244, 5131 5139. IN&E H. & LOWENSTE~NJ. M. (1972) Acetyl CoA carboxylase from rat liver-purification and demonstration of different subunits. J. hiol. Chem. 247, 4825-4832. JUNGAS R. L. (1970) Effects of insulin on fatty acid synthesis from pyruvate. lactate or endogenous sources in adipose tissue: evidence for the hormonal regulation of pyruvate dehydrogenase. Endocrinoloy~ 86, 1368-l 37.5. KORCHAK H. M. & MASORO E. J. (1964) Free fatty acids as lipogenic inhibitors. Biochem. hiophys. 4cttr 84, 750-753. LANE M. D., Moss J. & POLAKIS S. E. (1974) Acetyl coenzyme A carboxylase. In Cttrrent Topics in Cellular ReguIution (Edited by HORECKER B. L. & STADTMAN E. R.). Vol. 8, pp. 139-195. LEE K. H. & KIM K.-H. (1977) Regulation of rat liver acetyl coenzyme A carboxylase. J. hiol. Chrm. 252, 1748-1751. LEE K. H., THRALL T. & KIM K.-H. (1973) Hormonal regulation of acetyl CoA carboxylase--EtTect of insulin and epinephrine. Biochrm. hiophy,s. Res. Commun. 54, 1133.-1140.
LOWRY 0. H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the folin phenol reagent. J. bioL. Chem. 193, 265.-275. MATSUHASH~ M.. MATSUHASH~S. & LYNEN F. (1964) Biosyntheses der Fettsguren. V. Die acetyl CoA carboxylase aus rattenleber und ihre aktivierung durch citronensgure. Biochrm. Z. 340, 263-289. NUMA S., GOTO T., R~NGELMANN E. & R~EDEL P. (1967) Studies on the relationship between activity and structure of liver acetyl CoA carboxylase. Effect of magnesium ions. Eur. J. Biochem. 3, 124-128. RE~MANNE. M., WALSH D. A. & KREBS E. G. (1971) Purification and properties of rabbit skeletal muscle adenosine 3’5’-monophosphate-dependent protein kinases. J. biol. Chem. 246, 1986-1995. RYDER E., GRBG~L~N C., CHANG H.-C. & LANE M. D. (1967) Liver acetyl CoA carboxylase: insight into the mechanism of activation by tricarboxylic acids and acetyl CoA. Proc. natn Ad. Sci. U.S.A. 57, 1455-1462. SAGGERSON E. D. & GREENBAUM A. L. (1970) The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Biochem. J. 119, 193-219. STOLL E., RYDER E., EDWARDS J. B. & LANE M. D. (1968) Liver acetyl CoA carboxylase: activation of model partial reactions by tricarboxylic acids. Proc. nun Acad. Sri. U.S.A. 60, 986-991. WEISS L., LOFFLER G., SCH~RMANN A. & W~ELAND 0. H. (1971) Control of pyruvate dehydrogenase interconversion in adipose tissue by insulin. FEBS Lett. 15, 229-231. W~ELAND 0. H. & S~ESSE. (1970) Interconversion of phospho- and dephosphoforms of pig heart pyruvate dehydrogenase. Proc. nrztn Acnd. Sci. U.S.A. 65, 947~954.
Press.
TIME-DEPENDENT CARBOXYLASE
Printed in Greor Brirain
INACTIVATION OF ACETYL CoA BY ADENOSINE TRIPHOSPHATE
P. R. DESJARDINS and K. DAKSHINAMURTI* Department
of Biochemistry,
Faculty of Medicine, Winnipeg, Canada
(Received 30 September
University
of Manitoba,
1977)
Abstract-l. ATP and ADP but not AMP, cyclic AMP, GTP or phosphate caused a time-dependent inactivation of acetyl CoA carboxylase. 2. The protein phosphorylated during inactivation of partially purified acetyl CoA carboxylase in presence of y-32P-ATP was separated from acetyl CoA carboxylase on DEAE-cellulose. 3. The inactivation of acetyl CoA carboxylase by ATP has been shown to be directly dependent on the concentration of citrate in the medium. 4. These observations suggest that acetvl CoA carboxylase is not regulated by a phosphorylationdephosphorylation mechan&.
concentration of 30 mg/ml as previously described (Desjardins et al., 1972). Y-~*P-ATP was either purchased from Amersham/Searle, or synthesized by the method of Butcher (1971). Sepharose 4B was purchased from Pharmacia (Canada), Montreal. NaH“‘C0, was purchased from New England Nuclear Corporation, Montreal and acetyl CoA was purchased from Boehringer Mannheim Co., New York. Calf thymus histone (type II) and all other chemicals were purchased from Sigma Chemical Co., St Louis, Missouri.
INTRODUCTION
Acetyl CoA carboxylase (Acetyl CoA: CO* ligase; EC 6.4.1.2) catalyzes the first step in the series of reactions leading to the synthesis of long chain fatty acids from acetyl CoA. This enzyme has been recognized as the key regulatory enzyme of fatty acid synthesis because of its rate limiting nature (Ganguly, 1960; Numa et al., 1967, 1965; Matsuhashi et al., 1964; Korchak & Masaro, 1964). Although Lane and coworkers (Ryder et al., 1967; Stoll et al., 1968) established that citrate is an allosteric activator of acetyl CoA carboxylase, the in uiuo role of citrate as a regulator of acetyl CoA carboxylase and fatty synthesis has not yet been conclusively established (Lane et al., 1974). However, the role of palmityl CoA as a physiological inhibitor of acetyl CoA carboxylase seems to be well established (Goodridge, 1973; Goodridge et al., 1974). Inoue and Lowenstein (1972) have reported that pure rat liver acetyl CoA carboxylase contains phosphate. Following this, Carlson and Kim (Carlson & Kim, 1973; Lee et al., 1973; Carlson & Kim, 1974a, b; Lee & Kim, 1977) have presented evidence which would indicate that both rat liver and adipose tissue acetyl CoA carboxylase are regulated through phosphorylation and dephosphorylation of the enzyme to an inactive and active form respectively. These findings have been disputed by Lane et al. (1974) and Halestrap and Denton (1974) who claim that the results could be due either to disaggregation of the enzyme by ATP to its inactive form or carboxylation of the enzyme by endogenous bicarbonate. We have investigated that possible role of protein phosphorylation as a mechanism by which acetyl CoA carboxylase activity can be regulated.
Preparation of extracts Male Holtzmann rats (25G-300 g) fed ad libitum on Purina lab chow were sacrificed by decapitation and the livers removed and placed in an ice-cold solution containing 0.05 M Tris (HCl), 0.15 M KCI, and 0.1 mM EDTA, pH 7.2 at 25” (buffer A). The livers were minced with scissors and homogenized in a Potter-Elvehjem homogenizer with a Teflon pestle in 3 vol of buffer A. The homogenate was centrifuged at 10,000 g for 15 min. The pellet was discarded and the supernatant was further centrifuged at 105,oOOg for 1 hr. The 105,000 g supernatant was gelfiltered on a Sephadex G-25 column (2.5 x 45 cm). The fractions containing the protein fraction of the 105,OOOg supernatant protein were pooled. Assay of acetyl CoA carboxylase Enzyme activity was measured by the method previously described (Dakshinamurti & Desjardins, 1969). In all of these experiments, unless otherwise specified, the gel filtered enzyme was preincubated in the presence of citrate, bovine serum albumin and magnesium in order to activate it. Preincubation The gel filtered cytosol was incubated at 37” for 30 min in a mehium containing 60 mM Tris (HCl), pH 7.0, 3 mM elutathione (reduced form). a 8 mM M&I,, 0.1 mM EDTA, 5 rnh potassium &ate, 0.6 mg/m< bovine serum albumin and 0.5-1.0 mg protein from the gel-filtered cytosol per ml.
MATERIALS AND METHODS
Assay
Chemicals Vitamin-free casein (Fisher Canada) was dephosphorylated * To whom
all correspondence
Acetyl CoA carboxylase activity was measured at pH 7.0 and 37” in 0.5 ml of a reaction mixture containing the following: 60 mM Tris (HCl), 0.1 mM EDTA, 8 mM MgC12, 3 mM glutathione (reduced form), 2 mM ATP, 0.2 mM acetyl CoA, 5 mM potassium citrate, 0.6 mg/ml
Scientific Co., Toronto, and adjusted to a protein should
be addressed. 227
‘2X
P. R. DESJARDINS and K. DAKSHINAMUKTI
bovine serum albumin, 10 mM NaH14C0, (0.2 pc/flmole) and the enzyme. Blank tubes contained all of the components of the reaction mixture except acetyl CoA. The reaction mixture was incubated for either 2 or 5 min and the reaction was stopped by the addition of 0.1 ml 6N HCI. Aliquots (0.2 ml) of the reaction mixture were placed in a liquid scintillation vial and heated at 85” for 30 min. To this were added 0.2 ml water followed by 15 ml of a toluene-ethanol scintillator (Dakshinamurti & Desjardins, 1969) and the radioactivity was measured. Assq
of protein
kinase
Enzyme activity was assayed using the method previously described for cyclic AMP-dependent histone kinase (Desjardins et al.. 1975). Enzyme extracts were incubated at pH 7.0 and 306 in 0.1 ml of a reaction mixture containing the following: 50 mM Tris (HCl), 0.2 mM EDTA, 0.3 mM ethylene glycol his@ aminoethyl ether)-NJV’tetraacetic acid (EGTA), 6 mg/ml casein or 1.2 mg/ml calf thymus histone (type II), 0.1 mM Y-~‘P-ATP (20,~90,000 cpm/ mpmole), 1 mM magnesium acetate and enzyme. All reactions were started by the addition of enzyme, and initial velocities were determined over a 20 min period. All assays were done in duplicate. The reaction was terminated and “P determined by method B of Reithe protein-bound mann et al. (1971). This consisted of pipetting 0.05 ml of the incubation mixture on to filter paper squares (2cm x 2cm) and immediately placing them in ice-cold 10% trichloroacetic acid. The filter paper squares were washed in the following manner: 30 min in ice-cold 10% trichloroacetic acid, 20 min in ice-cold 5% trichloroacetic acid, 15 min in room temperature 5% trichloroacetic acid (repeated once), 10 min in 957; ethanol and 5 min in diethyl ether. The paper squares were dried, placed in liquid scintillation vials and the radioactivity was measured in a Beckmann liquid scintillation counter using as scintillator toluene containing 5.9 g/l 2,5-diphenyloxazole. Inactioation
qf acetyl
CoA
In order carboxylase
to investigate by ATP and
carboxylasr
the inactivation of acetyl CoA other nucleotides, the partially
$1 Q
, 10
Ttme,
, 20
min
purified acetyl CoA carboxylase or the gel-filtered htghspeed supernatant was first preincubated as previously dcscribed to activate the enzyme. The activated enzyme (1.0 ml) was then incubated (pH 7.0 and 37’) in 2.0 ml of a reaction mixture containing the following: 50 mM morpholino ethane sulfonic acid. 30 mM Tris (HCI). 1.5 mM glutathione (reduced). 14 mM magnesium acetate, 0.25 mM EDTA, 0.3 mM EGTA, 20 mM NaF. 1 mM aminophylline, 0.3 mg/ml bovine serum albumin, 5.0 mM potassium citrate, 2 mM ATP (when added) and the activated enzyme. The control had no ATP. At various time intervals a 0.2 ml aliquot was removed and the acetyl CoA carboxylase activity was measured in a 2 min incubation.
Rat liver acetyl CoA carboxylase was purified by the method of Inoue and Lowenstein (1972). Step I. Ten rats were sacrificed and their livers were removed and homogenized in 2 vol of buffer B (50mM Tris HCl, 20 mM potassium citrate, 1 mM EDTA, 10 mM 2-mercaptoethanol, pH 7.0) in a Potter-Elvehjem Teflon homogenizer. The resulting homogenate was centrifuged at 15,OOOg for 45 min. The pellet was discarded and the supernatant was centrifuged at 105,OOOg for I hr. Step 2. Ammonium sulfate (194 g/1000 ml) was added with stirring to the supernatant of Step 1. The pH was kept at 7.3 by cautious addition of 5N KOH. The resulting precipitate was collected by centrifugation at 14,OOOg for 60 min, and homogenized in 40 ml buffer C (10 mM potassium phosphate, 10 mM potassium citrate. 0.5 mM EDTA, 5 mM 2-mercaptoethanol, pH 7.5) and dialysed enzyme was then centrifuged at 105,OOOg for 30 min and the precipitate was homogenized in 20ml buffer C. The resulting suspension was centrifuged at 105,000 g for 30 min. The precipitate was washed once more and the supernatants were combined. Step 3. The combined supernatants from Step 2 were applied to a DEAE-cellulose column which has been equilibrated in 10 mM potassium phosphate, pH 7.4. After the enzyme solution has soaked into the DEAE-cellulose, the column was washed with 50ml buffer C. Elution of the
,“I
,
,
,
30
IO
20
30
Time,
mm
Fig. 1. ATP-dependent inactivation of acetyl CoA carboxylase activity. A. The acetyl CoA carboxylase activity of the gel-filtered high-speed supernatant was preincubated as described in Methods to activate the enzyme. The activated enzyme was incubated under the conditions described for Inactivation of acetyl CoA carboxylase, with no additions, W-0; 2 mM ATP, U; 2 mM ATP and 2.4 x 10m6 M cyclic AMP, W---M; 2.4 x 10e6 M cyclic AMP, O---O. Acetyl CoA carboxylase activity was measured in a 5 min incubation at various times (0, 10, 20 and 30min). B. The rate of endogenous protein phosphorylation was measured in the complete system. Conditions were identical to part A (U) except that 2mM cold ATP was replaced by 1 mM y-32P-ATP (2.0 x lo6 cpm/pmole ATP). The incubation was started by the addition of ATP and aliquots (0.1 ml) were removed at 0, 5, 10, 20, 25, 30 min and the protein-bound 32P was determined as described in the protein kinase assay.
Inactivation of acetyl CoA carboxylase Table 1. Effect of ATP concentration
229
on acetyl CoA carboxylase activity Acetyl CoA carboxylase activity* o/0original activity
Time ATP concn (mM)
0.01 118 f 9 41 f I
Zero time 5 min
0.1 84 _+ 5 25 + 6
0.05 94 _+ 3 30 f 4
* Conditions are identical to those described in Fig. lA, except that the ATP concentration was varied. The activity of the control without ATP was taken as 100%. Results are mean + standard deviation of 6 experiments. material absorbed on the column was conducted with a linear gradient from 350ml of buffer C and 350ml of a solution containing 150 mM potassium phosphate, 150 mM potassium citrate, 0.5 mM EDTA, and 5 mM 2-mercaptoethanol, pH 7.0 (buffer D). The resulting preparation had a specific activity of 1.1-1.3 units/mg protein. Purification of chicken liver acetyl CoA carboxylase
Chicken liver acetyl CoA carboxylase was purified according to the method of Hashimoto and Numa (1971). The resulting enzyme had a specific activity of 4 units/mg protein. Protein determination
Protein determinations were by the method of Lowry et al. (1951), using crystalline bovine serum albumin as standard. RESULTS ATP produced a time-dependent inactivation of the activated acetyl CoA carboxylase of the gel-filtered high-speed supernatant (Fig. 1A). Only 32% of the acetyl CoA carboxylase activity remained after 10 min when compared to the control with no additions. The inactivation was essentially complete by 10 min. Cyclic AMP at a concentration of 2.4 x 10m6 M had no effect on the time-dependent inactivation by ATP or on the enzyme alone. Phosphorylation of endogenous protein occurred concurrently with the time-dependent inactivation by ATP (Fig. 1B). Endogenous pro-
tein phosphorylation was essentially complete by 10 min. Although inactivation has been shown to occur at ATP concentrations of 2.0 mM (Fig. 1) ATP causes significant inactivation at concentrations as low as 0.01 mM. (Table 1). The greater the ATP concentration the greater the degree of inactivation. In Fig. 1, ATP inactivation was complete in 10 min. It can be seen (Fig. 2) that the rate of inactivation of acetyl CoA carboxylase is very rapid and almost complete after 2 min. Since ATP can be converted to ADP and AMP in crude enzyme preparations, the effects of equivalent concentrations of these nucleotides on the activity of acetyl CoA carboxylase were studied (Fig. 3). Surprisingly, 2 mM ADP inactivated acetyl CoA carboxylase in an identical time-dependent manner. AMP did not cause a time-dependent inactivation of acetyl CoA carboxylase, but rather inhibited the enzyme at all times, 0, 5 and 10 min. This inhibition was likely due to the carryover of the AMP into the assay. GTP and inorganic phosphate had essentially no effect on acetyl CoA carboxylase activity at these concentrations. The acetyl CoA carboxylase activity of the gel-filtered high-speed supernatant was inactivated in the
$1 a
, Time,
0
2
Time,
3
4
5
min
Fig. 2. Initial rate of ATP-dependent inactivation of acetyl CoA carboxylase. Conditions are identical to those described in Fig, lA, except that the ATP concentration was 0.05 mM. Aliquots were removed at various times from the inactivation incubation and acetyl CoA carboxylase activity was measured immediately.
, IO
5
min
Fig. 3. Effect of other nucleotides on acetyl CoA carboxylase. Conditions were identical to those described for Fig. 1A. The activated enzyme was then incubated as follows: O---O, no additions; M, 2mM ATP; 0-U; 2 mM ADP; m-m, 2 mM AMP; x-x, 2 mM GTP or A--A, 2 mM potassium phosphate. Acetyl CoA carboxylase activity was measured in a 2 min incubation at various times (0, 5, 10 min).
P.
R.
DESJAKIMN
and
K.
DAKSHINAMUI~I
Fractions
Fig. 4. DEAE-cellulose chromatography of phosphorylated high-speed supernatant. The gel-filtered high-speed supernatant (12 ml) was activated by preincubating at 37 for 30 min. The preincubated enzyme (12ml) was then incubated with 1 mM .!-“P-ATP i as described in Fig. I B for 15 min. The incubation was stopped by placing the reaction mixture in ice. The reaction mixture was made 65”i saturated with respect to ammonium sulphate by the addition of solid ammonium sulphate. The precipitate was resuspended and dialyzed against buffer C. The dialysate was then applied to a DEAE~cellulose column (1.5 x 30cm) under the conditions described in Methods for the purification of acetyl CoA carboxylase, except that a linear gradient of 400 ml was used. The molarities of the elution gradient are marked. Each fraction contained 5 ml. Aliquots (0.2 ml) were used for 3ZP counting and determination of acetyl CoA carboxylase.
of 1 mM Y-~‘P-ATP for 15 min. The reaction mixture was concentrated by ammonium sulfate precipitation and dialyzed. This removed excess 32Pi and Y-~‘P-ATP. The mixture was subjected to DEAEcellulose chromatography. (Fig. 4). All of the phosphoprotein was eluted ahead of the peak of acetyl CoA carboxylase activity. It is possible that the peak of phosphoprotein represents inactive acetyl CoA carboxylase. This is unlikely since phosphorylated acetyl CoA carboxylase would have additional negative charges and would require an eluting buffer of greater ionic strength to remove it from the column. Fractions 12-30 were pooled, concentrated. and mixed presence
with an equal amount of fresh gel-filtered high-speed supernatant. The mixture was then activated by preincubating in the presence of magnesium, citrate, and bovine serum albumin. The phosphoprotein peak itself contained no acetyl CoA carboxylase activity. Although phosphate was released during the activation, no additional acetyl CoA carboxylase activity could be detected over and above what was already present (results not shown). One possibility which cannot be excluded is that the phosphorylated enzyme was labile and irreversibly denatured. When the crude acetyl CoA carboxylase was inactivated in the presence of Y-~*P-ATP and chromato-
IO
1 -109
Fractions,
5ml
Fig. 5. Sepharose 4B chromatography of phosphorylated high-speed supernatant. The gel-filtered highspeed supernatant (6 ml) was activated by preincubation and then incubated with y-32P-ATP as described in Fig. 3. The phosphorylated protein was concentrated by ammonium sulfate precipitation, resuspended and dialyzed against buffer B. The dialysate was applied to a Sepharose 4B column (2.5 x 74cm) equilibrated with buffer B. The column was eluted with buffer B at room temperature. M, optical density 280 nm; O-O, “P cpm; m---m, acetyl CoA carboxylase activity.
Inactivation of acetyl CoA carboxylase
Fractions,
IO ml
Fig. 6. Separation of acetyi CoA carboxylase from protein kinase I and II on DEAE-cellulose. The partially purified acetyl CoA carboxylase preparation (40 ml) obtained from Step II in the purification was applied to a DEAE-cellulose column as described in Step III of the purification. m----m, acetyl CoA carboxylase activity; M, protein kinase activity without added substrate; w, protein kinase activity with added substrate (casein). graphed on Sepharose 4B rather than DEAE-celluIose, 2 peaks of radioactive phosphate were found (Fig. 5). The first peak co-chromatographed with the acetyl CoA carboxylase activity and the second peak co-chromatographed with standard ATP. The major portion of the protein in the gel-filtered high-speed supernatant was not phosphorylated. During the purification of rat liver acetyl CoA carboxylase by the method of Inoue and Lowenstein (1972), it was found that the DEAE-cellulose step separated the major portion of the acetyl CoA carboxylase activity from 2 protein kinases (Fig. 6). Using casein as substrate 2 peaks of protein kinase were detected, which we have designated as I and II. Both protein kinases were dependent on the addition of substrate for maximal activity. Without added substrate, protein kinase activity was still detectable, suggesting the presence of endogenous substrate within these fractions. A third peak of protein kinase activity chromatographed with the acetyl CoA carboxylase activity. It is not known whether this represents a distinct protein kinase or if it is a non-
231
specific interaction of protein kinase I or II with either acetyl CoA carboxyiase or another protein eluting in this region. The substrate specificity and cyclic AMP dependency of protein kinase I and II were investigated (Table 2). Protein kinase I phosphorylated casein more extensively than histone when compared to protein kinase II. Cyclic AMP did not stimulate the activity of protein kinase I when histone was the substrate and showed only a slight stimulation with casein as substrate. Protein kinase II was equally effective with either protein as substrate in the absence of cyclic AMP. Cyclic AMP stimulated the activity of protein kinase II when either casein or histone was the substrate. The stimulation by cyclic AMP was greater when histone was the substrate. It is possible that protein kinase I is the free catalytic subunit which has dissociated from protein kinase II, the holoenzyme (Reiman et nl., 1971). The effect of ATP and ATP in the presence of added protein kinase I or II on the purified acetyl CoA carboxylase were investigated (Fig. 7). ATP alone, at a concentration of 2 mM, inactivated the purified acetyl CoA carboxylase. The degree of inactivation by ATP was moderate when compared to the results in Fig. 1. The purified acetyl CoA carboxylase retained 65% of its activity after 10 min incubation with 2 mM ATP, compared to the control with no additions. The crude enzyme retained only 32% of its activity after 10 min under similar conditions. The addition of ATP and either protein kinase I or II enhanced neither the rate nor the degree of inactivation. The addition kinase I alone had no effect although the addition of protein kinase II alone caused inhibition of acetyl CoA carboxyiase at all three incubation times (0, 5 and 10 min). Since the purified enzyme contained citrate in the buffer, ATP-inactivation studies were done at a citrate concentration of 8.8 mM. Due to this we could not conclude that the purified enzyme was, inactivated to a lesser extent than the crude enzyme. The effect of citrate concentration on ATP-dependent inactivation of rat liver and chicken liver acetyl CoA carboxylase was investigated. Representative results of 3 separate experiments are given in Fig. 8. Both the crude gel-filtered enzyme and the DEAE-purified enzyme from rat liver were inactivated to a greater extent at low citrate (2.5 mM) than at high citrate concentration (20 mM). The crude enzyme was inactivated to a
Table 2. Cyclic AMP dependency and substrate specificity of protein kinase I and II
Substrate Casein Histone
Protein kinase activity (cpm,/assay) Protein kinase I Protein kinase II plus minus minus plus cyclic AMP cyclic AMP cyclic AMP cyclic AMP 328 234
510 214
1418 1302
2378 3146
The fractions from the DEAE-Sephadex column (Fig. 3) which contained protein kinase I and Ii were concentrated by ammonium sulfate precipitation (65%) as described in Fig. 6. Protein kinase activity was measured as described in Methods with casein or histone as substrate. The activity is expressed as cpm “P incorporated per assay in a 20 min incubation at 30”. Blank tubes contained all of the constituents except the enzyme.
Protein
I
klnase
I 5
0
I
I 10
Time ,
I 0
P. R.
DESJARUINS
Protein
klnase
I 5
and K. II
I IO
min
Fig. 7. Inactivation of purified acetyl CoA carboxylase by ATP with and without added protein kinase. The fractions containing protein kinase I, II and acetyl CoA carboxylase from the DEAE-cellulose chromatography were individually pooled and concentrated by ammonium sulfate precipitation. Protein kinase I and II were resuspended and dialyzed against a buffer containing 10 mM Tris (HCl), and 10 mM 2-mercaptoethanol, pH 7.0. Acetyl CoA carboxylase was resuspended and dialyzed against buffer D. The partially purified acetyl CoA carboxylase was incubated without preincubation under the conditions described for the inactivation of acetyl CoA carboxylase in the Methods. O-O, acetyl CoA carboxylase alone (2.4 mg/ml); O-O, acetyl CoA carboxylase with protein kinase I or II at a concentration of 0.26 and 0.59mg/ml respectively; m---m, acetyl CoA carboxylase with protein kinase I or II and 2 mM ATP; O--U, acetyl CoA carboxylase with 2 mM ATP without protein kinase. Incubation conditions were identical to those of Fig. 1 except that the citrate concentration was 8.75 mM. Acetyl CoA carboxylase was measured in a 2 min incubation.
The pure chicken liver enzyme behaved in the same manner as the rat liver enzyme except that the crude and purified enzyme were inactivated to the same extent at all citrate concentrations. Again greater inactivation was observed at low citrate concentration (2.5 mM) than at high citrate concentration (20 mM). If the cold ATP present during the inactivation in Fig. 8 was replaced with radioactive 1;-3ZP-ATP no significant protein phosphorylation occurred in the presence of pure chicken liver enzyme at any citrate concentration. Thus significant enzyme inactivation occurred at low citrate concentration, and yet no protein phosphorylation was observed. greater
extent
at all concentrations.
DISCUSSION
It is well established
that insulin stimulates the rate of fatty acid synthesis from glucose in rat epididymal fat-pads, whereas adrenaline inhibits fatty acid synthesis (Cahill et nl., 1960; Denton & Halperin, 1968; Saggerson & Greenbaum, 1970). Recently, Halestrap & Denton (1974) have provided evidence that insulin and adrenaline control fatty acid synthesis by regulating the concentration of fatty acyl CoA’s, citrate and ATP within the tissue. They suggest that these compounds regulate the activity of acetyl CoA carboxylase through polymer-protomer transitions.
DAKSHINAMURTI
Other investigators (Heimberg et al., 1969; Bricker & Levy, 1972; Allard & Ruehrig, 1973) suggest that insulin and adrenaline mediate their effect on fatty acid synthesis, through cyclic AMP. Kim and coworkers (Carlson & Kim, 1973; 1974u, h; 1977) have suggested that insulin and adrenaline control fatty acid synthesis through the interconversion of acetyl CoA carboxylase from a dephosphorylated active form to be phosphorylated inactive form. They have suggested that the phosphorylation-dephosphorylation system is analogous to that which occurs with pyruvate dehydrogenase (Jungas, 1970; Coore Ed rrl., 1971; Weiss ef al., 1971), since the phosphorylation of acetyl CoA carboxylase appears to be insensitive to cyclic AMP. Carlson & Kim (1973, 19740, b) have based their hypothesis primarily on their observations that (1) inactivation of crude rat liver acetyl CoA carboxylase in the presence of y-“P-ATP resulted in (a) the parallel incorporation of 32P into protein which copurified with the carboxylase and (b) 32P incorporation into the carboxylase-antibody precipitate using antibody prepared in rabbits against the rat liver enzyme, (2) inactivation of the enzyme was dependent on the presence of ATP and a crude protein kinase fraction (fraction K). We have shown that ATP produced a time-dependent inactivation of the enzyme, which was paralleled 1 30 -
A-Rat
SO-
.
liver
\
to-
‘L.
!O-
i I
B-
iO-
I
Chlcken
I
I
lwer
. L.
. IO-
!O -
I 5
I 10
Cltrote.
I 15
20
J
mM
Fig. 8. Effect of citrate concentration on ATP inactivation of rat and chicken liver acetyl CoA carboxylase. A. Acetyl CoA carboxylase from rat liver at 2 stages of purification. (04, crude gel-filtered high-speed supernatant: -0, DEAE-cellulose enzyme) were incubated with 0.2 mM ATP for 10 min as described in Methods. The citrate concentration during the inactivation was varied from 2.5 mM to 20 mM. Acetyl CoA carboxylase activity was measured al the end of each inactivation incubation. B. Same as A except that pure chicken liver enzyme was used. (ed, Crude, gel-filtered high-speed supernatant; O-0, pure enzyme preparation.
Inactivation of acetyl CoA carboxylase by endogenous protein phosphorylation. Surprisingly, the same concentration of ADP produced an identical time-dependent inactivation of the enzyme. It is possible that the ADP was converted to ATP by an ATP generating system. Adenylate kinase catalyzes such a reaction and would be present in the high-speed supernatant. Similar concentrations of AMP, phosphate and GTP did not produce a time-dependent inactivation of the enzyme. Cyclic AMP did not inactivate the enzyme nor did it enhance the time-dependent inactivation by ATP. This latter result may be misleading since conditions may not have been favorable for detecting an effect of cyclic AMP. It is also possible that the protein kinase responsible for the phosphorylation of acetyl CoA carboxylase is not stimulated by cyclic AMP. It is also to be noted that under conditions of maximal phosphorylation the crude enzyme preparation still retained 25-30x of the original carboxylase activity. In case of enzymes like pyruvate dehydrogenase where a phosphorylationdephosphorylation mechanism has been established, the maximally phosphorylated enzyme had no enzyme activity (Wieland L Siess, 1970). We have also shown that ATP can cause very significant inactivation of acetyl CoA carboxylase at a concentration as low as 0.01 mM. In earlier work Greenspan and Lowenstein (1967) showed that when 4 mM ATP was included in the pre-incubation medium the activation of the enzyme was reduced considerably. However, at this concentration ATP inhibits the carboxylation reaction itself (Desjardins & Dakshinamurti, 1970). Lane and coworkers (1966) studied the sedimentation rate of the chicken liver enzyme in sucrose density gradient medium containing 2 mM ATP, 8 mM Mg2+ and 10 mM KHCO,. The enzyme sedimented in the protomeric form. Similar results were reported by Numa et at. (1967). These authors have concluded that the ATP-Mg complex is responsible for dissociation of the enzyme which is present in the carboxylated state. This is supported by the observation that malonyl CoA which also carboxylates the enzyme produces a similar disaggregation of the enzyme. In our experiments, the incubation medium contained only ATP and Mg2+ and no added HCO;. Inactivation, reversible with citrate, occurred at a very low concentration of ATP in the preincubation medium. Although Carlson and Kim (1974) stated that the 32P incorporated from Y-~~P-ATP during inactivation of acetyl CoA carboxylase copurified with this enzyme, we were able to separate the 32P-labelled protein from the acetyl CoA carboxylase on DEAEcellulose. The 32P-labelled protein and acetyl CoA carboxylase were found to co-chromatograph on Sepharose 43. These latter results suggest that the labelled protein(s) is (are) of a high molecular weight although not necessarily acetyl CoA carboxylase. DEAE-cellulose chromatography of the partially purified rat liver acetyl CoA carboxylase separated this enzyme from the bulk of the protein kinase activity. Two major protein kinases were identified. Protein kinase I which phosphorylated casein rather than histone and which was basically unaffected by a cyclic AMP, and protein kinase II which was stimulated by cyclic AMP and showed some preference to histone as substrate over casein in the presence of cyclic
233
AMP. A small peak of protein kinase activity was associated with the peak of acetyl CoA carboxylase activity. Neither protein kinase I or II could enhance the ATP inactivation of the purified acetyl CoA carboxylase. Although Carlson and Kim (1974) showed that increasing amounts of protein kinase (fraction K) caused increased inactivation of acetyl CoA carboxylase in the presence of ATP they did not show what effect the protein kinase fraction alone (minus ATP) would have on a&y1 CoA carboxylase. We have shown that inactivation by ATP of acetyl CoA carboxylase in both purified and crude acetyl CoA carboxylase from rat and chicken liver is directly dependent upon the citrate concentration. This result would appear to indicate that citrate prevents the ATP induced disaggregation (and thus inactivation) of the enzyme, a fact which has long been established. We have offered evidence which suggests that the time-dependent ATP inactivation of acetyl CoA carboxylase may not be due to phosphorylation of the enzyme to an inactive form, but that this may be entirely due to the disaggregating effect of ATP on the enzyme. Our evidence suggesting the latter is basically the following: (1) ATP inactivation occurs at a very low concentration and is dependent on the citrate concentration. (2) The protein phosphorylated during the time-dependent inactivation can be separated from acetyl CoA carboxylast on DEAE-cellulose. (3) Addition of protein kinase I or IT to purified acetyl CoA carboxylase does not enhance the inactivation of the enzyme by ATP. The evidence presented here strengthens the suggestion of previous investigators (Lane et al., 1974; Halestrap & Denton, 1974), that the time-dependent ATP inactivation is due to the disaggregation of the enzymes by ATP, to its inactive protomeric
form.
Acknowledgements-This investigation was supported by a grant from the Medical Research Council of Canada. P.R.D. acknowledges a grant from the University of Manitoba. REFERENCES
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