THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 246, No. 2, Issue of January 25, pp. 348358, 1971

Printed in U.S.A.

On the Mode of Action of Lipid-lowering Agents

V. KINETICS OF THE INHIBITION IN VITRO OF RAT ACETYL COENZYME A CARBOXYLASE

(Received for publication, August 27, 1970)

MICHAEL E. MARAGOUDAKIS AND HILDA HANKIN

From the Ciba Phawzaceutical Company, Research Department, Summit, New Jersey 07901

SUMMARY

Rat liver acetyl coenzyme A carboxylase was purified about 200-fold and the inhibition of this enzyme by certain hypo- lipidemic drugs was studied. The inhibition was more pro- nounced if the drugs were added before rather than after the citrate activation of the enzyme. Kinetic analysis revealed noncompetitive inhibition of the drugs with respect to the substrates acetyl-CoA, ATP, and HCO,-, and competitive inhibition with respect to the activator, citrate.

Sucrose density gradient centrifugations showed that the drugs reverse the aggregating effect of citrate to form the active polymeric forms of the enzyme from the inactive monomers. Arrhenius plots and heat-inactivation studies suggest gross conformational changes of the enzyme protein in the presence of the drugs.

Relative aflinities of acetyl coenzyme A carboxylase for citrate and the drugs are expressed by the calculated disso- ciation constant for citrate and the inhibition constants of the drugs. Derangement of fatty acid synthesis in vivo is con- ceivable by competition of the drugs with the activation of acetyl-CoA carboxylase at low and physiologically possible concentrations of the drugs and citrate.

Acetyl-CoA carboxylase (acetyl-CoA-carbon dioxide ligase (ADP), EC 6.4.1.2) catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA. This reaction is the first committed step in fatty acid biosynthesis and has been considered as the rate- limiting reaction in the over-all process (1, 2). The enzyme is thought to be regulated by a feedback mechanism involving the end products, palmityl-CoA and stearyl-CoA (3), and is acti- vated by certain Krebs cycle intermediates such as citrate and isocitrate (4, 5). Under metabolic conditions, such as fat feed- ing or starvation, in which lipogenesis is depressed, acetyl-CoA carboxylase was found to be the locus of this depression (6, 7).

Our interest in acetyl-CoA carboxylase arose from our earlier findings that this enzyme is inhibited by certain hypolipidemic drugs (8-13). It was postulated that this inhibition might ac- count, in part at least, for the lipid-lowering properties of these compounds. Derangement of lipid biosynthesis at the stage of acetyl-CoA carboxylase would be an ideal mode of attack be- cause accumulated acetyl-CoA can be diverted to Krebs cycle and no undesirable intermediates are likely to accumulate. A correlation of elevated plasma lipids with atherosclerosis has

found wide acceptance in recent years and hypolipidemic drugs such as 2-methyl-2-[p- (1,2,3,4tetrahydro-l-napthyl)phenoxy]- propionate and 2-(p-chlorophenoxy)-2-methyl propionate have attracted considerable interest as chemotherapeutic agents in the treatment of hyperlipoproteinemias. However, the mechanism of action of these agents is not understood, although many hy- potheses have been advanced (14-20).

We have shown that a number of lipid-lowering agents inhibit acetyl-CoA carboxylase purified from avian (8, 10, 12), rat (9, lo), and monkey liver (13). TPIAr-treated rats showed sign& cantly lower levels of hepatic acetyl-CoA carboxylase than con- trols and on prolonged TPIA treatment and fat-free diet mice showed progressive reduction in carcass lipids and lipogenesis from acetate (11). This paper describes the isolation of rat acetyl-CoA carboxylase and the kinetics of the inhibition of this enzyme by TPIA and CPIB.

EXPERIMENTAL PROCEDURES

ATP, acetyl-CoA, glutathione, palmitoyl-Coil, and crystalline bovine serum albumin were obtained from Sigma Chemical Company; potassium citrate from J. T. Baker Chemical Com- pany, Phillipsburg, New Jersey; and fat-free diet from Nutri- tional Biochemicals. NaHr4C03 and 1, 3-r4C-malonyl-CoA were purchased from New England Nuclear.

CPIB is a product of Imperial Chemical Industries, England, and TPIA is an experimental hypolipidemic compound synthe- sized in our laboratories by Dr. W. Bencze.

PuriJication of Acetyl-CoA Carboxylase-Acetyl-CoA carboxyl- ase was extracted from fresh rat livers obtained from male white CFr strain rats, (Carworth Farms), which were fasted for 48 hours and refed a fat-free diet (Nutritional Biochemicals) for 48 hours prior to being killed in a COz chamber. This dietary al- teration is known to drastically increase the enzyme activity (21).

The enzyme purification was carried out as described by Matsuhashi, Matsuhashi, and Lynen et al. (22) with some modi- fications. Table I summarizes the purification steps involved to obtain 4.5 units of enzyme from 400 g of rat liver correspond- ing to a purification of about 200-fold. The specific activity of this enzyme is twice the one obtained by Matsuhashi et al. (22) under similar conditions. The enzyme is highly unstable after the DEAE-cellulose column stage and can only be preserved

l The abbreviations used are: TPIA, 2-methyl-2-[p-(1,2,3,4- tetrahydro-1-naphthyl)-phenoxyl-propionic acid. The com- pound is also known as Melipan. The corresponding potassium salt was used in this work. CPIB, 2-(p-chlorophenoxy)-a-methyl propionic acid as its potassium salt.

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TABLE I Acetyl-CoA earboxylase purijication from rat liver 100

Fraction Volume Protein

ml

Crude homogenate.. 1,200 Supernatant (80,000 X g) . 760 First ammonium sulfate precipi-

tate. . . . . . . . . . . . . . . . . . . . . . 186 Calcium phosphate gel plus sec-

ond ammonium sulfate pre- cipitate....................... 57

DEAE-cellulose column plus third ammonium sulfate pre- cipitate. 55

ms

67,200 36,100

6,100

171

17

TOtd LctivitJ

unitsa

90.5 52.4

24.7

12.3

4.5

(1 One unit is 1 pmole of labeled CO2 fixed into malonyl-( min under the assay conditions.

Specific activity

units/mg )rotein 3

60 0.0013 2 0.0014 5

$ 40

0.004 B g

i 20

0.072 aw ;

under liquid nitrogen in the presence of 20 mM citrate and 0.6 z 6. mg of albumin per ml. Under these conditions the enzyme is stable for at least 2 months.

e Enzyme at the second ammonium +

sulfate stage is relatively stable at -20” for about a month with- L 40

out any addition of citrate and albumin. 2

The rat enzyme, unlike the avian enzyme, requires preliminary 20 incubation at 37” with citrate for enhanced activity (1,2). The purified enzyme (third ammonium sulfate stage, Table I) has an absolute requirement for citrate, Mg++, albumin, ATP, and 0

acetyl-CoA for activity. Addition of the citrate at the carbox-

W AT PREINCUBATION 150=8.~~10-5M

LA AT CARBOXYLATION150 =1.7x10-4M

4

0 ,

I 2 3 4 5 6 7 6

TPIAxl04 M

CPIB

Q-0 AT PREINCUBATION 1~~=7.5xiO-~ M

A-A AT CARBOXYLATION 15o=l.8xlO-3 M

I 1

I 2 3 4 5 6 7 6 CPIB x103M

ylation stage, without prior preliminary incubation, yields only 50% of the activity under standard assay conditions.

Albumin is apparently essential at the preliminary incubation stage for stabilizing the enzyme during the 30 min incubation. Addition of albumin at the carboxylation stage does not restore the activity. The time dependence of the citrate induced ac- tivation of acetyl-CoA carboxylase is a rather complex process as discussed under “Kinetics of citrate activation.”

Acetyl-CoA Carboxylase Assay-The enzyme activity was assessed by measuring acid-stable radioactivity incorporated into malonyl-CoA from H14C03 (21). The reaction mixtures con- tained 40 Bmoles of Tris-HCI (pH 7.5), 12 pmoles of MgC&, 14 pmoles of potassium citrate, 0.8 p moles of 2-mercaptoethanol, 0.6 mg of bovine serum albumin, and enzyme in a total volume of 520 ~1. After preliminary incubation at 37” for 30 min, 1.4 pmoles of ATP, 0.1 pmoles of acetyl-CoA and 12 pmoles of KH14C03 (4.2 x lo5 cpm per pmole) were added, yielding a total volume of 660 ~1. After incubation for 5 min at 37”, the reaction was terminated by the addition of 0.1 ml of 6 N HCI. The reaction mixture, routinely contained in a glass ampoule, was placed into a liquid scintillation vial, briefly exposed to gaseous HCl, and dried under a stream of warm air at 40-50”. The HCl treatment ensures complete release of all unreacted H14C0a-. Water (0.5 ml) and scintillation mixture was added and the 14C activity was determined by the use of a liquid scintil- lation spectrometer (Tracerlab, Waltham, Massachusetts) under flat spectrum conditions (23).

The assay gives highly reproducible results provided that en- zyme levels and time of incubation are adjusted to keep H14C03-, incorporation within range limits in which it varies in direct proportionality with respect to enzyme concentration and incu- bation time. Under the assay conditions, activity increases

FIG. 1. Inhibition of rat hepatic acetyl-CoA carboxylase by the hypolipidemic drugs TPIA and CPIB. The drugs were added be- fore and after citrate activation, at the concentrations indicated. 150 indicates 50% inhibition.

linearly with amount of enzyme up to 2 x lo+ units and the reaction is of zero order for at least 8 min.

Fatty Acid Synthetuse-Fatty acid synthetase assays were per- formed by the method of Guchhait, Putz, and Porter (24) with 1, 3J4C-malonyl-CoA as the radioactive precursor. The enzyme was prepared from rat liver homogenates as described by Chang et al. (25).

Sucrose Density Gradient Centrifugation-Density gradient centrifugations were performed as described by Majerus et al. (21) and Gregolin et al. (26) with a linear 5 to 20% (w/v) sucrose gradient, prepared by a Spinco gradient maker; the total volume was 5 ml and 20 fractions were collected. The variable addi- tions are indicated in the legend of Fig. 6. The enzyme solution was applied to the top of the gradient, and the tube was centri- fuged at 50,000 rpm in a Spinco model L ultracentrifuge with an SW50 rotor for 200 min at O-2”. Fractions of 0.25 ml were collected and were numbered from the bottom of the tube. Ali- quots of 0.1 ml were assayed for acetyl-CoA carboxylase activity by the 14HC08- fixation assay.

RESULTS

Inhibition of Acetyl-CoA Carboxylase by 6-MethyM-[p-(1,2,3,4- tetrahydro-1-naphthyl)-phenoxyl-propionat and B-(p-chloro-

phenoxy)-.%methyl-prop&ate

The activity of acetyl CoA carboxylase was measured by the H14C08- fixation assay in the presence of increasing concentra-

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TABLE II E$ect of b-methyl-~-[p-(1,~,S,4-tetrahydro-l-naphl-

propionate concentration on acetyl-CoA carboxylase activity under constant molar ratio of inhibitor

to enzyme

Incubation mixtures contained the components of the assay as outlined under “Materials and Methods” and enzyme (16.4 mg per ml of protein, determined by the method of Lowry) diluted 1:20 with 0.04 M phosphate buffer in the control experiment and 1:20 with 0.5 mM of TPIA in 0.04 M phosphate buffer in the drug experiment.

Enzyme

rcl

0 5

10 20 30 40 50 60 70

-

.-

Enzyme activity

Control Cont&dlp

0 0 312 179 814 546

2088 1474 3439 2420 4659 3019 5422 3648 6624 3813 7846 4450

- - LAURYL SULFATE x 10’5Y

-

-

Inhibition

%

43 33 30 30 35 32 43 44

t,ions of TPIA and CPIB (sodium salts). As shown in Fig. 1, the enzymatic activity falls with increasing drug concentrations and this is evident at all stages of purification of the enzyme.

TPIA is at least 10 times more potent as an inhibitor of acetyl- CoA carboxylase than CPIB. It is of interest that TPIA in viva is hypolipidemic at a much lower dosage than is CPIB, and is comparable in viva as well as in the enzyme assay here dis- cussed to 2-[p-(p-chlorophenyl) phenoxy]S-methyl propionic acid, as the potassium salt, an agent newly described as lo-fold more active than CPIB (10).

Rat liver acetyl-CoA carboxylase requires preliminary incuba- tion with citrate for activation. The inhibitory effect is more pronounced if the drugs are added before preliminary incubation with citrate than when the drugs are added after the citrate ac- tivation stage of the enzyme assay. Under our experimental conditions, the concentration of TPIA which is required for 50% inhibition (Iso) is 8.5 X 10B6 M if added before citrate activation, or 1.7 X 10s4 M if TPIA is added after citrate activation. For CPIB, Iso = 7.5 X 10m4~ if drug is added before and 150 = 1.8 X 10-s M if CPIB is added after citrate activation. The depend- ence of inhibitor potency on the state of enzyme activation may be due to the disappearance of drug binding sites as the monomers aggregate. When the data of Fig. 1 are plotted as log V/V,,, - V versus log (I) according to the modified Hill equation (27), the interaction coefficients (n) obtained from the slopes suggest that there are more enzyme-drug interacting sites before than after citrate activation.

Mammary Gland and, Adipose Tissue Acetyl-CoA Carboxylase Inhibition by 2-Methyl-W[p-1 ,d,S,&tetrahydro-l-naph-

thyl)-phenoxyl-propionate and B-(p-chlorophenoxy)- 2-methyl-propionate

Partially purified acetyl-CoA carboxylase from lactating mam- mary glands of rats (28) was used to test the susceptibility of the enzyme to inhibition by TPIA and CPIB. A preparation puri-

%60- %60-

g IO- 4

I 0 0 2 2 4 4 6 6 6 6 IO IO 12 12 I4 14 I6 16 I I

FIG. 2. Inhibition of rat hepatic acetyl-CoA carboxylase by lauryl sulfate.

fied through the first ammonium sulfate stage with specific ac- tivity of 71 mpmoles per mm per mg of protein was inhibited by 50% when 3.8 X 10e6 M TPIA or 5.1 X 10e4 M CPIB was present in the standard assay mixture.

Rat adipose tissue acetyl-CoA carboxylase, obtained by the procedure employed by Dakshinamurti and Desjardins (29) is also inhibited to about the same extent as the mammary or the hepatic enzyme.

Possible Detergent Effect

Palmitoyl-CoA has been implicated in the control of various enzymes, including acetyl-CoA carboxylase (3).

Some authors have suggested that the effects of pahnitoyl-CoA are not physiological, but are merely a strong detergent action, because the binding with these enzymes is not site specific. Thus, Dorsey and Porter (30) showed that inhibition of fatty acid synthetase by palmitoyl-CoA is dependent on both the critical micellar concentration of palmitoyl-CoA and the molar ratio of inhibitor to enzyme.

In view of the hydrophobic-hydrophilic nature of the hypo- lipidemic compounds under investigation, we have considered the inhibition of acetyl-CoA carboxylase by these compounds as a possible detergent effect.

Table II indicates that at constant molar ratio of acetyl-CoA carboxylase to TPIA no drastic change in the inhibition is evi- dent with increasing drug concentration, suggesting no depend- ence of the extent of inhibition on critical micellar concentra- tion of TPIA.

Furthermore, comparison of the inhibition by TPIA (Fig. 1) with that by lauryl sulfate (Fig. 2) shows an abrupt drop of acetyl-CoA carboxylase activity at the critical micellar concen- tration of lauryl sulfate (about 0.1 mM), which is not evident in the TPIA-inhibited system. Moreover, Table III indicates that

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TABLE III TABLE IV

Mixed inhibition of acetyl-CoA carboxylase by B-methyl-d-[p- (i,d,3,4-tetrahydro-l-naphthyl)-phenoxy]-propionate and

lauryl sulfate

Effect of S-methyl-~-[p-(l,3,3,~-tetrahydro-l-naphthyl)-phenoxy]- propionate on fatty acid synthetase from rat liver

Assays for enzyme activity contained 66.7 pg of protein; all other conditions, components, and their concentrations as re- ported under “Materials and Methods.” Inhibitors were added before citrate activation.

TPIA concentration Fatty acid synthetase activitp

Inhibitor Enzyme activity

CM

Control....................................... TPIA, 7.6 X 10-b M. . . . . Lauryl sulfate, 3.78 X lo+ M.. . TPIA, 7.6 X lo-& M plus lauryl sulfate, 3.78 X

lo-+M......................................

6092 4212 5192

2391

M x 10”

1.25.. .................................... 2.50 ...................................... 6.25 ...................................... 12.50 ..................................... 25.00 .....................................

6549 6062 5781 2860

75

Control (no drug) ......................... 5150

the mode of inhibition by lauryl slufate and by TPIA are differ- ent because mixed inhibition is cumulative and not additive. Kinetic data demonstrating a different mode of interaction of TPIA and palmitoyl-Coil carboxylase have already been pre- sented (8).

a Fatty acid synthetase activity is measured as total radio- activity found in the saponifiable and petroleum ether-extractable lipid fraction from the reaction mixture: potassium phosphate buffer, pH 7.0, 100 pmoles; acetyl-CoA, 0.025 pmoles; 1,3-“C- malonyl-CoA, 0.01 pmole, 2 X lo5 cpm; TPNH, 1.2 rmoles; gluta- thione, 2pmoles; EDTA, 3 pmoles; rat liver enzyme, 65 fig of pro- tein (determined by the method of Lowry) in a total volume of 1.0 ml incubated at 37” for 10 min.

E$ect of d-Methyl-%[p-(I ,2,S,&strahydro-l-nuphthyl)-pherwxy]- propionate and d-(p-chlorophenoxy)-$-methyl-prop&&e on

Other Enzymes

TABLE V

Steady state kinetic parameters of inhibition of acetyl-CoA carboxylase by hypolipidemic drugs” present at

citrate-activation stage

A number of liver enzymes have been checked for their sus- ceptibility to inhibition by hypolipidemic drugs (8). At drug concentrations where acetyl-CoA carboxylase activity is virtu- ally 0, all enzymes tested are unaffected by the presence of these compounds.

Variable0 hhibitor Vm, kt S.E.) 1

.-

-

M x 10s c9m

Acetyl coenzyme A (0.03 to 0.30 rnM)

None. ................. 7902 zk 483 TPIA. ................. 7.60 4027 f 298 TPIA. ................. 11.38 3128 2~ 146 CPIB .................. 76.4 4779 zk 251 CPIB .................. 153.0 3354 f 201

ATP (0.1 to 3.0 mM) None .................. 3124 f 119 TPIA .................. 7.6 1437 Zk 34 TPIA .................. 11.38 1013 ZfZ 49 CPIB. ................. 76.4 1427 rt 33 CPIB .................. 153 1079 Zk 39

HCOE- (1.2 to 15.0 mM) None. ................. 3384 zk 91 TPIA .................. 7.60 1902 Zk 70 TPIA. ................. 11.38 1071 + 39 CPIB .................. 76.4 1645 ZIZ 35 CPIB .................. 153.0 946 f. 71

Fatty acid synthetase seems to be stimulated by TPIA and only at very high concentrations is there an abrupt drop in en- zyme activity, probably because of a detergent or salting out effect (Table IV). The same results were obtained with CPIB (data not shown).

0.216 zk 0.022 0.213 i 0.027 0.286 & 0.021 0.237 zb 0.020 0.242 rt 0.023

Kinetics of Acetyl-CoA Carboxylase Inhibition by S-Methyl- 8-[p-(1,1 ,S ,.&etrahydro-1 -naphthyl)-phenoxy]-propionte

and %(p-Chlorophenoxy)-Smethyl-propionate with Respect to Substrates Acetyl-CoA, ATP,

and HCOe-

0.220 zt 0.022 0.197 Zk 0.012 0.248 3~ 0.028 0.193 Zt 0.011 0.265 f 0.020

Since the inhibitory effect of TPIA and CPIB on acetyl-CoA carboxylase depends on the state of activation of the enzyme by citrate, rate measurements were performed with and without preliminary incubation of the enzyme with citrate. The steady state kinetic parameters were obtained from the initial velocity data by means of a digital computer program written for the Lineweaver-Burk (31) form of the Michaelis-Menten rate equa- tion. Data weighing and calculation of standard errors of the kinetic constants were done by the method of Wilkinson (32).

4.321 f 0.252 5.472 zt 0.047 4.768 rt 0.334 4.462 f 0.203 4.207 zk 0.539

0 Substrates were employed at 10 to 12 concentration levels within the limits given in each case. Other experimental condi- tions were as described under “Experimental Procedures.”

Table V shows the kinetic parameters from the double re- Previous results with avian acetyl-CoA carboxylase showed a ciprocal plots for variable concentrations of acetyl-CoA, ATP, competitive inhibition of this enzyme by TPIA and CPIB with and HC03 at two levels of TPIA and CPIB added before pre- respect to acetyl-CoA (8). In order to confirm this specie differ- liminary incubation of the enzyme with citrate. Table VI ence of acetyl-CoA carboxylase, we have obtained the Dixon shows the same data for the drugs added after 30 min of citrate plots, l/v versus (I)), for the rat enzyme by varying TPIA con- activation of the enzyme. Table VII summarizes the kinetic centration from 0 to 9 x 10T5 M, added at the time of preliminary constants calculated from these data. The inhibition was found incubation, at four levels of acetyl-CoA (33). This analysis to be noncompetitive for all three substrates, acetyl-CoA, ATP, confirms the conclusion to be drawn from Tables V and VI; i.e. and HCOa-. the inhibition is of the noncompetitive type with respect to

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acetyl-CoA with KJ = 7.98 x 10e5 M for TPIA. Noncom- of each inhibitor separately, and then in the simultaneous pres- petitive inhibition for acetyl-CoA was also revealed by plotting ence of both TPIA and CPIB. The data were analyzed by the data as V versus V/S. Parallel lines with slopes numerically Lineweaver-Burk plots and the slopes of each line, corresponding equal to K, were obtained. to Km/v, were obtained. Theory predicts (34) that such slopes

Mixed Inhibition Studies be numerically different for inhibitors acting upon one and the same or two separate sites. The experimentally found slope for

The question of independent or identical sites of action for the mixed inhibition with TPIA plus CPIB is in close agreement

TPIA and CPIB has not been answered from the kinetic data of with the line calculated on the bases of identical sites of action Tables V and VI. This question is of practical importance of the inhibitors (data not shown). The same conclusion has because the drugs might exert a synergistic effect in their ef- been obtained with the avian liver enzyme (8). fectiveness as lipid-lowering agents if they are bound at inde- pendent sites and if inhibition of acetyl-CoA carboxylase is, in Kinetics of Citrate Activation

fact, important in their mechanism of action. Mixed inhibition studies were performed with acetyl-CoA as

The kinetics of the citrate activation of acetyl-CoA carboxylase

the varying substrate, in the absence of inhibitors, in the presence are quite complex and not completely understood. As shown in Fig. 3, the enzyme has an absolute citrate requirement. Citrate

TABLE VI catalyzes the formation of active aggregates of the enzyme from

Steady state kinetic parameters of inhibition of acetyl-CoA inactive monomers (4, 26). This process is time dependent, as

carboxylase by hypolipidemic drugs added after citrate-activation stage

Substrates were varied at 10 to 12 concentration levels. Other experimental conditions as described under “Experimental Procedures.”

Variable

Acetyl coenzyme A (0.03 to 0.30 mM)

None. ................. TPIA. ................. TPIA. ................. CPIB .................. CPIB ..................

ATP (0.1 to 3.0 mnn) None. ................. TPIA. ................. TPIA .................. CPIB. ................. CPIB. .................

HC03 (1.2 to 15.0 mM) None. ................. TPIA. ................. TPIA. ................. CPIB .................. CPIB ..................

-

1

-

:nhibitor I’m,, kt S.E.)

M x 106

15.2 22.8

152 304

9663 zk 831 4560 f 471 3659 f 193 5375 zt 360 4915 f 114

15.2 22.8

152 304

2658 rk 57 0.232 f: 0.013 :

1351 zt 38 0.263 f. 0.019 : 937 4~ 60 0.249 * 0.037

1638 f 35 0.226 r!z 0.013 1135 zt 32 0.221 32 0.017

15.2 22.8

152 304

6050 A 117 2825 f 537 1853 i 194 3295 zt 169 2014 & 327

Inhibitor with variables Type of inhibition Apparenta Michaelis constant

TPIA Acetyl-CoA. ....... ATP .............. HC03. ...........

CPIB Acetyl-CoA ........ ATP .............. HCOs- ............

. . . . .

. . . . .

. . . . .

Apparent Km kt SE.1

2.8

??iM

f VI e

0.244 f 0.033 0.253 zk 0.040

g u

0.291 f 0.024 ; 0.208 rk 0.022 0.324 & 0.073

E <

5.758 i 0.233 3.938 zt 1.069 3.221 ZIZ 0.584 4.135 =t 0.382 CITRATE 3.236 i 0.805

(mM)

FIQ. 3. Citrate dependence of acetyl-CoA carboxylase activity

TABLE VII

Summary of kinetic constants

Noncompetitive 2.30 X lo-’ Noncompetitive 2.26 X lo-’ Noncompetitive 5.03 x 10-Z

Noncompetitive 2.3 X lo-’ Noncompetitive 2.26 X 1O-4 Noncompetitive 5.03 x 10-a

Inhibitor added before Inhibitor added after activation (Ki) activation (Ki)

24 I

M

7.85 X 1O-5 1.31 x 10-d 5.97 x 10-S 1.40 x 10-4

7.06 X 1O-5 1.16 X lo-’

1.19 x 10-Z 2.52 X 1O-3 7.25 X 1O-4 2.30 X 1O-3 6.82 X 1O-4 1.66 x 10-s

5 Apparent Km and Kc values are the average of the values obtained from the data of Tables V and VI.

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I 1 I 0 I I I I

4.0

t

- CONTROL M TPIA Z5xI0-*Y 0

I 0 IO 20 30 40 50 60 70

PREINCUBATION TIME (MIN)

TABLE VIII

Time dependence of citrate activation and 2-methyl-d-[p-(1,6,3,4- tetrahydro-l-naphthyZ)-phenozyl-propionate inhibition of

rat liver acetyl-CoA carboxylase

Enzyme activity was measured as described under “Experi- mental Procedures” but with citrate concentrations and times of preliminary incubation at 37” as indicated.

Concentration

Citrate 0 rnM

5.76 rnM.. .

8.48 mM.. 12.4 mM.. . . . . . 16.7 mM..

Citrate plus TPIA, 7.5 x 1o-5 M

0 rnM . . .

5.76 mM.. 8.48 mM...

FIG. 4. Time dependence of citrate activation and TPIA in- 12.4 mM.. _. hibition of rat liver acetyl-CoA carboxylase. Preliminary incu- 16.7 mM.. . bation times were as indicated; all other conditions as described under “Experimental Procedures.” TPIA at a concentration of 7.5 x 1Om6 was present in the preliminary incubation mixture.

shown in Fig. 4, and the inhibitor interferes with the activation rate of the enzyme. Citrate is also essential for the stability of acetyl-CoA carboxylase as stated above.

Kinetic parameters of time-dependent citrate activation and 2-methyZ-2-[p-(l,2,3,Q-tetrahydro-i-naphthyZ)-phenoxy]- propionate inhibition of rat liver acetyl-CoA carboxylase

Under our preliminary incubation conditions (37” for 30 mm), no appreciable inactivation of the enzyme occurs, even at sub- optimal citrate levels i.e. below 16.7 mM. However, prolonged incubation at 37”, in the absence of enough citrate, leads to drastic reduction in enzyme activity (Table VIII). At the saturation level of citrate, the enzyme not only is stable at 37” for 6 hours, but also shows a slow increase in activity.

Pre- Control (no drug) I TPIA, 7.5 X 10-S M

!%a~ I tiona time V,,, f S.E. K,,, f S.E. I’,,, =t SE. K,,, f S.E.

Table VIII shows that the presence of 7.5 X low6 M of TPIA in the preliminary incubation mixture does not seem to cause any time-dependent inactivation of the enzyme for at least 60 min. With longer periods of time, however, there is a definite inactivation of the enzyme due to TPIA, especially at low levels of citrate. This is probably a secondary effect due to competi- tion of drug with citrate for the enzyme, which renders citrate stabilization ineffective.

0. 2. 5..

10. 25. . 60..

7352 zk 533 6.47 i 1.06 4372 f 578 10.66 & 2.54 8077 f 768 8.34 zt 1.54 5832 f 678 12.40 zt 2.45 8355 zt 792 9.16 zt 1.62 4948 i 862 8.91 & 2.83 9415 f 85010.15 f 1.65 4329 f 760 4.43 f 1.89 6879 f 509 5.55 zt 1.00 7884 zk 370 13.68 + 1.13 9132 iz 302 7.54 rt 0.54 9959 rt 1715 23.08 zk 5.68

a Citrate was varied at nine concentration levels ranging from 3.5 to 16.7 mM, and TPIA was added to the assay mixture before the enzyme. All other components and conditions of the car- boxylation assay were as described under “Experimental Proce- dures.”

The inhibitory effect of TPIA or CPIB depends on the stage of citrate activation of the enzyme. Since the activation is time dependent, so must be the inhibition of acetyl-CoA car- boxylase by TPIA and CPIB. In Table IX are summarized the kinetic parameters of the TPIA-inhibited and noninhibited sys- tem at different times for activation (preliminary incubation) intervals. At zero time the inhibition by TPIA is of the mixed type with respect to the activator, citrate. After 60 mm of preliminary incubation, the enzyme is almost fully activated and the inhibition is competitive for citrate with K; = 3.65 X 10-e M. Under similar conditions Ki = 3.0 X NY4 for CPIB.

Kinetic and Thermodynamic Parameters from Hill Plots

can be used advantageously in obtaining additional information from kinetic data.

Equation 1 is the empirical Hill equation as modified by Changeux (35).

log VIVIU,, - V = n log (s) - log K (1)

where (.s) is substrate concentration, I’ is the reaction rate, V max is the rate attained when the enzyme is saturated with sub- strate, n is moles of substrate per enzyme molecule, and K is the product of the n dissociation constants of the enzyme-substrate complexes (Equation 2).

K = (E)(S)”

WS’n) (2)

-

--

-

30 min I 1

60min 1 120 min 1 240 min 1 360 min

c$vnfized inmalonyl-CoA

39 29 10 0 6 1261 1101 780 472 317 1536 1544 1351 865 919 2439 2392 2366 1927 2002 2909 3103 3478 3545 3594

14 12 5 2 0 474 412 251 143 35 783 565 398 226 78 983 861 668 517 336

1093 1008 942 522 767 -

Time of incubation with citrate

TABLE IX

-

The modified Hill equation as derived by Atkinson, Hathaway, and Smith (27) is a form of the Michaelis equation and as such it

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0.2

> 1

i 0.0

\=+ >

:: -1 -0.2

- 0.4

- 0.6

FIG. 5. Hill plot of the citrate activation of acetyl-CoA car- boxylase. Preliminary incubation time was 60 min; all other con- ditions are outlined in Table X. The regression equation de- scribing the straight line is log V/V,,, - V = 2.138 + 1.008

log 63.

TABLE X

Kinetic parameters obtained from Hill plots

I I K I Variable

I

?z

Acetyl-CoA ...... 0.986 ATP ............ 1.002 HCO,-. ......... 0.984 Citrate”. ........ 1.010

M x 10”

0.225 0.233 4.45 7.27

L

K, (From Liieweaver- Burk plot)

M x 10’

0.200 0.237 4.21 7.53

-

hf x 10,

0.216 0.220 4.32 7.54

0 Data for varying citrate concentrations were obtained from Table IX (60-min citrate activation without drug). The same excellent correlation of K, values was obtained for all time inter- vals of citrate activation. The data for varying acetyl-CoA, ATP, and HC03 concentration were obtained from Table VI. In all plots the slope was significant at p < 0.001.

The assumption involved in deriving Equation 1 as a form of the Michaelis equation is that the concentrations of all complexes containing fewer than n molecules of S are negligible. Plots of log ~/Vnl,, - V versus log (8) are straight lines with slopes numerically equal to substrate-binding sites and intercepts equal to -log K.

Fig. 5 is such a Hill plot for varying citrate concentration, when previously incubated for 60 min (data from Table IX). The number of citrate binding sites per protomer determined by this plot is 1 (n = l.OOS), and the dissociation constant, K = 7.27 x 1c3 M, indicates a rather tight enzyme-citrate complex.

The same parameters were obtained for varying acetyl-CoA and HC03- from the data of Table V by the least square method using program 310 on IBM computer 1130 (Table X). From the slopes the number of interacting sites obtained for acetyl- CoA, ATP, and HC03- is numerically equal to 1, which means that 1 mole of each of the substrates per mole of protomer is in-

7 WITH CITRATE 6

5 4

3 2

I

0 7 6 PLUS TPIA 1.33~10-~ N

;’ 2 4 6 6 10 12 14 16 I8 20

i

FRACTION NUMBER

FIG. 6. The effect of citrate and TPIA on the sedimentation pattern of acetyl-CoA carboxylase in sucrose density gradients. The enzyme, about 4 X 10-a units (purified after the second am- monium sulfate stage (see Table I)) was diluted to 0.1 ml with the same solution as the gradient medium (minus sucrose). The en- zyme solution was incubated at 37” for 10 min and then was applied to the top of the gradient, centrifuged, and analyzed for enzyme activity as described in “Experimental Procedures.” All gradi- ents contained 0.05 M phosphate buffer, pH 7.0, albumin, 0.6 mg per ml, manganese chloride, 0.5 mM, EDTA, 0.1 mM, and 2-mer- captoethanol, 0.5 mM. In addition, tube No. 2 contained 20 mM citrate and tube No. 3 contained 20 mM citrate plus 7.5 X 10-h M TPIA.

volved in the carboxylation reaction. The Km values calculated from the modified Hill equation (for V = +I’,,,, log X = log K, = log K/n) agree closely with those obtained from Line- weaver-Burk plots (Table X) .

The enzyme-citrate complex is not dissociable to products; therefore, Km is equal to K,,, a true thermodynamic constant. From this the free energy of binding of enzyme with citrate can be calculated and under the conditions of Fig. 4, AF = 3018 cal per mole.

Inhibition constants for the drugs are also true thermody- namic constants of dissociation of the enzyme-drug complex. The relative affinities, therefore, of the drugs and citrate to the enzyme can be calculated from the ratio of Kcitr/Ki for the drugs. This ratio is Kcitr/Ki = 7.27 X W3/3.65 X lo+ = 198 for TPIA and K,itr/K< = 7.27 X 10m3/3 X 10-* = 24.1 for CPIB. From these aflinity ratios the differences in the free energy of binding of citrate and drugs with the enzyme can be calculated. This difference in free energy of binding of citrate and TPIA is -3240 cal per mole. The same parameter for CPIB is -1950 cal per mole.

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Issue of January 25, 1971 M. E. Maragouclakis and H. Hankin 355

T”C

40 35 30 25 21 17 13 I

8 0-O CONTROLlNO DRUG)

A-A 7.6~lO-~M of TAP, AT PREINCIJBATION

22.5OC

4

315 320 325 330 335 340 345 350

f.105

FIG. 7. Influence of temperature on acetyl-CoA carboxylase activity and inhibition by TPIA. The reaction mixture is the standard H14C0a- fixation assay as outlined under “Experimen- tal Procedures.” Preliminary incubation temperature was 37” Enzyme activity was measured at each temperature (12-40”) in the absence and the presence of 7.5 X 10e6 M TPIA. Initial velocity is expressed as counts per min fixed within 5 min in each assay tube.

Sucrose Density Gradient Centrijugation

Variations in the molecular form of acetyl-CoA carboxylase in the presence and absence of citrate and presence of citrate plus TPIA are illustrated in Fig. 6, the sucrose gradient sedimentation patterns.

The enzyme is sedimenting in the large form in the presence of citrate as shown by the increase in sedimentation velocity. The simultaneous presence of TPIA reverses the aggregating effect of citrate.

Gregolin et al. (36) have shown conclusively in avian acetyl- CoA carboxylase that isocitrate-induced aggregation is associ- ated with formation of an active polymeric form of the enzyme. We have shown earlier (8) that TPIA, a competitive inhibitor for the activator isocitrate, prevents the isocitrate-catalyzed aggregation of the avian enzyme. The same conclusions are supported from the present study on the rat liver enzyme.

Arrhenius Plots-Detailed studies of the temperature depend- ence of acetyl-CoA carboxylase activity in the TPIA-inhibited and noninhibited systems are shown in Fig. 7. Under our condi-

TABLE XI

Heat inactivation of acetyl-CoA carboxylase from rat liver in pres- ence and absence of S-methyl-%[p-(1 $,S,C-tetrahydro-l-

naphthyl)-phenoxyl-propionate and d-(p-chloro- phenoxy)-$-methyl propionate

Reaction mixtures and assay conditions were as described in the HCOs- fixation assay under “Experimental Procedures.” Final concentrations of TPIA and CPIB were 7.6 X 10e6 M and 2.56 X 10e4 M, respectively, and were added before the citrate activation. At the stage of the assay indicated the reaction mixture was heat treated at 45’ for various times and the enzyme activity was measured. The inactivation pattern did not change materially from Experiment II if the drugs were added after the citrate activation.

Heat treatment and inhibitor

Experiment I Before citrate activation

Control.. . . . . . TPIA . . . . CPIB . . .

Experiment II After citrate activation

Control. . . TPIA................................ CPIB . . . .

Experiment III After addition of the carboxylation mix-

ture Control. . . . . . . . . . . . . . . . . . . TPIA . . . . . . . . . . . CPIB . . . . . . . . . . . . . . . . . . . .

Experiment IV After addition of the carboxylation mix-

ture minus acetyl-CoA Control.. . . . . . . . . . . . . . . . TPIA . . . . . . . . . . . . . . . . . CPIB . . . . . . . . . .

-

I

-

Tie required for inactivation

50% 90% nactivation Inactivation

ntin

4 12.4 2.8 9.4 3.5 11.8

3.2 12.8 2.0 7.0 2.5 9.0

>15 >15 1.3 >15 2.4 >15

1.0 7.0 0.9 5.3 0.8 6.1

tions the enzyme is not cold labile in either system (37) and the magnitude of the inhibitory effect does not change with tempera- ture. The temperature at which the activity is determined, however, has a very pronounced influence on the reaction rate in both systems. Fig. 7 reveals that both plots are polyphasic with points of discontinuity at 22.5”, 30.5”, and 36.5”. Below 22.5 there is an unusually high temperature dependence with tempera- ture coefficient (&lo) 7.2 and activation energy 34.2 kcal per mole. At 22.5” an abrupt change in slopes occurs and in the region, 22.5-33.5”, the temperature coefficient (&lo) is 2.9 and the activation energy is 18.6 kcal per mole. In the region, 30.5-36.5”, the temperature coefficient (&lo) is 1.7 and the activa- tion energy is 9.45 kcal per mole. Above 36.5’ there is a de- crease in enzyme activity with temperature coefficient (Q13 2.2 and energy of activation -15.4 kcal per mole.

The same results were obtained when TPIA was added at the carboxylation stage. The slopes of the lines within the several temperature regions and the transition points were the same, but the inhibition was less pronounced at every temperature. Re- sults obtained for CPIB were also the same (data not shown).

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C-

H CONTROL H CPIB 2.56 xIO-~M

M TPIA 7.6 x lO-5 M

2 4 6 B IO 12 I4 TIME (MINUTES)

FIG. 8. Heat inactivation of acetyl-CoA carboxylase from rat liver. Drugs were added at the preliminary incubation stage and heat treatment at 45” was performed before citrate activation. The incubation mixture contained the components of the H%OZ- fixation assay and the drugs at the concentrations indicated.

H CONTROL

Bd CPIB 2.56 x IO-4 M W TPIA 7.6 x10-5Y

IO

,~L-------1 2 4 6 8 IO I2 I4

TIME (MINUTES)

FIG. 9. Heat inactivation of acetyl-CoA carboxylase from rat liver. Drugs were added before citrate activation and heat treat- ment at 45’ was performed after the addition of carboxylation mixture.

The assumption underlying these experiments is that no drastic change in the Km value occurs as the temperature varies. Otherwise, the enzyme will not be saturated with substrates and the measured rate will have no relation to V,,, and the rate constant (V,,, = Ka (total enzyme)).

Earlier results with the avian hepatic acetyl-CoA carboxylase (8) also revealed a biphasic character in the Arrhenius plots and nondependence of the magnitude of the inhibition on tempera- ture. Below 22” the energy of activation was found 33.7 kcal per mole and above 22” 12.8 kcal per mole.

Heat Inactivation

The enzyme used in the heat treatment experiments was puri- fied through the second ammonium sulfate stage. Table XI summarizes time required for 50% and 90% inactivation under different conditions of heat treatment at 45”. Figs. 8 and 9 are graphic illustrations of differences in rates of inactivation de- pending on the presence and absence of drugs and other condi- tions.

Stability of acetyl-CoA carboxylase at 45” is greatly increased when the carboxylation mixture is added prior to heat treatment (Experiments I and III, Table XI). The component of the car- boxylation mixture, which has this protective effect on the en- zyme, is acetyl-CoA, as shown in Experiments III and IV of Table XI. Protection by acetyl-CoA is drastically reduced when either CPIB or TPIA is present in the reaction mixture.

The drugs enhance the initial rate of heat inactivation; it appears that by competing with citrate they favor the formation of a protomeric, and more heat labile, form of the enzyme.

DISCUSSION

Acetyl-CoA carboxylase has been considered as the pace setter in lipid biosynthesis (1, 2). In this and previous reports (8-13) we have studied the inhibition of this key enzyme by a number of clinically important hypolipidemic drugs. As shown by kinetic analysis, the drugs interfere with the activation process of the enzyme, brought about by tricarboxylic acids. The activation has an apparent regulatory significance (4) and is associated with gross conformational changes of the enzyme protein (36). Ki- netic evidence that the drug competes with the function of the activator to bring about these conformational changes is only circumstantial; rate variations are not directly referable to conformational changes.

More direct evidence on the competition of the activator, citrate, with TPIA were obtained from the sucrose gradient sedi- mentation experiments. The citrate-mediated aggregation of acetyl-CoA carboxylase does not take place when TPIA is pres- ent.

The inhibition of acetyl-CoA carboxylase is not unique for the hepatic enzyme. Acetyl-CoA caroxylases from rat adipose tissue and lactating mammary gland are equally susceptible to inhibition by hypolipidemic drugs.

The inhibitory effect on acetyl-CoA carboxylase appears to be quite specific. We are considering other enzymes as possible target molecules of these compounds, but at present no other enzyme tested is significantly affected by the drugs.

It seems unlikely that a detergent effect of the drugs in the assay mixture can account for the inhibitory effect on acetyl-CoA carboxylase. Lauryl sulfate, a strong detergent, causes 50% inhibition at a concentration of 8 x 10m6 M. At the critical micellar concentration of lauryl sulfate, (about 0.1 mM), how- ever, there is a precipitous drop in enzyme activity. Mixed inhibition studies of TPIA with both lauryl sulfate or palmitoyl- CoA show different modes of interaction with the activity of the enzyme.

The chemical nature of the interaction between TPIA or CPIB and the enzyme is very little understood. The drugs

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Issue of January 25, 1971 M. E. Maragoudakis and H. Hankin 357

presumably bind at the citrate binding site, or a site near enough to account for competitive inhibition with citrate, thus prevent- ing the activator from bringing about the aggregation of the inactive monomers of the enzyme.

The data contained in this paper lead us to believe that acetyl- CoA carboxylase inhibition by hypolipidemic drugs is a site- specific interaction and as such is probably physiologically im- portant.

Studies on the dependence of carboxylation rate on tempera- ture in the absence and presence of the drugs reveal that the inhibitory pattern does not change materially with temperature. The discontinuities of slope (Fig. 6) indicate that there are changes from one value of the activation energy to another at the transition temperatures. Since the over-all carboxylation reaction of acetyl-CoA involves several successive reactions, such as citrate activation of the enzyme, carboxylation of the biotin enzyme, and others, it is conceivable that these have different temperature coefficients. As the temperature increases, the reaction with the high coefficient will become the more rapid of the two and the over-all process will then be limited by the reaction with a temperature coefficient. The activation energy of the latter will then be the observed value. Other explanations, such as reversible inactivation at the higher temperatures, or the possible existence of the enzyme in more than one form with different activation energies, can be considered. This type of discontinuity in Arrhenius plots has been theoretically discussed by Stearn (38).

Arrhenius plots (Fig. 6) make it quite clear that the inhibitor does not materially affect the enthalpy of activation (AH*). Therefore, the rate decrease brought about by the inhibitor must be reflected in the entropy of activation (AF* = AH* - TAS*). The appreciable increase in the entropy term can be visualized as a conformational change in the protein when it becomes asso- ciated with the inhibitor. Entropy changes may further result from changes in hydration when the ionic inhibitors interact with the enzyme protein. The sedimentation data (Fig. 5) are evi- dence in support of gross structural changes of the enzyme in the presence of the inhibitory agent.

Contrary to the avian enzyme, the enzyme from rat liver is protected against heat inactivation by acetyl-CoA rather than by the activator, citrate. In the presence of the drug this pro- tective effect is altered. This is difficult to explain in view of the noncompetitive kinetic behavior of acetyl-CoA and CPIB or TPIA. Acetyl-CoA probably can protect the enzyme by means of an enzyme-acetyl-CoA complex with a stable conformation that is less susceptible to heat inactivation than either the proto- merit or the polymeric form of the enzyme.

Citrate-activated enzyme shows a biphasic rate of inactivation with a rapid initial rate, accelerated further by the presence of the drugs; this suggests a rapid denaturation of the nonaggre- gated fraction of the enzyme (Fig. 8).

From the kinetic, physical, and thermodynamic data available, it is suggested that TPIA and CPIB compete with citrate for the same or mutually interacting sites of the enzyme protein. The relative affinities of binding of citrate and TPIA or CPIB to the enzyme indicate that both drugs can displace citrate from the enzyme citrate complex at equimolar concentrations with a free energy gain of about 3240 cal per mole for TPIA and 1950 cal per mole for CPIB.

At the cellular level the contribution of enzyme-citrate and enzyme-drug complexes in the over-all carboxylation reaction will depend on the the relative concentrations of enzyme, citrate,

and drug. The levels of citrate in the cell are not known with certainty. Cleland (39) has suggested that as a general rule enzymes operate in the cell at substrate levels about 0.5 their Km value. Herrera and Freinkel (40) have found citrate levels in the rat liver, under normal feeding conditions, to be 0.38 pmoles per g of fresh liver. On the basis of probable citrate concentration in the liver cells and the relative affinities of citrate and TPIA for the enzyme, we suggest that very low intra- cellular concentrations of free drug would be required for the inhibition to become manifest, by interference with the activa- tion of acetyl-CoA carboxylase. Thus, hypolipidemic drugs may act as auxiliary feedback inhibitors applying a temporary and reversible block in the over-all sequence of fatty acid syn- thesis by inhibiting the rate-limiting carboxylation reaction. No undesirable intermediates are expected to accumulate since acetyl-CoA can be diverted to the Krebs cycle. Granzer and Schachtg have found an increase of acetyl-CoA of about a-fold and a 2- to a-fold increase in acetoacetate and pyruvate in the livers of TPIA-treated rats over that of controls.

TPIA or CPIB have no inhibitory effect on the activity levels of enzymes other than acetyl-CoA carboxylase, which is signifi- cantly lower in drug-treated animals (11). I f no other enzymes are affected by these drugs, only the fatty acids synthesis will be deranged by inhibition of acetyl-CoA carboxylase while the degradation would proceed unaffected, since different enzymes and intermediates are involved in these unidirectional processes. Inhibition of acetyl-CoA carboxylase by TPIA or CPIB should therefore result not only in inhibition of lipogenesis but also, on limited fat-intake, in reduction of lipid stores in the tissues (11). In agreement with that thought, long term treatment with CPIB has achieved certain regressions of lipid deposits (xanthomas) in humans (41). Kokatnur and Malcom (42) have observed little or no hypolipidemic effect of CPIB in the serum of rats receiving a high fat regimen.

In summary, the effectiveness of a lipid-lowering agent, such as TPIA or CPIB, may be related to its inhibitory potency on acetyl-CoA carboxylase. Inhibition of this critical enzyme makes malonyl-CoA unavailable for lipid biosynthesis while degradation proceeds unaffected. This may lead to a decrease not only of circulating lipids, but also of lipid stores in the tissues. It is, therefore, conceivable that treatment with this type of hypolipidemic agents can prevent, arrest, or reverse the athero- sclerotic processes resulting from high levels of circulating lipids.

Aclcnowledgments-The contribution of Mmes W. Marchetto and H. Sylwestrowicz in calculating the regression equations is gratefully acknowledged. Special thanks are due to Dr. H. Heymann for his suggestions in the preparation of the manu- script.

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Michael E. Maragoudakis and Hilda HankinINHIBITION IN VITRO OF RAT ACETYL COENZYME A CARBOXYLASE

On the Mode of Action of Lipid-lowering Agents: V. KINETICS OF THE

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