280

BBA 52205

Bzochtmrcu et Bu)physrcu Acta 876 (1986) 280-287

ElS3W

Acetoacetyl-CoA synthetase specific activity and ~oncen~ati~n in rat tissues

Masaki Ito, Tetsuya Fukui *, Terumi Saito and Kenkichi Tomita

Departmeni of Health Chemcstry. Faculty of Pharmaceutrcul Scrences, Kvoto LJnwersrty. Yoshrda, Sakyo-ku. Kvoto 606 (Jupanj

( Recetved May 20th. 1985)

(Revtsed manuscript received November 11 th, 1985)

Key words: Acetoacetyl-CoA synthetase; Antrbody: Enzyme-lmked tmmunosorbent assay: (Rat tissue)

An antibody against acetoace~i-CoA synthetase purified from rat liver was raised in rabbits. Utilizing the binding of anti~y-antigen complexes to a nitrocellulose membrane, a sensitive enzyme-linked immuno- sorbent assay was developed to estimate the enzyme concentration in rat tissues. The enzyme concentration (pg immunoreactive protein/mg protein) in rat liver cytosol was increased about 3-, 1.8- and 7-fold by feeding rats diets containing 5% cholestyramine, 0.2% ML-236B (compactin), and 5% cholestytamine plus 0.2% ML-236B for 4 days, respectively, and decreased about L&fold by fasting the animals or 1.3-fold by feeding them a diet confining 5% cholesterol. Changes in the enzyme activity were almost parallel to those in the enzyme concentration, suggesting the pbysiologicai role of this enzyme in cholesterol biosynthesis. immunoblotting of the hepatic cytosol also confirmed that the increase in enzyme concentration on cholestyramine and/or ML-236B feeding was due to an increase in an enzyme protein the same as the purified enzyme and not the isozymic protein. Among various rat tissues examined, the concentrations of imm~ologi~lly crossreactive enzyme were higher in lipogenic tissues, such as brain, adipose tissue and liver, than in other tissues. The enzymes in these three tissues were identical in molecular weight determined by gel filtration and immunoblotting.

Accumulated evidence indicates that acetoace- tate is utilized as a precursor of lipids [l] through

activation to acetoacetyl-CoA by acetoacetyl-CoA

synthetase (acetoacetate-CoA ligase, EC 6.2.1.16), which is present in the cytosolic fractions of vari- ous tissues of rats [Z-6], and effectively incorpo- rated into cholesterol and fatty acids synthesized de novo in rat brain [7,8] and liver [9]. This enzyme appears to play an important physiologi-

cal role especially in cholesterol biosynthesis, be-

cause the enzyme activity is enhanced by the administration of hypocholesterolemic agents, such

as cholestyramine [6,10] and/or mevinolin (111. to

rats. In order to investigate the alteration of the

acetoacetyl-CoA synthetase concentration under

various physiologica conditions, in the present study we have investigated this enzyme immuno- logically in various rat tissues.

Materials and Methods

* To whom correspondence should he addressed.

Abbrevtattons: Pipes. 1,4-p~pera~inediethanesulfonlc actd:

SDS. sodium dodecyl sulfate: Hepes. 4-(2-hydroxyethyl)-l-

ptperazmeethanesulfomc actd; PMSF. phenylmethylsulfonyl fluoride.

~u~~ri~~~. The materials used were purchased from the following sources: rats of the Sprague- Dawley strain (SIC. SD), Shizuoka Agricultural Cooperative for Experimental Animals (Hama-

0~5-2760/84/$03.50 qi’ 1986 Elsevier Sctence Pubhshers B.V (Btomedtcal Dtwsmn)

281

matsu, Japan); standard diet (Oriental M), NADf and NADH, Oriental Yeast Co. (Tokyo, Japan); CoA, Kyowa Hakko Co. (Tokyo, Japan); ATP and bovine serum albumin (Fr. V), Sigma Chem- ical Co. (St. Louis, MO, U.S.A.); DEAE-cellulose, Whatman (~aidstone, Kent, U.K.): Ultrogel AcA-44, LKB (Bromma, Sweden); Zeta-Probe, Bio-Rad Laboratories (Richmond, CA, U.S.A.): malate dehydrogenase and citrate synthase from pig heart, Boehringer (Mannheim, F.R.G.); horse- radish peroxidase conjugated with antirabbit IgG, Miles Laboratories (Elkhart, IN, U.S.A.); nitrocel- lulose sheet (Type TM-2, pore size 0.45 pm), Toyo Roshi Co. (Tokyo, Japan); and bovine serum, the Research Foundation for Microbial Diseases of Osaka University (Osaka, Japan). ML-236B (com- pactin) was kindly supplied by Dr. A. Endo (Tokyo Noko University, Tokyo, Japan), and cholestyra- mine by Bristol Banyu (Tokyo, Japan). Lithium acetoacetate was prepared by hydrolysis of ethyl acetoacetate with lithium hydroxide followed by recrystallization [lo]. Other chemicals of reagent grade were purchased from commercial sources.

Enzyme us.says. Acetoacetyl-CoA synthetase ac- tivity in crude extracts of tissues was assayed by following either (a) acetyl-CoA formation [lo], with the use of 0.1 mM CoA and 50 mM Pipes-NaOH (pH 7.0). or(b) NADH formation according to the method of Buckley and Williamson [4] but using high NADH/oxaloacetate ratios as suggested by Pearson [ 121 and Londesborough et al. [13]. The assay mixture (0.75 ml) contained 0.375 pmol lithium acetoacetate, 0.0375 pmol CoA, 0.75 pmol ATP, 3.75 pmol MgC12, 37.5 ymol KC], 5.0 pmol sodium malate, 2.0 pmol NAD+, 0.075 pmol NADH, 0.02 units acetoacetyl-CoA thiolase (EC 2.3.1.9) purified from Zoogloea ramigeru I-16-M [14], 6 units malate dehydrogenase (EC 1.1.1.37) from pig heart mitochondria, 0.88 units citrate synthase (EC 4.1.3.7) from pig heart, 75 pmol Tris-HCl (pH 8.0), and enzyme. The reaction was initiated by the addition of lithium acetoacetate, and the rate of NADH increase was measured at 340 nm and 30°C with a Shimadzu recording sp~trophotometer, model UV-240. One unit of acetoacetyl-CoA synthetase activity was defined as the amount of enzyme required for the conver- sion of 1 pmol of acetoacetate to the CoA ester in 1 min at 30°C. Protein was determined using the

Folin-Ciocalteu reagent with bovine serum al- bumin as the standard [15].

Purification of acetoacetvl-CoA svnthetase. Acetoacetyl-CoA synthetase was purified to elec- trophoretic homogeneity from livers of choles- tyramine-treated rats as described previously [lo].

Antiserum against acetoacety&CoA synthet~se. The purified enzyme (120 pg of protein) dissolved in 1 ml of 10 mM sodium phosphate (pH 7.5) buffered 0.85% saline was emulsified in 1 ml of complete Freund’s adjuvant and injected subcuta- neously twice with an interval of 2 weeks into the back of male white rabbits (about 3 kg). 2 weeks later the enzyme (30 pg) without adjuvant was intravenously injected. Each rabbit was bled 1-2 weeks after the last injection. The blood was al- lowed to clot, and the serum was separated by centrifugation was stored at -2O’C in the pres- ence of 0.1% sodium azide. For immunoblotting and enzyme-linked immunosorbent assays, the IgG fraction of the serum was obtained from the anti- serum by DEAE-cellulose chromatography as de- scribed by Fahey [16]. Double immunodiffusion in agar was performed according to the method of Ouchterlony [17].

Immunoblotting. The method of Gershoni and Palade [18] was used with a slight modification. Protein (5 mg/ml) was dissolved in a mixture of 2% SDS, 5% 2-mercaptoethanol, 10% glycerol and 0.05% Bromophenol blue, followed by heating at 100°C for 5 min. Aliquots (10 ~1) were subjected to electrophoresis on 10% polyacrylamide gel using Laemmli’s system [19]. After the gel had been equilibrated in 192 mM glycine/25 mM Tris (pH 8.3) for 20 min, the protein was transferred to a Zeta-Probe sheet in the same buffer by electro- phoresis at 200 mA for 3 h, the voltage being decreased from 42 to 27 V. The Zeta-Probe sheet was then incubated overnight in a blocking solu- tion, 5% bovine serum albumin in 20 ml of 20 mM Tris/HCl (pH 7.5) - buffered 0.5 M saline (buffer 1). The primary antibody was bound to the sheet on incubation for 6-8 h in 20 ml of IgG (0.1 mg/ml) in the blocking solution. After washing six times each with 40 ml of buffer 1 for 5 min, the sheet was again blocked for 15 min with 10% bovine serum in 20 ml of buffer 1. Then the sheet was reacted with horseradish peroxidase (EC 1.11.1.7) -conjugated anti-rabbit IgG at l/1000

282

dilution in 20 ml of 10% bovine serum in buffer 1

for 2 h. The sheet was finally washed six times

each with 40 ml of buffer 1 for 5 min. Peroxidase

activity was visualized with 4-chloro-1-naphthol,

which forms an insoluble blue product with the

peroxidase reaction, as substrate, as described by

Hawkes et al. [20].

Enzyme-linked immunosorbent assav (ELISA).

The ELISA described below utilized the binding

of antigen-antibody complex to nitrocellulose.

which was used by Jahn et al. [21] for dot-radio

immunobinding assay of synapsin I. The nitrocel-

lulose sheet, on which a grid of 6 X 6 squares

(1 X 1 cm) was drawn, was rinsed for 5 min in

distilled water and then dried. The crude tissue supernatant fraction (lo-12 mg protein/ml) was

diluted with an equal volume of 10 mM Tris-HCl

(pH 7.5) and aliquots (4 ~1) were applied to the

centers of the squares. After drying, the sheet was

fixed for 15 min in 40 ml of 10% acetic acid/25%

isopropanol, and rinsed several times with distilled

water and then with 40 ml of buffer 1 for 5 min.

The unbound surface of the sheet was blocked by

incubation for 1 h with 20 ml of 5% bovine serum

albumin in buffer 1, then it was incubated in 20 ml

of the primary antibody solution (0.05 mg/ml IgG

in buffer 1 containing 10% bovine serum and 0.1%

Triton X-100) overnight. After rinsing five times

each with 40 ml of buffer 1 for 5 min, the sheet

was again blocked with 20 ml of buffer 1 contain-

ing 10% bovine serum for 15 min. The sheet was

incubated for 2 h in horseradish peroxidse-con-

jugated anti-rabbit IgG solution ( x l/1000 dilu-

tion in 20 ml of buffer 1 containing 10% bovine

serum and 0.1% Triton X-100). Finally, it was

washed four times each with 20 ml of buffer 1 containing 0.1% Triton X-100 for 5 min and then

four times more each for 20 min. After each square was cut out separately, the peroxidase ac-

tivity was detected with o-phenylendiamine. which forms a soluble yellow product by peroxidase reac- tion. as substrate by the method of Palfree and Elliott [22].

Gelfiltration. The supernatant fractions of brain and adipose tissue of rats were concentrated with a Micro-ultrafiltration system (model 8 MC, Amicon Co.. Lexington, MA, U.S.A.) to 19.6 and 6.4 mg protein/ml, respectively, while the hepatic super- natant fraction was used without concentration.

These preparations (3 ml) were subjected to gel filtration on an Ultrogel AcA-44 column (1.6 cm

x 70 cm), preequilibrated with 50 mM Tris-HCl

(pH 7.5) containing 5% glycerol and 10 mM 2-

mercaptoethanol.

Dietar?, expenments. Female rats of the

Sprague-Dawley strain (about 6 weeks old) were

fed a standard diet (Oriental M) for 4 days, and

then divided into seven groups. A-G, of 6 rats

each. Group A was fed the standard diet for

another 4 days. Groups B to F were fed the

standard diet supplemented with 2% cholestyra- mine, 5% cholestyramine, 0.2% ML-236B, 5%

cholestyramine plus 0.2% ML-236B and 5% cholesterol. respectively. Group G was fed the

standard diet for 2 days and then fasted for 2

days. The rats were then killed by decapitation

and their livers were excised. All the following

procedures were carried out at 4°C. The livers

were homogenized in 4 vol. of buffer A (5 mM

Hepes-triethanolamine (pH 7.4) containing 250

mM mannitol. 70 mM sucrose, 1 mM EDTA and

10 mM 2-mercaptoethanol) by five strokes in a

Teflon-glass homogenizer. and then centrifuged at 100000 x g for 60 min. The supernatant fraction

obtained was dialyzed against buffer B (10 mM Tris-HCl (pH 7.5) containing 5% glycerol and 10

mM 2-mercaptoethanol) overnight, and then used

for immunological and enzyme activity assays.

Supernatant fractions of various other tissues

(brain, adipose tissue, heart. kidney. spleen and

lung) were similarly prepared from rats fed the

standard diet, except that 0.5 mM PMSF was

added to buffer A.

Results

Specificity of the rabblt antiserum

The rabbit antiserum against rat liver acetoace-

tyl-CoA synthetase inhibited the enzyme activity dose-dependently (Fig. 1). In the Ouchterlony double diffusion assay, the antiserum formed a single continuous precipitin line against the puri- fied enzyme and rat liver cytosol (Fig. 2). No reaction occurred between the antibody and the

acetoacetyl-CoA synthetase purified from Zo- ogloea ramigeru I-16-M [23] (data not shown). The specificity of the antibody was also confirmed by an immunoblotting assay. Band signals at the

283

100

2

550 ?

B

0

i.. . too 200

Antcserum(jJi)

Fig. 1. Inhibition of acetoacetyl-CoA synthetase activity by antiserum. After the purified enzyme (1.1 mu) had been in- cubated with antiserum (0) or control serum (0) in buffer containing SO mM Pipes-NaOH (pH 7.0), 5 mM MgCl, and 50 mM KC1 for 20 min at 30°C. the enzyme reaction was started by the addition of substrates and thiolase, and assayed by the acetyl-CoA formation method. Antiserum and control serum were used after dialysis overnight against phosphate-buffered saline (10 mM sodium phosphate (pH 7.9, 0.85% NaCl).

same position as the purified enzyme were stronger, without the appearance of any other immunoreactive protein bands, with the hepatic cytosols from cholestyramine- or cholestyramine plus ML-236B-treated rats than those from con- trol rats (data not shown).

Standardization of the enzyme-linked immunosor-

bent assay Fig. 3 is a typical titration curve for the purified

(PI (Cf

(A)

Fig. 2. Ouchterlony double diffusion test. The wells contained the crude supematant fraction of control rat liver (1.3 mg) (C), purified acetoacetyl-CoA synthetase (2.2 pg) (P) and anti- serum. (20 ~1) (A), respectively.

0 20 40 60 Enzymefng)

Fig. 3. Standard curve for the enzyme-linked immunosorbent assay. The purified enzyme dissolved m 10 mM Tris-Cl (pH 7.5) containing 2.5 mg/ml bovine serum albumin (0) or 2.5 mg/ml supernatant fraction of control rat liver (0). was spotted onto a nitrocellulose sheet (4 pl each).

enzyme with the newly developed enzyme-linked immunosorbent assay using nitrocellulose mem- brane filters. Prior to spotting, the purified enzyme was diluted in 10 mM Tris-HCI (pH 7.5) contain- ing bovine serum albumin (2.5 mg/ml) to mini- mize undesirable adsorption of the enzyme protein to the glassware. Bovine serum albumin did not affect the titration values. For blocking the un- bound surface of the membrane, 5% bovine serum albumin was used. The antiserum was diluted in 10% bovine serum. Under these conditions the background AdgZnrn value could be reduced to below 0.1. Fig. 3 also shows that the slope of the curve was not affected by the addition of crude hepatic supernatant fraction.

Activity and concentratron of acetoacetyl-CoA svn- thetase in rat liver

Of the two different assay methods employed, the NADH formation assay gave somewhat higher specific activities and standard errors than the acetyl-CoA formation assay (Table I). These high standard errors resulting from high blank values are probably due to the effects of endogenous substrates present in the supernatant fractions, which may affect NADH levels in this assay method based on continuous NADH formation. In contrast, the acetyl-CoA formation assay, which is performed in two steps (conversion of the acetoacetyl-CoA formed to acetyl-CoA by cou- pling with thiolase, and termination of the reaction by heating at 80% and then enzymatical assaying of acetyl-COG) gave only negligible blank values

284

TABLE I

SPECIFIC ACTIVITY AND CONCENTRATION OF ACETOACETYL-CoA SYNTHETASE IN LIVER SUPERNATANT

FRACTIONS OF RATS UNDER VARIOUS DIETARY CONDITIONS

Groups of six female rats of the Sprague-Dawley strain were fed the various diets or fasted as described m the text. The livers were

homogenized, and the supernatant fractions were dialyzed overnight and then assayed for the enzyme activity by two different

methods. Enzyme concentrations (gg lmmunoreactive protein/mg protein) were estimated by the enzyme-linked immunosorbent

assay as described in the text. * P < 0.01 compared to control data.

A. Control

B. 2% Cholestyramine

C. 5% ChoIestyra~ne

D 0.2% ML-236B

E 5% Cholestyramine

+ 0.2% ML-236B

F. 5% Cholesterol

Cr. Fasted

Specific activity

NADH formation acetyl-CoA formation

(munits/mg protein)

0.96 + 0.082 0.86 _t 0.039

3.8 +0.20 2.1 &O&I9

42 *0.35 3.3 -to.20

1.7 *0.53 1.3 +oXl

8.9 +0.41 6.5 +0.30

0.73 kO.12 0.61 *O.ll

0.37 + 0.057 0.30+0.021

Concentration

( pg immunoreac-

tive protein/mg)

0.66 + 0.040

1.6 kO.058

1.8 iO.083

1.2 kO.28

4.5 +0.20

0.49 + 0.041

0.37 f 0.028

Ratio

(munits/pg immuno-

reactive protein)

1.3 50.081

1.7 kO.067 *

1.8 *0.10*

1.2 io.034

1.4 &0.062

1.2 10.13

0.86 * 0.094 *

and lower standard errors than the NADH forma- tion assay. The specific activity (units/mg protein)

of the enzyme was increased about 4-, 1.5 and

S-fold by feeding rats diets containing 5% choles-

tyramine. 0.2% ML-236B and 5% cholestyramine

plus 0.2% ML-236B. respectively. In contrast, the

enzyme specific activity was decreased about 2.9-

fold by fasting rats or 1.6fold by feeding the

animals a 5% cholesterol-supplemented diet. Table

I also shows that changes in the enzyme con-

centration determined by ELISA (pg immuno-

reactive protein/mg protein) were almost parallel

to those in enzyme units/mg protein under these

various dietary conditions. However, the ratios of

the enzyme specific activities to enzyme con-

centrations (munits/~g i~unoreactive protein)

were not the same, they fluctuated slightly from

the control value (1.3) and were lowest (0.86) in

fasted rats, suggesting that the catalytic efficiency

of the enzyme may be altered by drug administra-

tion or by the nutritional state of animals.

I~~un~reuctzu~ty and the enzyme actiui<y in rat tissues

As shown in Table II, the specific enzyme activ-

ity of the cytosol fraction was high in liver, brain

TABLE 11

ACETOACETYL-CoA SYNTHETASE SPECIFIC ACTIVITY AND CONCENTRATION IN CYTOSOL FRACTIONS OF RAT

TISSUES

Tissues from six female rats were combined, homogenized and centrifuged, and then the supematant fractions were dialyzed and

assayed as to the enzyme activity and concentration. Data represent the mean of four determinations f SE. * The enzyme specific

activities and enzyme concentrations were correlated with r = 0.980.

Tissue Specific activity (munits/mg protein)

Concentration Ratio (munits/Fg (pg immunoreactive (immunoreactive

Liver 0.82 + 0.034 *

protein/m@

0.81 f 0.037 *

protein)

1.0 Bram 0.42 +0.005 * 0.33 +0.013 * 1.3 Adipose tissue 1.5 iO.048 * 1.1 +0.057 * 1.4

Lung 0.16 +0.003 * 0.19~0.011 * 0.84 Spleen 0.14 + 0.007 * 0.25 + 0.034 * 0.56 Heart 0.089 f 0.007 * 0.16f0.019 * 0.56 Kidney < 0.05 0.16 f 0.023 i 0.3

285

and adipose tissue, and the concentration of pro-

tein which was cross-reactive with the antibody was also high in these tissues. Lung, spleen, heart and kidney showed relatively low enzyme specific

activities and cross-reactivity with the antiserum.

Although the enzyme specific activities and con-

centrations in various rat tissues were generally

well correlated with the r value of 0.980, the

ratios of enzyme munits/pg immunoreactive pro-

tein were somewhat different among these tissues.

These results suggest that the enzyme, which is

immunologically cross-reactive with the hepatic

acetoacetyl-CoA synthetase and may be present in

different forms, is widely distributed in rat tissues,

especially in lipogenic ones.

Gel filtration and immunoblotting of the supernatant

fractions of rat tissues

The acetoacetyl-CoA synthetase activity present

in the cytosol of liver, brain and adipose tissue was

eluted from an Ultrogel AcA-44 column in almost

the same fraction (Fig. 4), indicating that the

enzymes in these three tissues have similar molecu-

lar weights. Furthermore, immunoblotting in the

presence of SDS of the active fractions obtained

30 40 50 60 70 0

::,- IrJ *&];I5 30 40 50 60 70

Fraction number

Fig. 4. Gel filtration of supernatant fractions of rat tissues.

105 000 x g 60 min supernatant fractions of liver (A), brain (B)

and adipose tissue (C) were subjected to gel filtration on an

Ultrogel AcA-44 column. 0, absorbance at 280 nm; 0,

acetoacetyl-CoA synthetase activity.

Fig. 5. Immunoblotting of the partially purified acetoacetyl-CoA

synthetase from rat tissues. The active fractions on Ultrogel

AcA-44 column chromatography were denatured by mixing

with an equal volume of 20% trichloroacetic acid, and then

centrifuged at 3000 rpm for 20 min. The precipitate was

washed with ethanol/diethyl ether (1: 1) several times, fol-

lowed by the addition of 2% SDS and 5% 2-mercaptoethanol.

After boiling until the precipitate had been dissolved, each

sample was subjected to SDS-polyacrylamide gel electrophore-

sis and immunoblotting. Lanes 1 and 5, purified enzyme (38 ng

each); lane, 2, liver (44 pg); lane 3. brain (45 pg); lane 4,

adipose tissue (22 pg).

on Ultrogel AcA-44 gel filtration showed that the immunoreactive enzymes in these tissues are simi-

lar in subunit structure (Fig. 5).

Discussion

A highly sensitive enzyme-linked immunosor- bent assay, newly developed in the present study,

for rat liver acetoacetyl-CoA synthetase, made it possible to measure the enzyme concentration in the cytosol of rat tissues directly and indepen-

dently of the assayable enzyme activity. Our re-

sults (Table I) show that fluctuations of the en-

zyme activity in rat liver under various dietary

conditions are mostly accounted for by changes in the enzyme concentration.

The greatest increase in the liver enzyme con- centration was observed in the rats which were fed the diet supplemented with two different hypo- cholesterolemic agents, cholestyramine, an anion exchanger and accelerator of bile acid excretion

286

[24], and ML-236B, a competitive inhibitor of hydroxymethylglutaryl-CoA reductase (EC 1.1.1.88), the rate-limiting enzyme in cholesterol biosynthesis 1251. Similar additive effects of these two agents on the concentration of acetoacetyl- CoA synthetase demonstrated in the present study as well as those reported for hydroxymethyl- glutaryl-CoA reductase 1261 in rat liver further suggest the physiological role of, this synthetase in cholesterol biosynthesis. The enzyme concentra- tion was decreased significantly by fasting rats, and only slightly by feeding the animals a high (5%) cholesterol diet. Therefore, effect of dietary cholesterol, as compared with that of restricted food intake, on the enzyme concentration might be of questionable physiological significance.

In these experiments, the ratios of the enzyme activity to enzyme concentration (munits/pg im- munoreactive protein) varied from, 0.86 to 1.8, being lowest in fasted rats and highest in choles- tyramine-treated rats. This is probably due to changes in the catalytic capacity of the enzyme as in the case of hydroxymethylglutaryl-CoA re- ductase, the activity of which was reported to be regulated by phosphorylation and dephosphoryl- ation of the enzyme molecule 1271, in addition to changes in enzyme concentration. Scallen et al. [28] reported that the total amount of liver hy- droxymethylglutaryl-CoA reductase was higher in cholestyramine-treated rats than in untreated rats, and also that the enzyme from cholestyramine- treated rats was catalytically more active than that from untreated rats 1281. Further investigation is required to elucidate whether or not the activity of acetoacetyl-CoA synthetase is similarly regulated.

Our results (Table II) also indicate that the acetoacetyl-CoA synthetase activity and the pro- teins which are cross-reactive with the antiserum against the rat liver enzyme are widely distributed in various lipogenic tissues other than the liver. Probably there is a variety of enzyme forms in different tissues which are cross-reactive with the antibody for the liver enzyme and which have identical native and subunit structures (Figs. 4 and 5) because the role of acetoacetyl-CoA syn- thetase may differ from one tissue to another.

Acetoacetyl-CoA synthetase in liver [4} and brain [29] was shown to participate mainly in cholesterol biosynthesis, while the enzyme in

adipose tissue [9], lung [8] and mammary gland [6] is apparently involved in fatty acid biosynthesis. It was also reported that acetoacetate may be utilized for acetylcholine synthesis in the cytosolic com- partment of rat brain 1301. The metabolism of acetoacetate in various rat tissues certainly re- quires further investigation.

We thank Y. Ueha for her skillful secretarial assistance. This work was supported in part by grants from the Ministry of Education, Science and Culture of Japan.

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