Conversion of acetoacetyl-coenzyme a into acetoacetate in subcellular liver preparations: Differentiation between the pathways of acetoacetate formation and their localization in the rat liver cell

CONVERSION ACETOACETATE

OF ACETOACETYL-COENZYME A INTO IN SUBCELLULAR LIVER PREPARATIONS:

DIFFERENTIATION BETWEEN THE PATHWAYS OF ACETOACETATE FORMATION AND THEIR LOCALIZATION IN THE RAT LIVER CELL IDS MULDER Laboratory

of Veterinary

AND

G. VAN

SIMON

DEN

Biochemistry, State University Utrecht, The Netherlands (Received

BERGH of Utrecht,

Biltstraat

172,

1 October 1976)

Abstract--- I. Both the direct enzymatic hydrolysis of acetoacetyl_CoA and the 3-hydroxy-3-methylglutaryl_CoA (HMG-CoA) pathway occur in mitochondria and cytosol prepared from rat liver. 2. Moreover, an appreciable non-enzymatic hydrolysis of acetoacetyl-CoA occurs. 3. An independant radioactive assay showed, that the HMG-CoA pathway is completely inhibited

by iodoacetamide. Iodoacetamide may be used to differentiate between the two enzymatic pathways. 4. The four systems for acetoacetate formation (2 pathways in each cell compartment) have about equal activities under our in vitro conditions. 5. Chanees in the activities of each of these systems capacity of the liver cell, e.g. during ketosis. _

may contribute

to alterations

of the ketogenic

homogenates, which was mainly caused by the deacylase. Sauer & Erfle (1966) established HMGXoA pathway activity in guinea-pig liver cytosol. Williamson et al. (1968) found 8-20x of the HMG-CoA pathway activity and most of the deacylase in the cytosol of rat liver cells. Recently, Clinkenbeard et al. (1975a) denied the presence of HMG-CoA lyase (EC 4.1.3.4), one of the enzymes of the HMG-CoA pathway, in the cytosol of rat and chicken liver, whereas a few years earlier Allred (1973) found about half of the activity of both HMG-CoA synthase (EC 4.1.3.5) and HMG-CoA lyase in the cytosol of chicken liver cells. In this paper it will be shown that both pathways for acetoacetate formation are present in the cytosol as well as in the mitochondria of rat liver and that

INTRODUCXON

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) pathway for the formation of acetoacetate from acetoacetyl
The

1.,,,..gz =I cn -CO-scoA

,i5====r

HiCO-Ct$-CO-SCOA

d=

* Ico*Jc ?H2-COOH H,-C-CHiCO-SW OH

r-l

HMG-CO/+

I

Fig. 1. Acetoacetate formation from acetoacetyl-CoA in liver cells. Enzymes: thiolase (acetyl
IDS MULDER AND SIMON G. VAN

228

the activities of the 4 systems are of the same order of magnitude, at least under our in vitro conditions. Parts of this work have been preliminarily reported in abstract form (Mulder & Van Rheenen, 1971; Mulder, 1972a). MATERIALS AND METHODS Chemicals

Enzymes and coenzymes were obtained from Boehringer, Mannheim, Germany; Sephadex from Pharmacia Fine Chemicals. Uppsala, Sweden; Ecteola cellulose from Merck. Darmstadt, Germany; [I-‘“Cland [2-14C]acetic anhydride from New England Nuclear, Boston, MA, U.S.A. All other chemicals were of the purest quality commercially available. AcetoacetylXoA was synthesized from diketene and CoA (Lynen, 1953) and purified by means of Ecteola cellulose column chromatography in order to remove acetoacetyl glutathione (Sauer & Exile, 1966). Acetoacetyl carnitine was a kind gift from Dr. A. van Tol, Erasmus University, Rotterdam. [2-‘4C]acetylpCoA was prepared from [2-‘4C]acetic anhydride and CoA (Ochoa, 1955). The contents of an ampoule of [2-“%]acetic anhydride (5 mg, 250 &i) were taken up in 1.5 ml of sodium-dried benzene. Half of this solution was added to an ice-cold solution of 38 mg CoA and 38 mg KHCO, in 3.8 ml of water in a stoppered test tube. After 30 min at 0°C with occasional shaking, the benzene layer was removed carefully and the other half of the acetic anhydride solution was added. After another 30min at 0°C with occasional shaking, the benzene layer was removed again. Addition of 0.75 ml of 2 M acetic acid, shaking with 3 ml of ether and discarding the ether removed most of the excess radioactivity which must have been present as acetate after the reaction. The addition of acetic acid was repeated once, the ether extraction thrice. Traces of ether were removed by blowing pure nitrogen over the solution, which contained at this stage 35 pmole of [2-“YZ]acetyl-CoA. Removal of acetic acid, which might still contain some radioactivity, was carried out by applying 0.5-ml portions of the solution to a 25 x 1.6 cm Sephadex-GlO column which was then eluted with distilled water. The peaks of acetate and of acetylCoA were fairly well separated. Acetyl-CoA solutions could be kept at pH 6 and -20°C for over 6 months. Occasionally up to 10% decomposition occurred, in which case the Sephadex purification was repeated. Unlabelled acetyl-CoA and [l-‘YJ]acetyl-CoA were prepared in the same way as described for [2-‘%Z]acetylCoA. in order to get fully comparable preparations. Fractionation

DEN

BERGH

To check possible contamination of the cytosol with mitochondrial material, the activity was determined of the mitochondrial marker enzymes 3-hydroxybutyrate dehydrogenase (EC I. 1.1.30)and glutamate dehydrogenase (EC I .4.1.2). Virtually no 3-hydroxybutyrate dehydrogenase activity could be detected in the cytosol. The cytosolic activity of glutamate dehydrogenase, calculated per mg of protein, was less than 1.3% of that of the mitochondrial activity. Although the bulk of the microsomes had been removed from the cytosol by centrifugation. traces of the microsomal fraction may still have been present.

Protein determinations were performed by the biuret method as described by Cleland and Slater (1953). Acetoacetyl-CoA and acetyl-CoA were assayed according to the methods of Decker (1970; see, however, Mulder. 1972h) and acetoacetate according to the method of Mellanby and Williamson (1970). Reactiorl

conditions

Incubations were carried out at 37°C and contained in a final volume of 1.5 ml: l-2 mg cell-fraction protein per ml, 50 mM Tris+HCl buffer, pH 7.4, 15 mM KCI, 2 mM EDTA, 5 mM MgCl, and, if indicated, 5 mM iodoacetamide. After 20min of pre-incubation, the reactions were started by the addition of acetoacetyl-CoA and acetylCoA to a final concentration of 250 and 25 PM. respectively. Reactions were stopped after 15 min by the addition of 0.15 ml 307” (w/v) HCIO,. Then acetoacetyl-CoA, acetoacetate and acetyl_CoA were assayed spectrophotometritally in the deproteinized. neutralized samples. In some experiments the added acetyl-CoA was labelled. It contained approx 0.2 PCi of [I-“Clor [2-14C]acetylm CoA. These radioactive incubations were carried out in open Thunberg tubes. After stopping the reactions with HCIO,, a small magnetic stirring bar and a glass vial containing a piece of filter paper impregnated with 0.1 ml IO M KOH were quickly added (Fig. 2) and the tubes were provided immediately with a hollow stopper containing 0.3 ml of a mixture of 2 ml hydrazine hydrate and 5 ml 88% (w/w) lactic acid (cf. Mayes & Felts, 1967). Before closing the tubes definitively, the inside pressure was lowered to approx 0.75atm in order to prevent leakage of radioactivity. The lower parts of the tubes were then heated to

qf’ liwr

Cell fractions were prepared from livers of 175-g female Wistar random rats. The liver was excised and placed in ice-cold 0.25 M sucrose immediately after bleeding the rat. After weighing the liver was minced and homogenized in 10 vol of 0.25 M sucrose in a Potter-Elvehjem homogenizer with a loosely fitting teflon pestle. Differential centrifugation was carried out at 0°C. After 5min at 775g, the sediment was discarded. Centrifugation of the supernatant for 10 min at 4500 g gave the crude mitochondrial fraction as a sediment. The supernatant of the foregoing centrifugation was centrifuged twice during 30 min at 48,000 g. Each time the fluffy layer and the sediment were discarded. The last supernatant was called the cytosol. The crude mitochondrial fraction was washed by suspending it in 7.5 vol of 0.25 M sucrose and centrifugation during 10 min at 12,600 g. The supernatant was discarded and the sedimented mitochondria were resuspended in a known volume of 0.25 M sucrose. The membranes were disrupted by 3 times freezing and thawing.

I lcm

b

Fig. 2. Glass vessel for experiments with radioactive acetyl-CoA. Decarboxylation of acetoacetate is brought about by heating the lower part of the tube to 60°C: acetone dlfhlses into the hydrazine solution: (a) 0.3 ml hydrazine lactate; (b) filter paper with 0.1 ml 10 M KOH; (c) 1.5 ml incubation mixture plus 0.15 ml 30:/i (w/v) HC104; (d) stirring rod.

Ketogenesis

in liver cell fractions

6tYC with gentle stirring during 40hr in order to decarboxylate the acetoacetate formed during the incubation and to enable the resulting acetone to diffuse into the hydrazine lactate. Control experiments proved that with this method over 964; of the radioactivity of added [2-‘“Clacetoacetate was recovered in the hydrazine-lactate solution in the hollow stopper and that acid radioactive products like CO, and acetate were held back almost completely by the KOH-filter paper. The contents of the hollow stopper were rinsed with 6 ml of water in several portions into a vial containing 10 ml of a commercial scintillation liquid. like Unisolve (Koch-Light Laboratories, Colnbrook, U.K.) or Instagel (Packard Instrument Co.. Downers Grove. IL. IJ.S.A.), shaken well and counted in a liquid scintillation counter. Blank and standard vials also contained 0.3 ml of hydrazine lactate and 6 ml of water. Parallel to each series of radioactive incubations, a corresponding series of incubations was carried out in which an equal amount of unlabelled acetyllCoA was substituted for the labelted acetyl--CoA, in order to determine the amounts of substrates and products enzymatically.

Contrary sum

229

to the first process,

Z[acetoacetyl-CoA]

in the second

+ Z[acetoacetate]

is no longer constant. CoA passing through ecule disappears, so:

process

+ [acetyl-CoA]

In fact. for each molecule of acetyll this second process, one such mol-

A acetyllCoA (4)

“’ = [acetyl-CoAj’

in which Aacetyl-CoA can be calculated from measurements of [acetoacetyl-CoA], [acetoacetate] and [acetyl-CoA] at the start (t = 0) and at the end (t = 15) of the incubations: AacetyllCoA

= (Z[acetoacetyllCoA]

+ Z[acetoacetate] + [acetyl-CoA]

- (2[acetoacety~CoA]

x, = 2.3 log ~

cacetyl-CoA],,,

= i[acetylCoA],=O + i[acetyi-CoA],=

15 + $Aacetyl-CoA.

(6)

After K and Z have been measured directly and x2 has been calculated from equation (4). xi can be calculated from equation (3) by trial and error or by computer. The activity of the HMG-CoA pathway follows then from equation (2).

RESULTS AND DISCUSSION

Incubations

H = x,. [acetyl-CoA].

(2)

However. the assumptions mentioned above are not always correct. Cleavage of acetoacetyl-CoA by thiolase (acetylCoA acetyltransferase, EC 2.3.1.9) which is present in both celi fractions ~Williamson et al., 1968; ~iddleton, 1973). generates unlabelled acetyl-CoA. At the same time acetvfCoA is decomposed. probably by enzymatic (Miziorko- et (II., 1975) or non-enzymatic hvdrolvsis. The sum of this generation and decomposition of- acetyl-CoA may be regarded as a second process, beside the HMG-CoA pathway. diluting the radioactivity of the acetyl-CoA. Therefore. equation (1) should be improved to: K s, + X2’ s in which .x2 is the fraction by this second process.

(5)

Spontaneous hydrolysis of acetoacetyl--CoA

in which Y, is the fraction of the acetyl-CoA which is ‘exchanged by’ or ‘gone through’ the HMG-CoA pool, K is the radioactivity (disjmin) of the added [Z-i4C]acetylCoA. and 2 is the radioactivity (disimin) of the resulting acetone. The total amount of acctyl&CoA which has diluted the radioactive pool is equal to the diluting fraction multiplied by the concentration of acetyl-CoA. If our assumptions are correct, this equals the amount of acetoacetate (H) generated by the HMG-CoA pathway. so:

K-Z_--

is.

In a number of cases the size of the acetyl-CoA pool was not constant during the incubations, In those cases [acetyl-CoA] in equations (2) and (4) has been replaced by an average [acetyl-CoA], which we assumed to be:

K

K - 2’

Xi + Xl = 2.3 log

), = 0

+ Z[acetoacetate]

+ [acetyl-CoA]),=

In experiments in which acetoacetate is formed from unlabelled acetoacetyl-CoA in the presence of [2-“‘ClacetylCoA, the contribution of the HMG-CoA pathway can be calculated from the radioactivity of the acetoacetate. which is measured after its decarboxylation to acetone (see above). This calculation is simple if we assume the size of the acetyllCoA pool to be constant and the HMG-CoA pathway to be the only process that dilutes its radioactivity. As the formation of each molecule of unlabelled acetyl-CoA via the HMG-CoA pathway is accompanied by the formation of one molecule of radioactive acetoacetate, the dilution of the acetyl-CoA poet may be expressed by the exponential equation:

the

(3)

,

of the acetyl-CoA

exchanged

of acetoacetyl-CoA

without

the addi-

tion of any enzyme or cell preparation result in a considerable hydrolysis (Fig. 3a). Generally the decrease in the acetoacetyl-CoA concentration corresponds with the increase in the acetoacetate concentration, as is to be expected. This non-enzymatic hydrolysis is enhanced by the addition of glutathione (GSH), the effect being roughly parallcl to the GSH concentration. Non-enzymatic deacylation of acetoacetyl-CoA. both in the absence and in the presence of GSH, is faster in a phosphate buffer than in a Tris buffer of the same pH and concentration (Fig. 3b). This finding led us to use Tris buffer in incubations for studying the enzymatic conversion of acetoacetyl-CoA to acetoacetate. From the observation that the stimulatory effect of GSH on the non-enzymatic hydrolysis of a~etoacetyl-boa is inhibits by iodoacetamide (Fig. 3c), we conclude that the SW group is essential for this effect. Indeed it was found that cysteine is active as a catalyst too (Table 1). Sometimes a discrepancy is found between the disappearance of acetoacetyl_CoA and the formation of acetoacetate when cataIysts are present. Perhaps thioi compounds act as catalysts by tem~rarily taking over the acetoacetyl group from acetoacetyl-CoA. a reaction already found by Drummond and Stern ( lY61). The consecutive release of the acetoacetyl group might then be the rate-limiting reaction. Not shown in the table are the slight inhibition of the non-enzymatic hydrolysis of acetoacetyl&CoA by 2 mM EDTA

230

IDS

MULDER

AND

SIMON

G.

VAN

DEN

BERGH

- IAA.GSH ;!A - GSH , GSH ;%A ’ IAA.GSH

time

(min)

Fig. 3. Non-enzymatic deacylation of acetoacetyl-CoA. Reactions were started after 20 min pre-incubation by the addition of approx 120 PM acetoacetyl-CoA. They were carried out at 37°C. l , AcetoacetylL CoA concentrations; 0, acetoacetate concentrations. (a) Incubations contained 50 mM phosphate buffer, pH 7.4, 2.5 mM MgClz and 0, 0.56 or 1.1 mM GSH. (b) Incubations contained 60 mM phosphate buffer, pH 7.4 or 60 mM Tris buffer, pH 7.4, 3 mM MgC12 and, if indicated, 1.3 mM GSH. (c) Incuba-

tions contained 60mM phosphate buffer, pH 7.4, 2.5mM MgC12 and, if indicated, 1.25 mM GSH and 6 mM iodoacetamide (IAA). and the lack of effect of MgCl, in the millimolar range. Both EDTA and MgCl, are included in the standard incubation medium for our enzyme studies. Also not shown is the very slight stimulatory effect of protein solutions, which was enhanced if these solutions had been heated to boiling for some time. Boiled cell fractions of rat liver also had a stimulatory effect on the hydrolysis of acetoacetyl-CoA, as shown for the cytosol in Table 2. In the presence of boiled cytosol there is a discrepance again between the decrease in acetoacetylKoA and the increase in acetoacetate concentration. Addition of iodoacetamide almost completely abolishes the effect of the boiled cytosol, so it is likely that most of the catalysing effect of boiled cytosol is due to thiol compounds. As we found that the non-enzymatic deacylation of acetoacetyl-CoA, (a) could make up an important part of the total acetoacetate formation in our incubations, (b) varied widely, and (c) was influenced by many substances that were present in our incubations, we concluded that in our enzyme studies in every experiment and for each variation in incubation conditions there should be a comparable blank, containing the corresponding boiled cell fraction. Moreover, Table

1. Non-enzymatic

for each incubation, including the blanks, there should be a corresponding zero-time control. Nonenzymatic deacylation of acetoacetylKoA can then be determined from the blank and its zero-time control, taking into account that in the corresponding enzymatic incubation the average acetoacetyl-CoA concentration is usually lower than in the blank and so the non-enzymatic deacylation is lower too, as non-enzymatic reaction rates are proportional to the concentration of the reactants. of acetoacetate

Enzymatic formation

Contrary to other protein solutions, unboiled rat liver cell fractions had a greater catalysing effect than the boiled cell fractions, as shown for the cytosol in Table 2. Iodoacetamide decreased thk catalytic effect of unboiled cell fractions, but not to the level of boiled cell fractions (Table 3). The conclusion must be that the cell fractions contain 2 heat-labile catalysts for the formation of acetoacetate from acetoacetyl-CoA, one of which is inhibited by iodoacetamide. It is stated in the literature that the HMG-CoA pathway is inhibited by iodoacetamide at the concentration used in our experiments (Burch & Triantafillou, 1968;

hydrolysis

of acetoacetyl-CoA

Additions

Disappearance of acetoacetyl-CoA (nmole/ml)

Formation of acetoacetate (nmole/ml)

None Iodoacetamide GSH GSH + iodoacetamide Cysteine Cysteine + iodoacetamide

45 57 98 45 127 60

43 54 77 38 65 47

The incubations contained 60mM phosphate buffer, pH 7.4 and 6 mM MgC12. Additions: 6mM iodoacetamide, 1.3 mM GSH and 1.3 mM cysteine. After 20min pre-incubation, the reactions were started by the addition of 174 PM acetoacetyl-CoA and ran for 40min at 37°C.

Ketogenesis Table

2. Effect of rat liver cytosol

on the deacylation Experiment

None Iodoacetamide Boiled cytosol Boiled cytosol Cytosol

Experiment

1

2 Formation of acetoacetate (nmole/ml)

Disappearance of acetoacetyl-CoA (nmole/ml)

91 91 93 90 153

92 91 124 100 217

+ iodoacetamide

of acetoacetylCoA

Formation of acetoacetate (nmole/ml)

Disappearance of acetoacetyl-CoA (nmole/ml)

Additions

231

in liver cell fractions

69 62 102 71 273

56 54 73 60 214

The incubations contained 50 mM phosphate buffer, pH 7.4 (experiment I) or 50 mM Tris buffer, pH 7.4 (experiment 2) 5 mM MgCl, and 2 mM EDTA. Additions: rat liver cytosol, 2mg protein per ml, 5 mM iodoacetamide. After 20 min pre-incubation, the reactions were started by the addition of 247 (experiment 1) or 295 PM acetoacetyl-CoA (experiment 2) and ran for 120min at 37°C. The boiled cytosol was heated to 95°C for 30min.

Williamson et al., 1968; Stewart & Rudney, 1966). If this is correct (see next section). the heat-labile catalyst which is not inhibited by iodoacetamide should be acetoacetyl-CoA deacylase. Differentiation between the two enzymutic pathways of acetoacetate formation In order to check whether used to differentiate between

iodoacetamide the contributions

can be of the

HMG-CoA pathway and the deacylase to the total enzymatic formation of acetoacetate, experiments have been carried out with [2-r4C]acetyl-CoA (25 PM) and unlabelled acetoacetylCoA (250 PM) (Fig. 4). In such experiments, radioactivity will be incorporated into acetoacetate if this compound is formed via the HMGCoA pathway (Lynen et al., 1958; Rudney & Ferguson, 1959). The deacylase will produce unlabelled acetoacetate, provided that no labelled acetoacetyllCoA is formed through the action of thiolase operating in the synthetic direction (see below). The radioactivity of [2-“Vlacetoacetate can be assayed specifically after decarboxylation of this compound to acetone (see Methods section). Table

3. Calculation

of HMGCoA

pathway

From the experiment shown in Table 3, it is clear that 5 mM-iodoacetamide completely blocks the HMGCoA pathway. In incubations with iodoacetamide, both with cytosol and with mitochondria, the radioactivity of the recovered acetone was not significantly higher than in the blank. From these results it may be concluded that the heat-labile formation of acetoacetate in the presence of iodoacetamide is catalysed by the deacylase. It is also clear that in the presence of iodoacetamide no acetoacetylKoA is formed from acetylCoA. This is to be expected, since it is known that thiolase is also inhibited by iodoacetamide (Stewart & Rudney, 1966). Thiolase operating in the synthetic direction would have produced labelled acetoacetyl-CoA, from which labelled acetoacetate would have resulted through enzymatic or non-enzymatic deacylation, the latter being especially rapid during the 40-hr heating period at 60°C in an acid medium. In the absence of iodoacetamide, acetoacetate is rapidly labelled by both cell fractions (Table 3). To conclude from these results that the HMGCoA pathway is operating and to calculate its rate from the

from radioactivity iodoacetamide

measurements

Formation

Fraction Boiled cytosol Boiled cytosol Cytosol Cytosol Boiled mitochondria Boiled mitochondria Mitochondria Mitochondria

Iodoacetamide (5 mM)

Disappearance of acetoacetyl-CoA (nmole)

+ _ + _

9 13 53 234

+

4

_ + _

12 42 187

Formation of acetyl-CoA (nmole)

Total (nmole)

Nonenzymatic (nmole)

-6 197

16 18 15 12

-1

12

12

7 -2 147

26 52 98

26 12 20

7

the

presence

and

absence

of

of acetoacetate

16 18 53 83

-2

in

Enzymatic (nmole)

Radioactivity in acetone (dis/min)

HMG-CoA pathway (nmole)

38 71

60 132 12 523

0.06 33

40 78

73 98 16 683

0.05 28

Incubations and pre-incubations were carried out as described in the Methods section. The 25 PM [2-‘4C]acetyl-CoA added contained 95,075 dis/min. Incubations (1.5 ml) contained 2.18 (cytosol) or 1.97 (mitochondria) mg protein and at time = 0, 352 (cytosol) or 350 (mitochondria) nmole acetoacetyl-CoA. Boiled cell fractions were heated to 95°C for 30min.

232

IDS MULDER AND SIMON G. VAN DEN BERGH

-CO-&i. eoe-----

j’ cn

rYilkJ2 cn

-co-en-coon

Fig. 4. Acetoacetate formation from labelled acetylLCoA and unlabelled acetoacetyl-CoA, Incorporation and detection of label. radioactivity measured in the resulting acetone, it should be demonstrated that, also in the absence of iodoacetamide, no synthesis of acetoacetyl-CoA from acetyl
=

F? acetoacetyl-CoA

CacetoaceQ-CoAl. CCoAl

+ CoA. =

2x

1o_5

[acetyl_CoA]’ (Stern et al., 1953) makes the synthetic route very slow when appreciable amounts of acetoacetyl-CoA are present, as in our incubations. Secondly, from the results published by Gehring et al. (1968) it can be concluded that the exchange of acetyl-groups between acetyl_CoA and acetoacetyl_CoA is at least 3000 times slower than the thiolytic cleavage of acetoacetyl-CoA. (These authors indicate that 1 enzyme unit of thiolase splits 21,200 nmole of acetoacetylL CoA per min at 20°C whereas it exchanges 1.Onmole of acetylKoA per min at 0°C.) Nevertheless. we investigated the extent to which incorporation of radio-

activity from acetylKoA into acetoacetyl-CoA occurred under our conditions. Synthesis of acetoacetyl-CoA from acetyl_CoA can be measured if [1-14C]acetylkCoA is used in the incubation of rat liver cell fractions with unlabelled acetoacetyl-CoA (Fig. 4). Under these conditions the HMG-CoA pathway only causes carbon atom 1 of acetoacetate to become labelled, which gives no label in acetone. Synthesis of acetoacetylLCoA by thiolase would cause label in both carbon atoms 1 and 3 of acetoacetate, which would result in labelled acetone. Parallel experiments were performed in which equal amounts of [ I-‘4C]- or [2-14C]acetyl--CoA were used. With [ l-‘4C]acetylLCoA the resulting radioactivity in acetone was only 2.8% and 2.00,b of that with [2-14C]acetylLCoA for mitochondria and cytosol, respectively. Therefore, it may be concluded that under our conditions synthesis of acetoacetyl-CoA by thiolase is negligible. The exchange of acetyl groups by thiolase was checked by measuring the radioactivity of acetoacetyl-CoA after the usual incubation of cell fractions with unlabelled acetoacetyl-CoA and [2-‘4C]acetylL CoA. After the addition of HClO,, the deproteinized samples were neutralized with KOH and centrifuged. In an aliquot of the supernatant (1.5 ml, containing 64”; of the original incubation) the excess (radioactive) acetylLCoA was converted into citrate. In a total volume of 2 ml this aliquot was incubated for 45 min at X’C with 0. I M Tris buffer, pH 8.1. 2 mM oxaloacetate and 0.13 unit citrate synthase (EC 4.1.3.7) per ml. CoA esters were then separated from compounds with a lower molecular weight on a Sephadex G IO column as described in the Methods section. Unlabelled acetoacetyl-CoA was added to the sample in order to facilitate the enzymatic detection of the acetoacetyl-CoA peak in the eluate. As the amount of radioactivity in these acetoacetylLCoA peaks was negligible, we conclude that the exchange of radioactive acetyl groups fro’m [2-14C]acetylLCoA into acetoacetylLCoA under our conditions is insignificant. Culculation of the contribution of the HMG-CoA wuy jiiom radiouctivity rneasurrments

Since both the synthesis of acetoacetyl-CoA from acetyl-CoA and the exchange of acetyl groups between acetylLCoA and acetoacetylLCoA may be neglected, the rate of the HMG-CoA pathway may be calculated from the radioactivity recovered in the

Table 4. Comparison of the two methods to determine the contributions the two enzymatic pathways of acetoacetate formation

Fraction

Method

Cytosol Cytosol Mitochondria Mitochondria

iodoacetamide radioactive iodoacetamide radioactive

puth-

HMGCoA pathway (nmole) 33 33 38 28

of

Deacylase

(nmole) 38 38 40 50

The data given in this table are calculated from the experiment shown in Table 3. In lines 1 and 3 the contribution of the HMG--CoA pathway is calculated as the iodoacetamide-sensitive part of the total acctoacetate formation. In lines 2 and 4 it is calculated by the radioactive method, described in the Methods section.

733

KetoQencsis in liver ccl) fractions Tahlc 5. Rates of acctoacetate formation by rat liver cell fractions “..ll.” .~__-

---“--

Fractwn

ISMG-CoA pathway (radioactive method) (nrn~)l~~rninper mg protein)

(radioactive method) (~rn~le~rnin per mg protein)

C’stosol Mitochondria

0.79 _ri:0.09 I .03 t 11.22

1.38 2 0.10 131 I 03

The

.._”

_.- -~

LhC$W

__“~

“-

I.I

Deacg last (with i~?~i~;lcet~irnid~j (nrn~~lelrni~lpr mg protein)

~.

I.16 * 0.07 0.21

I..31 I

_.“_“--

values given III the table are means of X lcytosol) of 6 (mitochondria) expermlcnts with the standard error

of the man. The rate of the deac~lae has dctermincd either as the total enzymatic a&it> mmus the calculated rate of the HMGCoA pathway (rad&ctiw method) or as the total enzymatic activity in the presenceof i{~d~acetamide. acctonc. as indicated in the Methods s&on. In the last column of Table 3 the results of such calculations arc shown. It is clear that we now have two possibilities to determine the contributions of the two enqmatlc pathways to the total ~icet~~~~cetat~ f~~rmation. The contributi(~n of the dcacylase may be deduced from the rate of acetoacetate formation in the presence of iodoacctamide, whereas the rate of the HMG-CoA pathway can be calculated from radioactivity measurements. In Table 3 the results of both types of determinations are compared for the experiment shown in Table 3. From the fair agrccmcnt between the results obtained with the two methods. WC tcntativcl! conclude that our method of calculating the rate of the HMMG Co/\ pathway is correct and that the dcqlasc is not appreciably inhibited by 5 mM iodoacetamide. In Table 5 the results of a number of experiments arc compiled. Althoilgh the differences between the rates of the deacylsse. as determined by both methods are not fully significant. the values from the direct measurements in the presence of iodoacetamide tend to bc the lower ones. This could meati that the deacylHSC is slightly inhibited by 5 m.M iodoacctamide. ‘in accordance with findings of Hurch and Wcrtheim (1973). However, on the basis of additi~lial experimental evidence, in the a~o~i~~t~ing paper a more plausible explanation will be developed.

From Table 5 it is clear that in our experiments the mte of acetoacetate formation from acetoacetyl-<‘oA in both cell c~~m~artments is of the same order of magnitude and that the contribution of the two enqmatic pathways is comparable, with the dcacylase tending to be the most active. These results may. howcvcr. not reflect the situation itr riro. As indicated below. the activity of the HMG-CoA pathwab is prc+abl? lltlder-estini~~ted in our cxperinient~, 6nco the lcvcl of added acetoacctvl Co,4 will lead to substrate inhibition of fiMG i’oA synthasc (Stewart 6t Rudney, 1966). WC felt. however. that conclusions could not be drawn from results obtained with other, known methods of mqmc assay. Measurement of the HMG. CoA pathway lvith a s\;stcm in which acetoacetyl-CoA is being generated hg a feeding ~lech~~nisrn (e.g. through the reaction scqusnw: acetyl phosphate, acetyl CoA. acctoacetyl C‘oA ; Williamson 6’1 ol.. I%%) makes differentiation between this pathway and acetoacctyl CoA deac!lase impossible. In such measurements the resulting figures include the dencylase and the non-cnqmatio hydrolysis of acetoacctyl CoA. Determination of the deacylnse by

measuring the drop in absorbance at 3 I3 nm has the disadvantage of being rather unspecific. Figures obtained by such an assal may include some thiolase activity. which must start as soon as some CoA is set free h> the dencylasc itself or b> non-enzymatic l+rolysis, and even some HMG- C‘oA pathway acttvlty. which starts as soon as acetyl-.Coi\ is generated by thiolase. Therefore. we decided for the experimental set-up as described in the Methods section. As all our experiments were performed under completclq comparable conditions. the draw-backs mentioned above against our set-up can in no way interfere with the f~~llo~~~in~ conclusions: First. significant deacylase activity is present in both cell fractions. Secondly, the total capacity of the cytosol to form acctoacetatefrom acetoacetyl-CYo.4 is only slightly less than that of the mitochondria. The recent suggestion of Clinkenbeard CI ctl. (197%; .see also Barth, 1977). that the HMG CoA pathway activity found in the cytosol is due to leakage of HMG Co,4 lyase from the ~iit~h~~n~iri~l to the cytosol during cell fractionation. is not in agreement with our results and with those of others (Williamson LV al.. 196X: Allrcd. 1973). The mitochondrial contamination of the cytosol. determined to be at the most IY;, in our studies. cannot account for the HMGCoA pathway activity which we found in the cytosol. Even if the ratio of synthnse to Ipase activity in the mitochondria is about f :5 (Clinkcnbcard (+r cd.. 1975~: Williamson er rrl., 196X). the qtosolic HMG -CoA pathway activit) in our csperiments cannot be more than 6.5”, of that in the mitochondria unless the q tosol contained original Iyase activit>. The low lyase activity found in the qtosol by Clinkenbeard cf al. (197%) may have been due to lack or complcxing of some activator like c‘a ‘ ’ ions. We found that S- 10 mM C’a . acti\atcs the HMG-CoA pathway in whole rat li\cr homogcnatcb. Another argument in favour of our suggestion that in a rat liver cytosol, as prepared in the usual way. HMG-CbA lyase is i~ihibitcd (or not (~p~irn~~ll~~activated). may be deduced from csperimonts in which wc tcstcd a possible substrate inhibition of WMG CoA synthase (Stewart 6t Rudney. 1966). A lowering of the steady-state concentration of acctoncctyl-CoA. brought about by replacing the added substrate with a substr~~t~-feeding qstem consisting of ncctoacetyl carnitine, camitinc acctyl-fr;insfrnlse (EC” 23.1 .J) and CoA. led to a &fold stimulation of the activity of the HMG -CoA pathway in the mitochondria. but to only a slight stimulation in the cl tosol. Even from the data of Clinkenbcard (‘r trl. (197%) the conclusion may bc drawn that it is improbable that leakage of HMG <‘oA lqase from the mitochon-

234

IDS MULDERAND

dria can explain the cytosolic

acetoacetate

SIMON G. VAN DEN

formation.

These authors established means to distinguish between mitochondrial and cytosolic HMG-CoA synthases. However, in the cytosol preparations which are supposed to contain a lot of fyase which has leaked from the mitochondria, they did not find any mitochondrial synthase. As the possible role of an enzyme itz viva depends in part upon its K, value as compared with the concentration of the substrate, the K, of the unpurified deacylase towards acetoacetyl-CoA was determined, using dilute mitochondrial suspensions or cytosol. Initial reaction rates were calculated from measurements of the disappearance of acetoacetyl_CoA and the formation of acetoacetate in the presence of iodoacetamide. The mean K, value observed in 3 experiments was 0.28 k 0.05 mM for the mitochondriai and 0.25 f 0.05 mM for the cytosolic enzyme. Reported values for the acetoacetyl-CoA concentration in rat liver cells are much lower (Greville & Tubbs, 1968; Williamson & Hems, 1970). Even concentrations of less than one molecule per mitochondrion have been postulated. As reported values for the estimated apparent K, for the HMG-CoA pathway are very low too (Lee & Fritz, 1972; Sugiyama et ul., 1972), one might conclude that the role of the deacylase in uiuo can be small only. However, this conclusion from the in vitro value of the K, of the deacylase is premature until we know more about the in uioo conditions, like adsorption, binding or complex formation of the substrate, or compartmentalization in the case of the ~t~hondr~a. The conclusion of this paper is that in rat liver

4 pathways exist for the formation of acetoacetate from acetoacetyl-CoA, a mitochondrial and a cytosolit HMG-CoA pathway as well as a mitochondrial and a cytosolic deacylase. As each of these pathways could play a part in the regulation of ketogenesis,

an investigation into shifts under ketotic conditions wilt be presented in the a~omp~ying paper (Mulder et al., 1977). Acknowledyements--The expert technical assistance of Mrs. E. A. de Vries-Akkerman, Mr. A. Lankhorst and Mr. A. van der Molen is gratefully acknowledged, This work was supported in part-by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization’for the Advancement of Pure Research (ZWO). REFERENCES ALLREDJ. B. (1973) Properties and subcellular distribution of enzymes required for acetoacetate biosynthesis in chicken liver. Biocb~m, ~i~p~ys. Acta 297, 22-30. BAIRDG. D., HIBBITTK. G. & LEE J. (1970) Enzymes involved in acetoacetate formation in various bovine tissues. Biochem. .T. 117. 703-709. BAKTHC. A. (1977) Compartmentation of ketone body synthesis in rat liver. In Biochemical and Clinical Aspects qf Ketone Body Metabolism (Edited by Siiling H. D.). Thieme Verlag, Stuttgart. In press. BUCHER N. L. R., OVERATHP. & LYNN F. (1960) j%Hydroxy-~-methylglutaryl coenzyme A reductase, cleavage and condensing enzymes in relation to cholesterol formation in rat liver, Biochim. biophys. Acta 40, 491-501. BURCHR. E. & CURRANG. L. (1969) Hepatic acetoacetylCoA deacylase activity in rats fed ethyl chlorophenoxyisobutyrate (CPIB). J. Lipid Res. 10, 668673.

BERGH

BURCH R. E. & TRIANTAFILLOU D. (1968) Acetoacetylcoenzyme A deacylase activity in liver mitochondria from fed and fasted rats. Biochemistry 7. 100%1013. BUHCHR. E. & WERTHEIM A. R. (1973) Subcellular localization of acetoacetyl-CoA deacylase and its role in acetoacetate synthesis. Am. J. Cfin. Nutr. 26, 814-822. CALDWELLI. C. & DRUMMONDG. I. (1963) Synthesis of acetoacetate by liver enzymes. J. hiol.‘Chem. i38, 64-68. CLELANDK. W. & SLATER E. C. (1953) Respiratory granules of heart muscle. Biochem. J. 53, 547-556. CL&KENBEARD K. D., REEDW. D., MWNEY R. A. & LANE M. D. (1975~) Intracellular localization of the 3-hydroxy~3-met~yl~lutaryl coenzyme A cycle enzymes in liver. J. biol. Gem. 250, 3108-3116. CLINKENBEARD K. D., SUGIYAMA T., REEDW. D. & LANE M. D. (1975b) Cytoplasmic 3-hydroxy-3-methylglutaryl coenzvme A svnthase from liver. J. biol. Chem. 250, 3114 313.5. . DIXKER K. (1970) Acctacetyl-foenzyme A. und acetyl&enzyme A. u.v.-Spektrophotometrische Bestimmung. In ~ethode~ der enzymatischen Analyse (Edited by BERGMEYER H. U.) 2nd edn. pp. 1922.-1927, 1935-1938. Verlag Chemie, Weinheim. DRUMMOND G. I. & STERNJ. R. (1960) Enzymes of ketone body metabolism---II. Properties of an acetoacetate-synthesizing enzyme prepared from ox liver. J. biol. Chem. 235,318-325. - . DR~MMO~DG. I. & STERNJ. R. (19611 The enzvmic hvdrotysis of glutathione thioesteis. Akhs Biociem. Bibphys. 95, 323-328. GEHRINGU.. RIEPER~NGERC. & LYNENF. (1968) Reinigung und Kristallisation der Thiolase, Untersuchungen zum Wirkunzsmechanismus. Eur. J. Biochem. 6, 264-280. GREVILLEG. D. & TUBBS P. K. (1968) The catabolism of lonrr chain fatty acids in mammalian tissues. In Essays in Biochemistry (Edited by CAMPBELL P. N. & GREV~L~E G. D.) Vol. 4. no. 155-212. Academic Press, London. LEEL. PI K. & +R&Z I. B. (1972) Factors controlling ketogenesis by rat liver mitochondria. Can, J. Biochem. 50, 120-127. LOPES-CARDOZO M., MULDERI., VAN VUGT F., HERMANS P. G. C. & VAN I)EN BERGH S. G. (1975) Aspects of ketogenesis: control and mechanism of ketone-body formation in isolated rat liver mitochondria. M&c. cell. Biochem. 9, 155.-173. LYNENF. (1953) Functional group of coenzyme A and its metabolic relations, especially -in the fatty acid cycle. F&r,. Proc. Fedn. Am. Sot. esa. Biol, 12. 683-691. LYNEN F.. HENNINGU., BLIBLITZ’C., !%RBOB. & KRBPLINRUIZFFL. (1958) Der chemische M~hanismus der Acetessig~urebildung in der Leber. Biochem. Z. 330, 269-295. MAYES P. A. & FELTS J. M. (1967) Determination of 14C-labelled ketone bodies by liquid-scintillation counting. Biochem. J. 102, 23&235. MELLANBYJ. & WILLIAMSON D. H. (1970) Acetacetat. In ~ethode~ der eff~~~rnatische~ Anatyse (Edited by BERGMEYERH. U.) 2nd edn. pp. 1776-1779. Verlag Chemie, Weinheim. MIUDLETONB. (1973) The oxoacyl-soenzyme A thiolases of animal tissues. Biochem. J. 132. 717-730. MIZIORKOH. M., CLINKENBEARD K. D., REED W. D. & LANE M. D. (1975) 3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetvlation. J. biol. Chem. 250, 5768-5773. MULD~KI. (1972uj Pathways and functions of acetoacetate formation in rat liver cytosol. In Abstr. Commun. 15th lnt. Conf Biochem. Lipids, p. 74. MIJI.DER I. (1972b) Determination of acetyl coenzyme A. Interference by a contaminant in malate dehydrogenase. J. Lipid Rex t3, 552-554.

Ketogenesis

in liver cell fractions

MULDER I., DE VRIES-AKKERMAN E. A. & VAN DEN BERCH S. G. (1977) Conversion of acetoacetyl-coenzyme A into acetoacetate in subcellular liver preparations. Shifts in enzyme activities in liver cell fractions of ketotic rats and cows. 1111.J. Biochrm. 8. 237-241. MULDER I. & VAN RH~ENEN D. L. (1971) Deacylation of acetoacetyl
235

STERN J. R., @ON J. J. & DEL CAMPILLO J. (1953) Acetoacetyl coenzyme A as intermediate in the enzymatic breakdown and synthesis of acetoacetate. J. Am. &em. sot. 75, 1517-1518. STERN J. R. & MILLER G. E. (1959) On the enzymic mechanism of acetoacetate synthesis. Biochim. hiophys. Acta 35, 576-577. STEWART P. R. & RUDNEY H. (1966) The biosynthesis of 3-hydroxy-3-methylglutaryl &enzyme A in beast-III. Purification and’ properties of the condensing enzyme thiolase system. J. biol. Chem. 241, 1212-1221. SUGIYAMA T., CLINKENBEARD K. D., Moss J. & LANE M. D. (1972) Multiple cytosolic forms of hepatic b-hydroxyp-methylglutaryl coenzyme A synthase: possible regulatory role in cholesterol synthesis. Biochem. biophys. Res. Commun. 48, 255-261. WILLIAMSON D. H., BATESM. W. & KREBS H. A. (1968) Activity and intracellular distribution of enzymes of ketone-body metabolism in rat liver. Biochem. J. 108, 353-361.

WILLIAMSXND. H. & HEMS R. (1970) Metabolism and function of ketone bodies. In Essays in Cell Metabolism (Edited by BARTLEY W., KORNBERG H. L. & QUAYLE J. R.) pp. 257-281. Wiley-Interscience, London.