Mitochondrial acetyl-CoA acetyltransferase in various organs from rat: form patterns and coenzyme-A-mediated modification

274

Biochimica etBiophysica Acta 830 (1985) 274-281 Elsevier

BBA32278

Mitochondriai acetyl-CoA acetyltransferase in various organs from rat: form patterns and coenzyme-A-mediated modification Walter H u t h * a n d F r a u k e Alves lnstitut fur Biochemie, Fachbereich Medizin, Georg-A ugust Universiti~t, Humboldtallee 23, D -3400 G6ttingen (F. R. G.) (Received May 2lst, 1985)

Key words: Acetyl-CoAacetyltransferase;CoenzymeA; Ketone body synthesis; Ketone body degradation; (Rat mitochondria) The mitochondrial acetyI-CoA acetyitransferase (acetyl-CoA: acetyl-CoA C-acetyltransferase, EC 2.3.1.9), which is involved in the biosynthesis or degradation of ketone bodies, was directly demonstrated in organ extracts applying a two-step chromatography-immunoelectrophoresis method. In liver, the enzyme can be shown in at least three forms: in an unmodified state, designated as AAT, and in the CoASH-modified forms AI and A2, in amounts of 51.5-1-5.0%, 39.4 +4.8% and 9.1 + 2.7% (areas of immunoprecipitation), respectively. This pattern, which could not be altered by a treatment with glutathione, resembles that of mitochondrial acetyI-CoA acetyltransferase in extrahepatic tissues. However, the proportion of the unmodified enzyme (AAT) is lower as compared to those in other tissues such as brain (81.5 + 4.4%). CoASH-modification and transformation into modified forms, which equal naturally occurring forms, can be demonstrated in vitro with acetyI-CoA acetyltransferase from both liver and brain. Thus CoASH-modification of mitochondrial acetyI-CoA acetyltransferase seems to be a process of general importance.

Introduction Mitochondrial acety!-CoA acetyltransferase (acetyl-CoA : acetyl-CoA C-acetyltransferase, EC 2.3.1.9) catalyzes the first step in the biosynthesis of ketone bodies in liver and is involved in the degradation of ketone bodies in extrahepatic tissues [1]. It can be regarded as the rate-limiting enzyme in the biosynthetic pathway [2-4]. Its activity seems to be mainly, controlled by the product inhibitor, CoASH [4]. Obviously CoASH-mediated control is the molecular mechanism of metabolic phenomena characterized by the correlation of ketogenic rates with the ratios of acetyl-CoA to CoASH in hepatocytes [5]. Interestingly, no change * To whom correspondenceshould be addressed. Abbreviations: GSH, glutathione (reduced form); CoASH, coenzymeA.

in the total amount of immunreactive enzyme protein was observed under metabolic conditions with differing rates of ketogenesis [6]. CoASH has a dual effect on acetyl-CoA acetyltransferase in rat liver. Apart from product inhibition, the enzyme can be chemically modified and inactivated by CoASH [7-10]. Both CoASH binding and inactivation can be simultaneously reversed by treatment with GSH. CoASH-modification of acetyl-CoA acetyltransferase results in the formation of additional enzyme forms, which resemble the naturally occurring forms of the enzyme. The aim of the present study was to quantitate the relative amounts of the naturally occurring forms of mitochondrial acetyl-CoA acetyltransferase functioning in the biosynthesis and degradation of ketone bodies in various organs from rat. In addition, the effects of GSH and CoASH on the form patterns have been investigated.

0167-4838/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)

275 Materials and Methods

Unless stated otherwise, the reagents used were from Boehringer-Mannheim (Mannheim, F.R.G.) or Merck (Darmstadt, F.R.G.). Phosphocellulose was purchased from Schleicher and Schi~ll (Dassel, F.R.G.), agarose A- and protein A-Sepharose CL4B from Pharmacia (Freiburg, F.R.G.). Freund's adjuvant was obtained from Behring (Marburg, F.R.G.) and membrane filters (HAWP, 0.45 ~m) from Milipore (Molsheim, France). AcetoacetylCoA was prepared, purified and assayed as previously described [3].

Preparation of antibodies Antibodies against the rat liver mitochondrial acetyl-CoA acetyltransferase were raised in rabbits. The enzyme was injected initially at multiple sites with 50 /~g enzyme emulsified in complete Freund's adjuvant. Booster injections of 30 /~g enzyme in incomplete Freund's adjuvant were made at 2-week intervals. One week after the third and final injection blood was collected from the rabbits. The immunoglobulin G fraction was isolated from antisera by ammonium sulfate precipitation (final saturation 50%) and further purified on protein A-Sepharose. Antibodies were completely retained by protein A-Sepharose and subsequently eluted with 0.2 M glycine-HCl (pH 2.7)/ 0.14 M NaC1. The specificity of the antibodies was verified by double immunodiffusion [11].

Tissue preparation 2 g of frozen rat liver, or whole organs, such as heart, kidneys or brain, were used for the preparation of crude organ extracts by homogenization in 0.05 M potassium phosphate buffer (pH 7.2). Hearts were homogenized with an Ultra Turrax (4 x 30 s; Jahnke u. Kunkel, Staufen, F.R.G.). Cell debris and nuclei were removed by centrifugation at 120 x g for 10 min, and the pellets were washed twice. For total liver extracts mitochondria in the postnuclear supernatant were solubilized (60 min at 4°C under stirring) in 12 ml of a medium containing 0.05 M potassium phosphate buffer (pH 7.2), 0.02 mM EDTA, 20% (w/v) glycerol, 0.02 mM GSH and 0.5% (w/v) Triton X-100 under stirring at 4°C for 60 min. This material was then centrifuged at 105000 x g for 60 min. The

resulting supernatant represented the total liver extract. It was also used for studying the effects of GSH on the form patterns. For the preparation of a mitochondrial extract the liver (2 g) was homogenized (1/8, w/v) in 0.3 M sucrose/5 mM Tris-HC1 (pH 7.4)/1 mM EDTA. After removal of the cell debris and nuclei, the mitochondria were sedimented at 26650 x g for 10 rain. The mitochondrial pellet was gently resuspended in the buffer used for homogenization and centrifuged at 26 650 x g for 15 min. The resulting mitochondrial pellet was subsequently subjected to sonication (3 x 15 s, Branson sonifier) at 0°C in 0.05 M potassium phosphate buffer (pH 7.2), or alternatively treated with Triton X-100. A mitochondrial extract was obtained by centrifuging these suspensions at 105 000 x g for 60 min. The preparation of a freeze-stopped liver in non-aqueous media was performed according to Soboll et al. [12]. 0.6 g of freeze-dried liver powder was sonicated in a mixture of heptane/carbon tetrachloride. The homogenate was subsequently filtered through a column (2 x 5 cm) containing glass beads of 0.5 mm diameter and subsequently through a column with glass beads of 0.3 mm diameter. The resulting filtrate was centrifuged at 9200 × g for 10 min. The supernatant was discarded and the pellet was lyophilized. The lyophilized material was extracted by homogenization in 7 ml 0.05 ml potassium phosphate buffer (pH 7.2) and centrifuged at 36900 x g for 10 min. After filtration of the supernatant through membranes (pore size 0.45 ~m) the effluent was transferred to a phosphocellulose column.

Chromatography and fused rocket immunoelectrophoresis Extracts from total liver or liver mitochondria were adjusted to a potassium concentration of 0.17 M and subsequently transferred to a phosphocellulose column (80 x 6 mm), which had been equilibrated with 0.1 M potassium phosphate buffer (pH 7.2)/20% (W/V) glycerol/0.02 mM EDTA. The various forms of acetyl-CoA acetyltransferase were eluted by a K ÷ gradient (0.17 M to 0.65 M). The effluent was collected in 0.5 ml fractions. These were analyzed for immunoreactive protein by fused rocket electrophoresis using 14.4/~g IgG anti acetyl-CoA acetyltransferase/cm2 gel as de-

276

scribed previously in Ref. 6. The areas of immunoprecipitations were calculated by graphical integration using a 7-fold photographic enlargement. Incubation with C o A S H

According to the immunochemical profile of phosphocellulose chromatography, fractions were pooled which contained the unmodified enzyme (AAT) in a partially purified state. The enzyme solution was dialyzed against 0.05 M potassium phosphate buffer (pH 7.2)/20% (w/v) glycerol/ 0.02 mM GSH. For studying the effects of CoASH (0.2 mM), the reaction mixture, in a sealed flask under nitrogen, contained the enzyme, 0.1 M potassium phosphate buffer (pH 7.2), 20% (w/v) glycerol and 0.02 mM GSH. The enzyme activity was monitored in the direction of acetoacetyl-CoA cleavage as previously described [10]. After incubation the reaction mixture was transferred to chromatography on phosphocellulose and again the fractions were analyzed for immunoreactive protein in a fused rocket immunoelectrophoresis as described above. Results and Discussion

As had been shown in experiments performed in vitro with homogeneous unmodified acetyl-CoA acetyltransferase (AAT), the formation of the

modified forms A1 and A2 is due to the binding of CoASH [8,9]. Presumably the heterogeneous forms result from different amounts of CoASH bound to the enzyme. Unmodified and modified forms of the enzyme can be demonstrated directly in liver extracts by a two-step chromatography immunoelectrophoresis method using monospecific antibodies. Mitochondrial acetyl-CoA acetyltransferase hassbeen shown to be immunologically distinct from mitochondrial acetyl-CoA acyltransferase (EC 2.3.1.16) (3-ketoacyl-CoA thiolase), the cytosolic acetyl-CoA acetyltransferase [7] and from the peroxisomal acetyl-CoA acyltransferase [13]. The immunoelectrophoretic method employed produces values with a reproducibility of 3.9 + 2.7% (area of immunoprecipitation). Fig. 1 shows the unmodified enzyme and modified forms in amounts of immunoprecipitation area of 44.9% for AAT, 41.7% for A1 and 13.4% for A2. However, form A2 often could not be detected by this procedure. This could have been due to 'demodifications' during partial purification and analysis. We therefore became interested in the relative amounts of the unmodified and modified enzyme as analyzed by the chromatography-immunoelectrophoresis method. Form A2 is missing, when a total liver extract was used (Table I) and contrary to the in vitro CoASH-modified forms the amount of the in vivo modified enzyme forms could not significantly be

TABLE I RELATIVE AMOUNTS OF RAT LIVER MITOCHONDR1AL ACETYL-CoA ACETYLTRANSFERASE A1 A N D A 2 I N D E P E N D E N C E O N T H E M E T H O D S O F L I V E R P R E P A R A T I O N

(AAT) AND FORMS

Details o f the p r o c e d u r e s a r e given u n d e r M a t e r i a l s a n d M e t h o d s . G l u t a t h i o n e ( G S H ) effects w e r e s t u d i e d b y i n c u b a t i n g a total liver e x t r a c t in p r e s e n c e o f 5 m M G S H at 3 0 ° C for 120 m i n . V a l u e s are m e a n s + S.D. o f 5 - 7 d e t e r m i n a t i o n s each. M o d e of liver p r e p a r a t i o n and additional treatment

Total extract T o t a l extract, i n c u b a t i o n without GSH with GSH Mitochondrial extract T o t a l e x t r a c t in non-aqueous media " Form A2 was not detected.

Relative a m o u n t s (% a r e a of i m m u n o p r e c i p i t a t i o n )

A m o u n t of m o d i f i e d e n z y m e (%) (A1 + A 2 )

AAT

A1

A2

63.2 + 2.5

36.8 _+2.5

_ a

36.8 _+ 2.5

66.9 + 2.5 60.8+6.1 51.5 + 5.0

33.1 + 2.5 39.2 + 6 . 1 39.4 + 4.8

- a _ a 9.1 + 2.7

33.1 + 2.8 39.2+7.0 48.5 + 3.8

5 3 . 9 + 8.8

39.1 + 3.6

7 . 0 + 5.5

46.1 + 4 . 5

277 Liver I/1 °

~

ttl

A2

A1

AAT

2 0 r"

E E Chromatography

=

Fig. 1. Form patterns of acetyl-CoA acetyltransferase inliver. The mitochondrial extract of.2 g liver was chromatographed on phosphocellulose. The immunochemical elution profile was performed with fused rocket immunoelectrophoresis. For details see Materials and Methods.

lowered by GSH [9]. The reason for this phenomenon is not yet known. On the other hand, GSH exerts a stabilizing effect in that the amount of modified forms remained at about 39%. However, by using a liver mitochondrial extract, not only was form A2 always detected (Table I), but also the amount of modified forms increased (up to 49%). It seems noteworthy to mention that the increased modified enzyme forms in mitochondrial extracts cannot have been caused by the method of solubilization. Both sonication and treatment with Triton X-100 result in virtually identical amounts of modified and unmodified enzyme forms. On the other hand, enzyme demodification may result from dissociation of CoASH from the enzyme in aqueous extracts of liver, provided the chemical modification is realized by binding of CoASH on specific high-affinity sites of the enzyme [9] but not by covalent binding [9,10]. In order to test this hypothesis, a freeze-stopped liver was fractionated in a non-aqueous medium. Applying this method modified enzyme amounted to about 46%. Obviously, this preparation does not result in a significant enhancement of the modified enzyme forms. As seen earlier, demodification by GSH of a homogeneous enzyme preparation modified in vitro by CoASH [9] does not work with naturally

occurring modified forms. Thus, the question arises as to whether CoASH-modification and inactivation occurs with partially purified unmodified enzyme (AAT). As demonstrated in Fig. 2 (lower left panel) CoASH caused an inactivation of 48.6% and, in addition to the unmodified AAT, gave rise to forms A1 and A2. Total immunreactive protein remained unchanged during this modification. The pattern of different forms resembles that in liver extract (Fig. 1). In the absence of CoASH, the enzyme was neither modified to yield additional forms nor was it inactivated (Fig. 2, upper left panel). When inactivation was low, only form A1 was observed which amounted to 40% (Fig. 2, right panel). Inactivation of about 80% corresponded to the appearance of forms A1 and A2 (43% and 35%, respectively). These findings suggest that the activity depends on the amount of the unmodified enzyme, and that the modified forms A1 and A2 are virtually inactive. However, forms A1 and A2, obtained by purification of the enzyme, reveal only a decrease in specific activities by 24.7% and 52.9%, respectively [9]. These contradictory findings remain to be clarified. However, it should be realized that crude enzyme preparations were used in the above experiments and some as yet unknown factors could have interfered with the activity but not with the immunochemical profile. In order to study the form patterns of mitochondrial acetyl-CoA acetyltransferases of various rat organs that have different functions in ketone body metabolism (liver, kidney, heart and brain), fractions obtained after phosphocellulose chromatography of the organ extracts were analyzed by a fused rocket immunoelectrophoresis. The antibodies against the liver enzyme were used in these experiments, since mitochondrial acetyl-CoA acetyltransferases from these various organs exhibit immunological relationships [7]. Rocket immunoelectrophoresis (Fig. 3; Table II) shows the different form patterns of acetyl-CoA acetyltransferases from fed rats. Organ extracts have been used in these experiments instead of mitochondrial extracts which would have given slightly higher values for the modified enzyme forms. In liver and kidney the enzyme was modified to amounts of 49% and 37% (area of immunoprecipitation), respectively. In both organs

278

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Chromatography forms A1 a n d A 2 were observed. In liver, in which the f l - h y d r o x y - f l - m e t h y l g l u t a r y l - C o A p a t h w a y is the m a j o r route of a c e t o a c e t a t e f o r m a t i o n [1,14], o n l y 51.1% of i m m u n o r e a c t i v e p r o t e i n exists in the u n m o d i f i e d , a p p a r e n t l y most active form ( A A T ) . A c e t y l - C o A acetyltransferase is u n d e r a tight control by the p r o d u c t , C o A S H [4], a n d is s u p p o s e d to be rate-limiting in the biosynthesis of k e t o n e b o d i e s [2-4]. The i n h i b i t i o n b y the C o A S H [4] offers a r e a s o n a b l e e x p l a n a t i o n for the o b s e r v a t i o n that the rate of ketogenesis does n o t correlate with the a c e t y l - C o A c o n c e n t r a t i o n b u t with the acetylC o A : C o A S H ratio in h e p a t o c y t e s o b t a i n e d from fed or fasted rats [5,15]. f l - H y d r o x y - f l - m e t h y l -

Fig. 2. Effect of CoASH on the activity of liver mitochondrial acetyl-CoA acetyltransferase (AAT) and on its form patterns, as analyzed by chromatography on phosphocellulose and fused rocket immunoelectrophoresis. (Upper left panel.) Control was performed by incubating 5.54 units AAT at 30°C for 2 h. 5.38 units were obtained after this incubation, yielding an immunoprecipitation area of 1235 mm2. (Lower left panel.) 5.54 units AAT were incubated with 0.2 mM CoASH at 30°C for 2 h; 2.85 units were obtained after incubation, yielding an immunoprecipitation area of 1258 mm2. The relative amounts (% area of immunoprecipitation) of AAT, A1 and A2 were 30.7%, 42.5% and 26.8%, respectively. (Right panel) Relative amounts of AAT (O) form A1 (©) and form A2 (zx) as a function of CoASH-mediated inactivation. Aliquots of 3.2-5.5 units of AAT were incubated with 0.2 mM CoASH at 30°C for 1, 2 or 3 h. The results represent the values of ten experiments.

g l u t a r y l - C o A synthase ( E C 4.1.3.5) is also a s s u m e d to be rate limiting in ketogenesis [16]. However, this enzyme (avian liver source) is not affected b y C o A S H [17]. It is q u e s t i o n a b l e whether in kidneys, the f l - h y d r o x y - f l - m e t h y l g l u t a r y l - C o A p a t h w a y c o n t r i b u t e s to f o r m a t i o n of a c e t o a c e t a t e [14,19]. T o a large extent a c e t o a c e t a t e f o r m a t i o n seems to b e due r a t h e r to direct d e a c y l a t i o n of acetoacetylC o A [14]. T h e a m o u n t of u n m o d i f i e d a c e t y l - C o A acetyltransferase in k i d n e y s increased up to 63%. Hence, the a m o u n t of the m o s t active enzyme a p p e a r s to d e p e n d on the functioning of the fl-hyd r o x y - f l - m e t h y l g l u t a r y l - C o A p a t h w a y . This ass u m p t i o n c o u l d be s u p p o r t e d b y the form p a t t e r n s

279

Liver

Ki~

A2

AI

AAT

A2

A!

AAT

( -

Ul Ul

Heart

Brain

b,-

0

A1

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AI

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Fig. 3. Form patterns of mitochondrial acetyl-CoAacetyltransferasein liver, kidney, heart and brain, as analyzed by chromatography on phosphocellulose and by fused rocket immunoelectrophoresis.Extracts from total organs were prepared in presence of 0.02 mM glutathione as described under Materials and Methods. o b t a i n e d with enzymes from heart a n d brain, which degrade ketone bodies. I n brain, m i t o c h o n d r i a l acetyl-CoA acetvltransferase for instance was pre-

sent with up to 82% in the u n m o d i f i e d form. Besides this only the modified form A1 was detected.

280 TABLE II RELATIVE A M O U N T S OF M I T O C H O N D R I A L ACETYLCoA ACETYLTRANSFERASE (AAT) A N D FORMS A1 A N D A2 IN VARIOUS ORGANS OF THE RAT The table summarizes the experiments demonstrated in Fig. 3. Values are means-+ S.D. of 10-12 determinations each. JOlT Organ

Liver Kidney Heart Brain

Relative amounts of AAT and forms (% area of immunoprecipitation) AAT

A1

A2

50.6 _+3.6 62.9_+3.9 78.9 _+2.1 81.5 +4.4

39.6 _+4.6 29.3+2.5 21.1 _+2.1 18.5 _+3.5

9.8 _+2.9 7.8_+2.1 - ~ _ a

a Form A2 was not detected.

From these results it became evident that, in all organs studied, m i t o c h o n d r i a l acetyl-CoA acetyltransferase exists in various forms. Organs in which the mitochondrial fl-hydroxy-fl-methylglutaryl-CoA pathway works exhibit relatively less active, i.e. unmodified, enzyme. In contrast, in organs in which ketone bodies are degraded the relative proportion of the most active unmodified enzyme is higher. The amounts of modified forms A1 and A2 apparently correspond with the degree of a modification by CoASH. As was already shown with the unmodified acetyl-CoA acetyltransferase from liver, the unmodified enzyme from brain was similarily inactivated and modified to the form A1 by CoASH (Fig. 4). The degree of inactivation (26.1% _+ 1.5%) corresponded to the appearance of form A1 (38.5 + 1.8%) (areas of immunoprecipitation). Form A2 was not detected. Immunochemical elution profiles after in vitro modification by CoASH of enzymes from liver and brain resemble those of the naturally occurring forms (Figs. 1-4). This suggests that the natural forms of acetyl-CoA acetyltransferase are identical with the forms obtained after an in vitro modification by CoASH. Further support for this assumption comes from isoelectrofocusing of unmodified and [3H]CoASH-modified forms [8] and from a demonstration of an in vivo labelling of the enzyme with [14C]CoASH (Huth et al., unpublished data). The modification of mitochondrial acetyl-CoA acetyltransferase by CoASH seems to be a process of general importance, which proceeds (but to a

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Fig. 4. Effect of CoASH on activity and form patterns of brain mitochondrial acetyl-CoA acetyltransferase (AAT), as analyzed by chromatography on phosphocellulose and by fused rocket immunoelectrophoresis. Aliquots of 7.1 units of AAT were incubated without (upper panel) and with 0.2 mM CoASH (lower panel). Total areas of immunoprecipitation were 1372 mm 2. Five experiments.

different degree) in the mitochondria of liver, kidney, heart and brain. The amount of the naturally occurring unmodified enzyme and thus of actual activity could not be increased by GSH in total liver extracts. This was, however, possible with enzyme preparations modified in vitro. In addition, the amount of unmodified enzyme is not increased

281 in m e t a b o l i c states w i t h i n c r e a s e d rates of k e t o g e n esis (e.g., s t a r v a t i o n , a c u t e d i a b e t i c ketosis) [6]. H e n c e , the a c c o m p a n y i n g i n a c t i v a t i o n u p o n m o d i f i c a t i o n b y C o A S H s e e m s n o t to b e the m a i n f u n c t i o n o f this process. M o d i f i c a t i o n m a y r a t h e r i n f l u e n c e the h a l f - l i f e - t i m e of this m i t o c h o n d r i a l enzyme.

Acknowledgements W e are g r a t e f u l to U l r i k e M611er for e x p e r t t e c h n i c a l a s s i s t a n c e a n d to H a n s K u n z e for h e l p in p r e p a r i n g the m a n u s c r i p t . T h i s w o r k was supp o r t e d by the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t .

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5 Siess, E.A., Brocks, D.G. and Wieland, O.H. (1976) FEBS Lett. 69, 265-271 6 Menke, R. and Huth, W. (1980) FEBS Lett. 19,19-32 7 Schwabe, D. and Huth, W. (1979) Biochim. Biophys. Acta 112-120 8 Huth, W. (1981) Eur. J. Biochem. 120, 557-562 9 Quandt, L. and Huth, W. (1984) Biochim. Biophys. Acta 784, 168-176 10 Quandt, L. and Huth, W. (1985) Biochim. Biophys. Acta 829, 103-107 11 Ouchterlony, O. (1949) Acta Pathol. Microbiol. Scand. 26, 507-511 12 Soboll, S., Seitz, H.J., Siess, H., Ziegler, B. and Scholz, R. (1984) Biochem. J. 220, 371-376 13 Miyazawa, S., Osumi, T. and Hashimoto, T. (1980) Eur. J. Biochem. 103, 589-596 14 Brady, P.S., Scofield, R.S., Ohgaka, S., Schumann, W.C., Bartsch, G.E., Margolis, J.M., Kumaran, K., Horvat, A., Mann, S. and Landau, B.R. (1982) J. Biol. Chem. 257, 9290-9293 15 Siess, E.A., Brocks, D.G. and Wieland, O.H. (1978) Hoppe-Seyler's Z. Physiol. Chem. 359, 785-798 16 Dashi, N. and Ontko, J.A. (1979) Biochem. Med. 22, 365-374 17 Reed, W.D., Clinkenbeard, K.D. and Lane, M.D. (1975) J. Biol. Chem. 250, 3117-3123 18 Baird, G.D., Hibin, K.G. and Lee, J. (1971) Biochem. J. 177, 703-709 19 Ohkagu, S., Brady, P.S., Schumann, W.C., Bartsch, G.E., Margolis, J.M., Kumaran, K., Landau, S.B. and Landau, B.R. (1982) J. Biol. Chem. 257, 9283-9289