Mitochondrial acetyl-CoA acetyltransferase in liver and extrahepatic tissues: role of modification by coenzyme A

350

Biochimica etBiophysicaActa 870 (1986) 350-356 Elsevier

BBA 32476

M i t o c h o n d r i a l a c e t y l - C o A acetyltransferase in liver and extrahepatic tissues: role of modification by c o e n z y m e A I v o n n e Balzer a n d W a l t e r H u t h * lnstitut fi~r Biochemie, Fachbereich Medizin, Georg-August Universiti~t GOttingen, Humboldtallee 23, D-3400 GOttingen (F.R.G.) (Received October llth, 1985)

Key words: Acetyl-CoA acetyltransferase; Coenzyme A; Clofibrate; Di(2-ethylhexyl)phthalate; Ketone body synthesis; Ketone body degradation; (Rat)

The influence of clofibrate and di(2-ethylhexyl)phthalate on mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9), the rate-limiting ketogenic enzyme, which can be modified and inactivated by CoA, was investigated. In fed rats, both compounds induced a doubling of ketone bodies in the blood and, moreover, an increase by about 13% in the hepatic relative amount of the unmodified, i.e., the most active form of the enzyme (immunoreactive protein). This shift would account for an elevation of overall enzyme activity by about 5% only. Thus, the CoA modification of mitochondrial aeetyl-CoA acetyltransferase did not explain the entire augmentation of ketone bodies. However, clofibrate and di(2-ethylhexyl)phthalate also increased the immunospecific protein and enzyme activity by approx. 2and 3-fold, respectively. These effects were observed in liver, but not in several extrahepatic tissues.

Introduction Mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA : acetyl-CoA C-acetyltransferase, EC 2.3.1.9) has been shown to represent the rate-limiting enzyme in ketogenesis and is involved also in the degradation of ketone bodies [1-3]. In hepatocytes and liver mitochondria from rats, synthesis of ketone bodies correlates with the acetyl-CoA/CoA ratio [4,5]. An analysis of this metabolic phenomenon at molecular level revealed that the actual activity of mitochondrial acetylCoA acetyltransferase is controlled by the product inhibitor CoA [3]. Moreover, CoA was demonstrated to modify the enzyme chemically and, thereby, to inactivate it [6-9]. This modification leads to additional enzyme forms with lower specific activities [7], both in liver [6,9-13] and To whom correspondence should be addressed.

extrahepatic tissues [11] of rats. The ratio of unmodified to modified enzyme proved to be unchanged in metabolic situations with increased rates of ketogenesis [10], but revealed pronounced differences between liver and extrahepatic tissues [11]. Thus, the degree of CoA modification of mitochondrial acetyl-CoA acetyltransferase seems to be related to the physiological function of the enzyme, i.e., synthesis or degradation of ketone bodies rather than to the actual rate of ketogenesis. However, clofibrate treatment of rats results in a marked increase of ketogenesis in hepatocytes [14]. In addition, this hypolipidaemic drug and also di(2-ethylhexyl)phthalate, an industrial plasticizer, are known to enhance mitochondrial and peroxisomal capacities for t-oxidation of fatty acids in liver [12,15,16]. Therefore, in the present paper, the effects of clofibrate and di(2ethylhexyl)phthalate on mitochondrial acetyl-CoA

01o/-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

351 acetyltransferase of liver and extrahepatic tissues were analyzed. Materials and Methods

Chemicals and assays. Unless otherwise noted, reagents were from Boehringer-Mannheim (Mannheim, F.R.G.) or from Merck (Darmstadt, F.R.G.). Other chemicals were purchased from the following sources: phosphocellulose: Schleicher and Schi~ll (Dassel, F.R.G.); agarose A and protein A Sepharose CL-4B: Pharmacia (Freiburg, F.R.G.); Freund's adjuvant: Behring (Marburg, F.R.G.); Triton X-100: Serva (Heidelberg, F.R.G.); clofibrate: Sigma (Taufkirchen, F.R.G.); di(2-ethylhexyl)phthalate: EGA-Chemie (Steinheim, F.R.G.). Centrisart-cups were from Sartorius (GtSttingen, F.R.G.). Acetyl-CoA and acetoacetylCoA were prepared, purified and assayed as previously described [2]. Antibodies against rat liver mitochondrial acetyl-CoA acetyltransferase were induced in rabbits and prepared as d~scribed by Huth and Alves [11]. DNA was determined as in Ref. 17. Ketone bodies in blood were, in general, estimated according to Williamson et al. [18]; however, 3-hydroxybutyrate was quantitated in a reaction mixture comprising 100 mM glycine-NaOH buffer (pH 9.4)/8 mM NAD+/0.45 units 3-hydroxybutyrate dehydrogenase. The activity of acetyl-CoA acetyltransferase was assayed according to Ref. 8. The activity with 3-ketohexanoylCoA as substrate was determined as described in Ref. 19. Animals. Male Wistar rats (150-180 g) were treated with clofibrate by subcutaneous injections of 600 mg/kg body weight daily, for 3 days, or were fed with a diet containing 2% (w/v) di(2-ethylhexyl)phthalate for 4 weeks. Tissue preparation. 2 g of liver, or the whole organs, such as kidneys (1.4-1.6 g), brains (1.0-1.2 g) or hearts (0.6-0.8 g) were used for preparations of crude organ extracts in a medium comprising 0.05 M potassium phosphate buffer (pH 7.2)/0.02 mM EDTA/0.02 mM GSH, as described in Ref. 11. In addition, the total heart extracts were frozen at - 3 0 ° C for 24 h before centrifugation at 105 000 x g. Chromatography and fused rocket immunoelectrophoresis. Both methods were performed as

described in Refs. 9 and 11. The immunoelectrophoretic method employed produces values with a reproducibility of 3.5 + 2.6% (area of immunoprecipitation). Rocket immunoelectrophoresis. The protein amount of mitochondrial acetyl-CoA acetyltransferase including all forms was quantitated by rocket immunoelectrophoresis. This was done according to Ref. 10 using 22.9 ~tg IgG anti-acetyl-CoA acetyltransferase/cm2 gel. Each plate contained a standard row; 5 #1 of suitable dilutions of purified mitochondrial acetyl-CoA acetyltransferase and of total organ extracts were added to the wells. For quantitation of acetyl-CoA acetyltransferase in brain, total brain extracts were concentrated by centrifugation in centrisart cups. This procedure was accompanied by a loss of acetyl-CoA acetyltransferase activity of 17.2 + 12.3%, as was shown by immunotitration experiments. The rockets were calculated using a 7-fold photographic magnification. Immunotitration. Total immunoreactive activity of mitochondrial acetyl-CoA acetyltransferase was estimated by immunotitration in whole organ extracts. An excess amount of antibodies (276 ~tg IgG, about 2-times excess of equivalence amount) was used in a reaction mixture (0.5 ml) comprising 100 mM potassium phosphate buffer (pH 7.2)/100 mM NaC1/1 mM GSH/100 mU enzyme activity. Activities were determined by the use of acetylCoA or acetoacetyl-CoA as substrates. After incubation with antibody for 60 min at room temperature, immunoprecipitates were collected by centrifugation. The sediments were washed three times with 0.5 ml 0.05 M potassium phosphate buffer (pH 7.2)/1 mM GSH, and in the supernatants, the remaining activities with acetyl-CoA or acetoacetyl-CoA as substrates were measured. Activities of mitochondrial acetyl-CoA acetyltransferase were calculated for the direction of either acetoacetyl-CoA synthesis (liver) or of acetoacetyl-CoA cleavage (kidney, heart, brain). Results

The concentration of ketone bodies in the blood of fed rats was practically doubled by administration of clofibrate or of di(2-ethylhexyl)phthalate (Table I). In order to analyse this phenomenon,

352 TABLE I EFFECTS OF CLOFIBRATE AND DI(2-ETHYLHEXYL)PHTHALATE ON THE KETONE BODY CONCENTRATIONS IN BLOOD OF FED RATS

A!

For assay of ketone bodies, see Materials and Methods. Values are means± S.D. of 5-6 determinations each. Statistically significant values (total ketone bodies), as compared to untreated fed animals, are indicated by the P values below the individual data. Treatment

Ketone bodies (# mol/ml blood) acetoacetate

hydroxybutyrate

total ketone bodies

None Clofibrate

0.07 ± 0.03 0.14 ± 0.05

0.16 ±0.06 0.26 ± 0.1

Di(2-ethylhexyl)phthalate

0.12 + 0.08

0.32 ± 0.09

0.23 ± 0.05 0.40 + 0.06 P < 0.001 0.44 ± 0.09 P < 0.001

t h e effects of c l o f i b r a t e a n d o f d i ( 2 - e t h y l h e x y l ) p h t h a l a t e o n m i t o c h o n d r i a l a c e t y l - C o A acetylt r a n s f e r a s e w e r e i n v e s t i g a t e d in liver a n d e x t r a h e p a t i c tissues u s i n g m o n o s p e c i f i c a n t i b o d i e s a g a i n s t this e n z y m e f r o m liver. T h i s was possible, since rat liver m i t o c h o n d r i a l a c e t y l - C o A a c e t y l t r a n s f e r a s e e x h i b i t s an i m m u n o l o g i c a l c r o s s - r e a c t i v i t y w i t h mitochondrial acetyl-CoA acetyltransferase from e x t r a h e p a t i c tissues [20], b u t is i m m u n o l o g i c a l l y

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E E TABLE 11 EFFECTS OF CLOFIBRATE AND DI(2-ETHYLHEXYL)PHTHALATE ON THE PATTERN OF FORMS OF MITOCHONDRIAL ACETYL-CoA ACETYLTRANSFERASE IN LIVER OF FED RATS Analysis by fused rocket immunoelectrophoresis as shown in Fig. 1. Values are means ± S.D. of five or six determinations each. Statistically significant values, as compared to untreated animals, are indicated by the P values below the individual data. Treatment

None Clofibrate Di(2-ethylhexyl)phthalate

Relative amounts (% area of immunoprecipitation) unmodified enzyme

modified form 1

58.92 ± 3.41 75.18 ± 1.51 P < 0.001 68.20 + 3.2 P < 0.001

41.08 ± 3.41 24.82 ± 1.51 P < 0.001 31.80 + 3.2 P < 0.001

Chromatography Fig. 1. Pattern of forms of mitochondrial acetyl-CoA acetyltransferase in liver of untreated (control) and clofibrateor di(2-ethylhexyl)phthalate (DEHP)-treated fed rats. The pattern was analyzed by chromatography on phosphocellulose and by fused rocket immunoelectrophoresis. 2 g of liver were prepared as described under Materials and Methods. AAT, unmodified; A1, modified form 1. u n r e l a t e d to the m i t o c h o n d r i a l a n d p e r o x i s o m a l a c e t y l - C o A a c e t y l t r a n s f e r a s e ( E C 2.3.1.16; 3-ketoa c y l - C o A thiolase) [20,12] a n d to t h e c y t o s o l i c a c e t y l - C o A a c e t y l t r a n s f e r a s e [20]. M o d i f i c a t i o n o f liver m i t o c h o n d r i a l a c e t y l - C o A a c e t y l t r a n s f e r a s e b y b i n d i n g o f C o A results in a d e c r e a s e of its specific activity. H e n c e , the r a t e o f k e t o g e n e s i s m a y b e r e l a t e d to t h e m o d i f i c a t i o n a l p a t t e r n c h a r a c t e r i z e d m a i n l y b y the u n m o d i f i e d

353 enzyme, and the modified forms 1 and 2 (Fig. 1, control). Among the forms mentioned, modified form 2 is the less abundant and often not detected in total liver extracts. This pattern, however, was significantly altered in favor of the unmodified, most active form by clofibrate or di(2-ethylhexyl)phthalate (Fig. 1, Table II). The 16% increase of unmodified enzyme (immunoreactive area) upon treatment with clofibrate results in an elevation of the total actual activity of about 5.0% only (calculated from the specific activities of the various forms [7] and the immunoreactive area). Obvi-

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ously, the observed enhancement of the most active, unmodified enzyme cannot account for the doubling of ketone body level in blood. Thus, the total immunospecific activity of the mitochondrial acetyl-CoA acetyltransferase was quantitated by immunotitration. Quantitative immunoprecipitations of the purified enzyme from rat liver (Fig. 2A) and of the enzyme in a total liver extract (Fig. 2B) were demonstrated. In liver, 44.6 + 3.4% of activity with acetoacetyl-CoA were immunoprecipitated (Fig. 2B). F r o m immunotitrations in total liver extracts, it became evident that the activity of mitochondrial acetyl-CoA acetyltransferase (direction of acetoacetyl-CoA synthesis) was significantly elevated, by 2- and 3.3-fold, when clofibrate or di(2-ethylhexyl)phthalate were administered to fed rats, respectively (Table III). This enhancement of the activity was apparently not caused by changes in liver weight, since the activity values were similarly augmented when using D N A content as reference. The further analysis of the effects of clofibrate or di(2-ethylhexyl)phthalate on the enzyme by rocket immunoelectrophoresis (Fig. 3) revealed a significant increase in the immunospecific enzyme protein, the extent of which would essentially explain the enhanced activity observed (Table III). Since mitochondrial acetyl-CoA acetyltransferase of extrahepatic tissues is involved in the degradation of ketone bodies and, therefore, influences the balance of these metabolites in blood, this enzyme was also investigated in kidney, heart and brain. Immunoreactive protein, enzyme activ-

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Fig. 2. Immunotitration of mitochondrial acetyl-CoA acetyltransferase in total fiver extracts. The immunotitration was performed as described in Materials and Methods. (A) Various amounts of purified rat liver enzyme were added to a fixed amount of antibodies (2.9 nag). The precipitate was washed three times and dissolved in 1 ml of 0.05 M NaOH. ×, Protein precipitated shown by absorbance at 280 nm. O, Activity with acetoacetyl-CoAas substrate. (B) Various amounts of antibodies were added to a fixed volume of total fiver extract. Activity with acetoacetyl-CoA(e) or 3-ketohexaonyl-CoA(©).

Fig. 3. Assay of mitochondrial acetyl-CoA acetyltransferasein a total liver extract by rocket immunoelectropfioresis.Sets of five wells on the left and right contained 5/L1 each of suitable dilutions of liver extracts. The five wells in the middle contained 17.8-133.5 ng of purified acetyl-CoA acetyltransferase from liver.

354 T A B L E III EFFECTS O F C L O F I B R A T E A N D D I ( 2 - E T H Y L H E X Y L ) P H T H A L A T E ON T H E T O T A L I M M U N O S P E C I F I C ACTIVITY A N D PROTEIN A M O U N T O F M I T O C H O N D R I A L A C E T Y L - C o A A C E T Y L T R A N S F E R A S E IN LIVER AS A N A L Y Z E D BY IMMUNOTITRATION AND ROCKET IMMUNOELECTROPHORESIS Immunochemical analysis were carried out as described in Materials and Methods. Activity was measured with acetyl-CoA as substrate. Values are m e a n s ± S . D , of five or six determinations each. Statistically significant values, as compared to untreated animals, are indicated by the P values below the individual data. Treatment

None Clofibrate Di(2-ethylhexyl)phthalate

Immunoreactive acetyl-CoA acetyltransferase activity (acetoacetyl-CoA synthesis)

prqtein

( u n i t s / g liver wet wt.)

(units/mg DNA)

( # g / g liver wet wt.)

(# g / r a g D N A )

2.17+0.3 4.29+ 1.09 P < 0.001 7.12 ± 2.22 P < 0.01

0.95±0.17 2.09±0.64 P < 0.01 3.37 ± 1.24 P < 0.01

235.1± 29.4 504.5 ± 80.0 P < 0.01 794.9 ± 194.9 P < 0.01

102.8± 11.9 243.5 + 42.8 P < 0.01 383.0 5:130.2 P < 0.01

TABLE IV EFFECTS O F C L O F I B R A T E A N D D I ( 2 - E T H Y L H E X Y L ) P H T H A L A T E ON T H E T O T A L I M M U N O S P E C I F I C ACTIVITY A N D PROTEIN A M O U N T OF M I T O C H O N D R I A L ACETYL-CoA A C E T Y L T R A N S F E R A S E IN E X T R A H E P A T I C TISSUES AS A N A L Y Z E D BY I M M U N O T I T R A T I O N A N D R O C K E T I M M U N O E L E C T R O P H O R E S I S Immunochemical analysis were performed as described in Materials and Methods. Activities were measured with acetoacetyI-CoA and 3-ketohexanoyl-CoA, respectively. Values are means of five or six determinations each. Treatment

lmmunoreactive acetyl-CoA acetyltransferase activity (acetoacetyl-CoA cleavage) ( u n i t s / g tissue wet wt.)

None Clofibrate Di(2-ethylhexyl)phthalate

protein ( # g / g tissue wet wt.)

kidney

heart

brain

kidney

heart

brain

15.66± 3.7 14.7 ±3.4

17.53±4.8 16.66±6.5

1.82±0.6 1.45±0.6 1.29 ± 0.5

622.5+168.5 594.8+218.0 -

454.08+138.5 359.59± 85.11 -

134.4±68.3 165.7±46.6 116.1 + 33.0

TABLE V E F F E C T S O F C L O F I B R A T E A N D D I ( 2 - E T H Y L H E X Y L ) P H T H A L A T E ON T H E P A T T E R N M I T O C H O N D R I A L ACETYL-CoA A C E T Y L T R A N S F E R A S E IN E X T R A H E P A T I C TISSUES OF RATS

OF

FORMS

Analysis by fused rocket immunoeloetrophoresis as shown in Fig. 1. Values are means of five or six determinations each. Treatment

Relative amounts (% area of immunoprecipitations) kidney

None Clofibrate Di(2-ethylhexyl)phthalate

heart

brain

unmodified enzyme

modified form 1

modified form 2

unmodified enzyme

modified form 1

unmodified enzyme

modified form 1

67.6 ± 6.4 70.3 ± 9.0 -

26.8±6.9 24.1±6.6 . .

7.3±3.7 6.6±2.7 .

83,4±4.0 78.0±4.6

16.6±4.0 22.0±4.5

86.8±4.1 84.2±4.9 83,6±7.6

13.2±4.1 15.8±4.9 16.4±7.1

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that clofibrate and di(2-ethylhexyl)phthalate had no effect, either on protein amount or on activity of the enzyme. Moreover, the modificational pattern of mitochondrial acetyl-CoA acetyltransferase in the three extrahepatic organs remained unchanged upon treatment with these agents (Table V).

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[mmunoglobulin G added (pl] Fig. 4. I m m u n o t i t r a t i o n of mitochondrial acetyl-CoA acetyltransferase in total extracts of extrahepatic tissues. Various amounts of antibodies were added to a fixed volume of tissue extracts. (A) Brain, (B) heart, (C) kidney. Activities were measured with (e) acetoacetyl-CoA and ( O ) 3-ketohexaonylCoA. For further details see Materials and Methods.

ity (direction of acetoacetyl-CoA cleavage), and the modificational pattern were determined using antibodies against the enzyme from liver. The enzyme was quantitated directly in total organ extracts. In kidney, heart and brain, 78.6_ 8.3%, 87.4 _ 12.7% and 89.1 __+7.2%, respectively, of the activity with acetoacetyl-CoA were immunotitrated (Fig. 4). In parallel experiments, however, the activity with 3-ketohexanoyl-CoA proved to be unaffected by the antibodies used. The amounts of protein of extrahepatic mitochondrial acetyl-CoA acetyltransferase were quantitated by rocket immunoelectrophoresis. Kidney and heart contained more enzyme protein than brain (Table IV). However, it became evident

The CoA modification of mitochondrial acetylCoA acetyltransferase can be demonstrated in vitro [6-8,11] and seems to be related to the physiological function of the enzyme [11]. In liver, about 50% (immunoreactive protein) of the enzyme appeared in the modified forms 1 and 2, i.e., approx. 40 and 10%, respectively, whereas in brain about 15% only were shown to be in the modified state (Tables II and V). Modification lowers specific activity - forms 1 and 2 are less active by 24.7 and 52.9%, respectively [7] - and consequently, overall enzyme activity. Thus, with respect to the ratelimiting function of mitochondrial acetyl-CoA acetyltransferase in ketogenesis [3], the CoA modification may represent a mode of control. Clofibrate and di(2-ethylhexyl)phthalate induced a moderate ketosis in fed rats, which was presumably caused by increased rates of ketogenesis, since increased blood concentrations of ketone bodies are usually a consequence of a slight imbalance between rates of synthesis and of degradation [21]. With respect to clofibrate, this conclusion has been confirmed, as this drug increases the rate of ketogenesis from oleate, both in hepatocytes and perfused liver [14,22]. In our study, it became evident that in liver the CoA modification of the mitochondrial acetyl-CoA acetyltransferase was not involved in the control of ketogenesis. The significant increase of the immunoreactive amount of unmodified enzyme caused by clofibrate or di(2-ethylhexyl)phthalate could account for an elevation of overall activity of about 5% only. However, liver mitochondrial acetyl-CoA acetyltransferase activity was enhanced more prominently (2-3-fold), which corresponded to an increased amount of immunospecific protein, as evidenced by rocket irnmunoelectrophoresis (Table III). Thus, the elevated activity of the rate-limiting enzyme renders the possibility for an enhanced

356 ketogenesis, provided that the a c e t y l - C o A / C o A ratio is elevated (cf., action of p r o d u c t inhibitor, CoA; Ref. 3). F r o m a calculation of specific activities (direction of acetoacetyl-CoA synthesis) of liver mitochondrial acetyl-CoA acetyltransferase (9.23, 8.50 and 8.96 u n i t s / m g protein in untreated, clofibrate- and di(2-ethylhexyl)phthalate-treated rats, respectively), it is obvious that the increased activity is solely due to an increase of the immunospecific protein and not caused by a demodification of modified enzyme forms. The higher a m o u n t of enzyme protein is presumably the consequence of an elevated rate of synthesis. As has been demonstrated by Oszasa et al., the translational activities of m R N A s for mitochondrial enzymes, including acetyl-CoA acetyltransferase, were induced by di(2-ethylhexyl)phthalate [24]. C o n t r a r y to liver, clofibrate and di(2-ethylhexyl)phthalate have no effect on mitochondrial acetyl-CoA acetyltransferase in kidney, heart and brain. Neither the ratio of unmodified to modified enzyme forms, nor the immunospecific activities nor protein amounts were affected by these compounds. In heart, L u n d and Bremer also could not demonstrate an effect of clofibrate on carnitine acetyltransferase (EC 2.3.1.7) [25]. The degree of C o A modification of the enzyme is obviously organ-specific, whereas no indication exists for an adaption to the actual rate of ketone b o d y degradation. The results presented also give the first information on the amounts of immunospecific mitochondrial acetyl-CoA acetyltransferase in extrahepatic tissues of fed rats. Specific activities (direction of acetoacetyl-CoA cleavage) of 25.16, 38.61 and 13.54 u n i t s / m g for kidney, heart and brain, respectively, were calculated. These values were virtually not increased by clofibrate, indicating that the degree of modification was not altered. In a d d i t i o n , the activities o f this mitochondrial enzyme, as quantitated by immunotitration, confirm data obtained from measurements in crude tissues extracts using various substrates and activation by potassium [9]. The physiological implications of the C o A modification of mitoehondrial acetyl-CoA acetyltransferase, which can be demonstrated in vivo (Huth, W. et al., unpublished data), remain to be further analyzed. In liver, a shift in favor of the u n m o d ified enzyme seems to be related to the rates of protein synthesis.

Acknowledgements We are grateful to Dirk Schwabe who performed parts of immunotitrations (Fig. 4) and to Ri~diger Hardeland for reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft.

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