Relationship between intramitochondrial citrate and the activity of carbomoyl-phosphate synthase (ammonia)

13

Biochimica et Biophysica Acta, 500 (1977) 13--26

@)Elsevier/North-Holland Biomedical Press

BBA 28359 RELATIONSHIP BETWEEN I N T R A M I T O C H O N D R I A L CITRATE AND THE ACTIVITY OF CARBAMOYL-PHOSPHATE SYNTHASE (AMMONIA)

ALFRED J. MEIJER and G.M. VAN WOERKOM Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, Plantage Muidergracht 12, Amsterdam (The Netherlands)

(Received April 4th, 1977)

Summary The possibility of control of the activity of carbamoyl-phosphate synthase (ammonia) (EC 2.7.2.5) in rat-liver mitochondria by variation in the intramitochondrial free Mg 2+ concentration has been investigated. Carbamoyl-phosphate synthase activity was measured by coupling the formation of carbamoylphosphate to the synthesis of citrulline in a reaction mixture containing ammonia, bicarbonate, a source of ATP, and ornithine. The synthesis of citrulline was inhibited by lowering the concentration of intramitochondrial free Mg 2+. This could be achieved n o t only by depleting the mitochondria of Mg 2+ (by adding the ionophore A23187), b u t also by increasing the intramitochondrial concentration of citrate. Under various conditions an inverse relationship between the rate of citrulline synthesis and the magnitude of the intramitochondrial concentration of citrate was observed. Inhibition of citrulline synthesis by intramitochondrial citrate could be partly reversed by addition of Mg 2+ in the presence of A23187. Possible implications of the regulation of carbamoyl-phosphate synthase (ammonia) activity by intramitochondrial citrate for nitrogen metabolism in the liver are discussed.

Introduction Synthesis of carbamoylphosphate in mitochondria is absolutely dependent on the presence of the cofactor N-acetylglutamate [1]. According to McGivan et al. [2] the concentration of N-acetylglutamate in the mitochondria changes only very slowly. Control of flux through carbamoyl-phospnate synthase (ammonia) (EC 2.7.2.5) by N-acetylglutamate is considered to be a long-term regulation which presumably occurs by variations in the diet [2]. Carbamoyl-phosphate synthase is also dependent on the presence of Mg 2+.

14 The e n z y m e requires MgATP as a substrate and free Mg 2÷ as an essential cofact o r [3--5]. The present study was under t a k en in order to find out whether short-term regulation of flux through carbamoyl-phosphate synthase can take place via changes in the mitochondrial free Mg 2÷ concentration. In these studies isolated rat-liver m i t o c h o n d r i a were used and the intramitochondrial free Mg 1÷ c o n cen tr atio n was manipulated by vary'ing the level of citrate in the mitochondria, citrate being a p o t e n t chelator of Mg 2. ions [6]. Materials and Methods Liver m i t o c h o n d r i a from fed Wistar rats weighing 200--250 g were prepared according the m e t h o d of H o g e b o o m [7] as described by Myers and Slater [8] using 250 mM mannitol as the isolation medium. Incubations were carried out in 25 ml E rl enm eyer flasks in a medium containing the following standard c o m p o n e n t s : 75 mM Tris • HC1 (pH 7.4), 15 mM KC1, 5 mM potassium phosphate, 16.6 mM KHCO3, 10 mM ornithine, 10 mM NH4C1 and 25 mM mannitol (derived from the mitochondrial suspension). The reason for using mannitol rather than sucrose for isolating the m i t o c h o n d r i a is that sucrose interferes with the citrulline assay. The incubation volume was 1-5 ml. After being gassed with 95% 02 plus 5% CO2 the flasks were stoppered and allowed to equilibrate for 15 min at 25°C in an incubator before the addition of mitochondria. The incubations were terminated by rapid centrifugation of the m i t o c h o n d r i a from a 0.75 ml aliquot of the suspension through a layer o f silicone oil (Wacker Chemie AR 100) into a layer of 14% (w/v) HC104. Centrifugation was carried out in an E p p e n d o r f microcentrifuge Model 3200. Immediately after centrifugation a 0.6 ml aliquot of the extramitochondrial fluid was acidified with HC104 to a final c o n c e n t r a t i o n of 3% (w/v). After careful removal of the remainder of the supernatant the mitochondrial pellet was resuspended in order to obtain full extraction of the mitochondria. After removal of the protein by centrifugation, all samples were neutralized with KOH, the KC104 being removed in the cold. Citrulline was measured according to the m e t h o d of Hunninghake and Grisolia [9]. In experiments where m i t o c h o n d r i a were separated from the suspension medium by rapid centrifugation the citrulline assays were perform ed on the extramitochondrial fluid. In many cases the intramitochondrial c o n t e n t of citrulline was also determined. However, these values are n o t given in the tables and figures for two reasons: firstly, the a m o u n t of citrulline present in the m i t o c h o n d r i a was only a small part of the total citrulline form ed (less than 3%) and secondly, the assay was n o t accurate enough to det ect with great precision the small amounts of citrulline present in the m i t o c h o n d r i a under the conditions o f the experiments. All other metabolites were det er m i ned according to standard enzymic procedures [10,11], using either an E p p e n d o r f fluorimeter (for small amounts of metabolites) or the semiautomatic Micromedic MS II s p e c t r o p h o t o m e t e r . Intramitochondrial metabolites are always expressed as the a m o u n t of metabolite present inside the m i t o c h o n d r i a of 1 ml of incubation medium. Enzymes and nucleotides were purchased from Boehringer (Mannheim, Germany). Atractyloside was a gift f r om Prof. V. Sprio. Oligomycin was obtained

15 from Sigma (St. Louis, U.S.A.), DL-fluorocitrate from Calbiochem. (Lucerne, Switzerland) and the ionophore A23187 from Lilly (Indianapolis, U.S.A.). Results Effect o f citrate on citrulline synthesis by sonicated rat-liver mitochondria Fig. 1 shows that the synthesis of citrulline in sonicated mitochondria was dependent on the a m o u n t of Mg 2÷ added, as expected. Increasing the initial ATP concentration from 7 to 14 mM strongly inhibited the reaction rate at low but not at high Mg 2÷ concentration. This result, which is in agreement with the observations by others on isolated carbamoyl-phosphate synthase [3--5], clearly demonstrates that carbamoyl-phosphate synthase needs free Mg 2÷ ions for activity, in addition to MgATP [5]. Addition of 20 mM citrate in the presence of either 7 or 14 mM ATP strongly inhibited citrulline synthesis at the lower but not at the higher Mg2÷ concentrations. The slight inhibition of activity of carbamoyl-phosphate synthase at low ATP and high Mg ~÷ concentration, noted by Kerson and Appel [4], was also seen in our experiments (see Fig. 1) and is, as suggested by these authors, undoubtedly due to inhibition of the enzyme by high concentration of free Mg 2÷ ions.

]/

i1

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S 5

10

~5

20

25

Mg'* (mM)

F i g . 1. E f f e c t o f A T P a n d c i t r a t e o n t h e a c t i v a t i o n o f e a r b a m o y l - p h o s p h a t e s y n t h a s e b y M g 2+ i n s o n i c a t e d mitoehondria. Sonicated mitochondria (3.8 mg protein]ml) were incubated for 10 min in a medium cont a i n i n g 7 5 m M T r i s • HC1 ( p H 7 . 4 ) , 1 6 . 6 m M K H C O 3 , 1 0 m M o r n i t h i n e , 1 0 m M N H 4 C l , 1 0 m M N - a c e t y l g l u t a m a t e , 10 p g l m l o l i g o m y c i n , 1% e t h a n o l , 25 m M m a n n i t o l ( d e r i v e d f r o m the m i t o c h o n d r i a l s u s p e n s i o n ) a n d t h e c o n c e n t r a t i o n s o f M g 2+ i n d i c a t e d i n t h e f i g u r e . T h e gas p h a s e w a s 9 5 % 0 2 a n d 5% C O 2. F u r ther additions were: • • 7 mM ATP; • A 7 m M A T P a n d 2 0 m M c i t r a t e ; ~; ~, 1 4 m M A T P ; P: a, 14 m M A T P a n d 20 m M c i t r a t e .

16

The effect of the ionophore A23187 on flux through carbamoyl-phosphate synthase in intact rat-liver mitochondria In order to find out w he t he r changes in the intramitochondrial free Mg 2÷ c o n c e n t r a t i o n can control the activity of carbamoyl-phosphate synthase in intact rat-liver mitochondria, we first studied the effect of the i onophore A23187 on citrulline synthesis. Addition of this i o n o p h o r e has the effect of depleting the m i t o c h o n d r i a of Mg 2÷ [1'2]. The results of a typical experi m ent are shown in Fig. 2. Mitochondria were incubated in the standard medium with 10 mM glutamate as the oxidizable substrate in order to provide ATP for citrulline synthesis (see ref. 13). E G T A was present in order to prevent uncoupling by cyclic m o v e m e n t of endogenous Ca 2÷ across the mitochondrial inner membrane [12]. In order to diminish leakage of ATP from the mitochondria during the long period of incubation atractyloside was added [14]. The use of atractyloside rather than ext r a m i t ochondr i a l ATP for this purpose [14] allowed accurate meas u r em ent of intramitochondrial ATP w i t h o u t the need for correction of adherent ATP. In the e x p e r i m e n t of Fig. 2 the a m o u n t of extramitochondrial ATP was only 10--15% of that f o u n d in the mitochondria, indicating that only very small amounts of ATP leaked from the m i t o c h o n d r i a under the conditions o f the experiment. Addition of A23187 inhibited citrulline synthesis strongly ( >8 5% at a c o n c e n t r a t i o n of the i o n o p h o r e of 2 pg/ml). A23187 at a c o n c e n t r a t i o n of 2 pg/ml caused a 70% decrease in mitochondrial Mg 2÷ (results n o t shown). The inhibitory effect of A23187 on citrulline synthesis was completely prevented by the presence of 5 mM Mg 2÷ in the incubation medium. Reed and Lardy [12] have shown that oxidation of glutamate is impaired by Mg depletion of the mitochondria, presumably because of inhibition of aoxoglutarate dehydrogenase (EC 1.2.4.2). Since the inhibition of citrulline synthesis by A23187 in the absence of Mg 2÷ might have been due to lack to ATP the intramitochondrial c o n c e n t r a t i o n of ATP was also measured. Fig. 2B shows

A xJX~}(

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B

-\

E

E

0 I

A23787 (~g Imt )

2

1 2 ,e,23'r87 (;,,.g Ira0

F i g . 2. E f f e c t o f t h e i o n o p h o r e A 2 3 1 8 7 o n c i t r u l l i n e s y n t h e s i s i n i n t a c t r a t - l i v e r m i t o c h o n d r i a a n d o n m i t o chondrial ATP. Liver mitochondria (4.3 mg protein/ml) were incubated for 20 rain in a medium containi n g t h e s t a n d a r d c o m p o n e n t s p l u s ] m M E G T A , 1 0 m M g l u t a m a t e , 1 m M a t r a c t y l o s i d e , 1% e t h a n o l a n d A23187 at the concentrations indicated. • e Mg2 + a b s e n t ; X ×, 5 m M M g C l 2 p r e s e n t .

17 that a small decrease of ATP was observed not only in the presence of A23187 alone, but also when Mg 2÷ was added together with the ionophore. In the absence of Mg 2÷, the ATP level was about 80% of that in its presence, yet citrulline production was inhibited by more than 85%. Exactly analogous results were obtained with proline as substrate, in which case two oxidation steps not involving a-oxoglutarate dehydrogenase can be utilized (results n o t shown). It is unlikely, therefore, that the inhibition of citrulline synthesis by A23187 was due to lack of ATP.

Intrarnitochondrial citrate and flux through carbamoyI-phosphate synthase The experiment of Fig. 2 suggested that in principle the activity of carbamoyl-phosphate synthase can be controlled by changes in the intramitochondrial Mg 2÷ concentration. We therefore decided to manipulate the mitochondrial free Mg2÷ concentration in a more physiological manner, by bringing about variations in the intramitochondrial concentration of citrate. The mitochondria were loaded with citrate in two ways: either by generating citrate inside the mitochondria metabolically or by adding it to the mitochondrial suspension. In the experiments of Figs. 3 and 4 citrate was formed from pyruvate added either alone or together with malate. Fluorocitrate was added to prevent further metabolism of citrate [15]. The results of an experiment with different concentrations of fluorocitrate in the presence of pyruvate plus malate as substrates is shown in Fig. 3. As the concentration of fluorocitrate was increased citrate accumulation increased, both inside and outside the mitochondria. At the same time citrulline synthesis declined to about 30% of its control value at 20 pM fluorocitrate. In the experiment of Fig. 4 mitochondria were incubated with pyruvate alone. Under these conditions citrate can only be formed after carboxylation

B Citrate

A CitruUine 2

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FI uorocitrate (/U.M) Fig. 3. E f f e c t of f l u o r o c i t r a t e o n c i t r u U i n e s y n t h e s i s i n rat-liver m i t o c h o n d r i a s u p p l i e d w i t h p y r u v a t e a n d m a l a t e as r e s p i r a t o r y s u b s t r a t e s . L i v e r m i t o c h o n d r i a ( 5 . 1 m g p r o t e i n / m l ) w e r e i n c u b a t e d f o r 3 0 r a i n i n a medium containing the standard components plus 1 mM EDTA, 3 mM ATP, 10 mM pyruvate, 10 mM malate and the concentrations of fluorocitrate indicated. The values for intramitochondrial citrate (Fig. 3B) are c o r r e c t e d for t h e a m o u n t of citrate p r e s e n t in t h e sucrose space (the v o l u m e of the sucrose space was 1.2 # l / m g m i t o c h o n d r i a l p r o t e i n ) .

18

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Time (rain) F i g . 4 . E f f e c t o f f l u o r o c i t r a t e o n c i t r u l l i n e s y n t h e s i s i n r a t - l i v e r m i t o c h o n d r i a s u p p l i e d w i t h p y r u v a t e as s u b s t r a t e . L i v e r m i t o c h o n d r i a (5.3 m g p r o t e i n / m l ) were i n c u b a t e d in a m e d i u m c o n t a i n i n g the s t a n d a r d c o m p o n e n t s p l u s 1 m M E G T A , 3 m M A T P a n d 10 m M p y r u v a t e . I n t r a m i t o c h o n d r i a l citrate was c o r r e c t e d f o r c i t r a t e p r e s e n t i n t h e s u c r o s e s p a c e . M o r e t h a n 9 0 % o f t h e k e t o n e b o d i e s u n d e r all c o n d i t i o n s c o m p r i s e d a e e t o a c e t a t e , o-• Control;X--X,+3 pM fluorocitrate; . ,+6 pM fluorocitrate.

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F i g . 5. E f f e c t o f M g 2+ p l u s A 2 3 1 8 7 o n t h e i n h i b i t i o n o f c i t r u l l i n e s y n t h e s i s b y f l u o r o c i t r a t e i n r a t - l i v e r m i t o c h o n d r i a s u p p l i e d w i t h p y r u v a t e as s u b s t r a t e . L i v e r m i t o c h o n d r i a ( 2 . 8 m g p r o t e i n / m l ) w e r e i n c u b a t e d for 20 m i n in a m e d i u m c o n t a i n i n g t h e s t a n d a r d c o m p o n e n t s p l u s I m M E G T A , 3 m M ATP, 10 m M p y r u r a t e , 1 5 m M MgC12, 1% e t h a n o l a n d f l u o r o c i t r a t e a t t h e c o n c e n t r a t i o n s i n d i c a t e d . I n t r a m i t o c h o n d r i a l citrate was corrected for citrate present in the sucrose space. X X, C o n t r o l ; , 2 pg/ml A23187 present;O o 4pg/ml A23187 present.

19 of pyruvate. In the absence of fluorocitrate the characteristic time-lag in the production of citrulline was seen, in agreement with the observations of Charles et al. [ 1 3 ] . Both intra- and extramitochondrial citrate were very low under these conditions. With fiuorocitrate present a strong inhibition of citrulline formation was observed. At the same time, citrate accumulated both inside and outside the mitochondria (Figs. 4B and C). Fluorocitrate had n o effect on the production of ketone bodies (Fig. 4D) nor had it any appreciable effect of the intramitochondrial ATP concentration (results nor shown). The inhibitory effect of fluorocitrate on citrulline synthesis in the presence of pyruvate could partly be prevented by allowing added Mg 2÷ to enter the mitochondria in the presence of A 2 3 1 8 7 (Fig. 5A). Flux through carbamoylphosphate synthase could n o t be completely restored by Mg 2÷ + A 2 3 1 8 7 . This must have been due to the fact that the increase in mitochondrial Mg > was offset by a higher accumulation of citrate in the mitochondrial matrix under these conditions (Fig. 5B). In the experiment of Fig. 6, citrate was not generated metabolically inside the mitochondria but was added to the mitochondrial suspension. In one series of incubations, increasing amounts of citrate were added in the presence of isomalate as non-metabolizable activator of citrate entry into the mitochondria

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Fig. 6 . E f f e c t o f c i t r a t e o n c i t r u l l i n e s y n t h e s i s i n r a t - l i v e r m i t o c h o n d r i a . L i v e r m i t o c h o n d r i a ( 3 . 9 m g p r o tein/ml) were incubated for 20 min in a medium containing the standard components plus 1 mM EGTA, 3 m M A T P a n d , if p r e s e n t , 1 0 m M m a l a t e , 1 0 m M i s o m a l a t e o r 1 0 m M g l u t a m a t e . P o t a s s i u m c i t r a t e w a s added in the concentrations i n d i c a t e d i n F i g . 6 A . T h e K + c o n c e n t r a t i o n in t h e v a r i o u s i n c u b a t i o n s w a s k e p t c o n s t a n t w i t h K C l . I n F i g . 6 B t h e i n t r a m i t o c h o n d r i a l c i t r a t e ( s e e T a b l e I) is p l o t t e d a g a i n s t c i t r u l l i n e p r o d u c t i o n u n d e r t h e i n c u b a t i o n c o n d i t i o n s o f Fig. 6 A . s~ /~, I s o m a l a t e p r e s e n t ; o isomalate plus glutamate present; • e, malate present.

20

[16]. In the absence of citrate citrulline synthesis was low (Fig. 6A), presumably because the intramitochondrial synthesis of ATP was rate-limiting (see refs. 13,17). In this c o n t e x t it is i m p o r t a n t to not e that the extramitochondrial ATP present in the incubation medium cannot be used for citrulline synthesis because of the very slow transport of exogenous ATP into the m i t ochondri a under these conditions [17]. In the presence of isomalate, citrulline synthesis was maximal at an external citrate c o n c e n t r a t i o n of 5 mM. At higher citrate concentrations citrulline p r o d u c t i o n declined. In a second series of incubations citrate was added in the presence of malate to activate its entry into the m i t o c h o n d r i a [16]. In the presence of malate alone ab o u t 1.2 p m ol / m l of citrulline were synthesized (Fig. 6A). As increasing amounts of citrate were added a progressive inhibition of citrulline synthesis was observed (Fig. 6A). At 25 mM citrate the inhibition was almost 90%. In a third series of incubations, different concentrations of citrate were added together with isomalate plus glutamate. In the absence of citrate, citrulline synthesis was higher with glutamate than with malate, in confirmation of the observations of Charles et al. [13]. On adding increasing amounts of citrate citrulline synthesis was progressively decreased. Between 10 and 25 mM citrate, identical curves were obtained in the presence and absence of glutamate. Measurement of intramitochondrial citrate levels under the three sets of conditions revealed a relatively low level in the presence of isomalate and a very high level in the presence of malate (Table I). Thus there again appeared to be an inverse relationship between the rate of citrulline f o r m a t i o n and the concentration o f intramitochondrial citrate. This relationship is plotted in Fig. 6B. TABLE

I

METABOLISM ORNITHINE

OF CITRATE

IN RAT-LIVER

MITOCHONDRIA

INCUBATED

WITH AMMONIA

T h e e x p e r i m e n t is t h a t d e s c r i b e d i n F i g . 6 . I n t r a m i t o c h o n d r i a l c i t r a t e is c o r r e c t e d the sucrose space. Glutamate and aspartate were measured in the extramitochondrial Additions

Malate

Citrate

Intramitochondrial

A glutamate

A aspartate

citrate (nmol/ml

(~mol/ml)

(Dmol/ml)

2.5 5 10 15 25 Isomalate

Isomalate + glutamate

for citrate present in fluid.

added (mM) 0

0

medium)

2

0.06

0.25

28 71 91

0.13 0.17 0.19

0.44 0.49 0.49

107 224

0.24 0.43

0.66 1.25

4

0.01

0.02

2.5 5 10 15 25

10 28 33 -39

1.30 1.46 1.55 1.70 1.65

0.31 0.30 0.27 0.40 0.31

0 2.5

8 17

---

0.54 0.60

-40 46 51

-----

0.45 0.45 0.52 0.45

5 10 15 25

AND

2]

T A B L E II M E T A B O L I S M O F C I T R A T E IN R A T - L I V E R M I T O C H O N D R I A

INCUBATED WITH AMMONIA

L i v e r m i t o c h o n d r i a (4.1 m g p r o t e i n / m l ) w e r e i n c u b a t e d f o r 20 m i n in a m e d i u m c o n t a i n i n g 75 m M T r i s • HC1 ( p H 7 . 4 ) , 15 m M KC1, 5 m M p o t a s s i u m p h o s p h a t e , 10 m M N H 4 C l , 1 m M E G T A , 3 m M A T P , 6.8 m M c i t r a t e a n d , w h e r e p r e s e n t , 10 m M m a l a t e , 10 m M i s o m a l a t e , 10 m M o r n i t h i n e a n d 16.6 m M K H C O 3 ( o r 16.6 m M e x t r a KC1 w h e n K H C O 3 w a s a b s e n t ) . T h e gas p h a s e w a s as i n d i c a t e d in t h e T a b l e . A b b r e v i a t i o n s : citrull, c i t r u l l i n e ; glu, g l u t a m a t e ~ i s o c i t , i s o c i t r a t e . V a l u e s : p m o l / m l . Additions

A eitruU

_ A citrate

A glu

A isocit

H C O 3 , 5% C O 2 , 9 5 % CO 2 p r e s e n t None Malate Isomalate Ornithine Ornithine + malate Ornithine + isomalate

0.00 0.00 0.00 1.68 0.64 1.57

0.52 1.47 2.71 0.82 1.45 2.95

0.08 0.24 1.96 0.25 0.33 2.05

0.07 0.31 0.03 0.07 0.33 0.03

100% O 2 present None Malate Isomalate Ornithine Ornithine + malate Ornithine + isomalate

0.07 0.00 0.02 0.38 0.16 0.61

0.60 1.15 2.67 0.44 1.16 2.54

0.12 0.12 1.95 0.23 0.32 1.98

0.08 0.35 0.06 0.10 0.39 0.07

The question arises of why the level of intramitochondrial citrate is so much higher in the presence of malate than in the presence of isomalate. At first sight it is tempting to say that the rate of penetration of citrate into the mitochondria is faster with malate than with isomalate present (see ref. 18) which then results in a higher steady-state intramitochondrial citrate concentration. However, this explanation must be incorrect for the following reason. In the presence of ammonia, glutamate can be synthesized from a-oxoglutarate produced from citrate. Table I shows that synthesis of glutamate from citrate and ammonia was much lower in the presence of malate than in the presence of isomalate. This difference could not be accounted for by a conversion of glutamate to aspartate in the presence of malate (Table I). These observations suggest that, in fact, malate inhibits citrate metabolism under these conditions. Table II demonstrates that this effect of malate was also seen in the absence of ornithine and/or bicarbonate. Under all conditions malate inhibited both citrate consumption and glutamate synthesis. This inhibition was accompanied by an accumulation of isocitrate. It must be concluded, therefore, that the high concentration of citrate inside the mitochondria in the presence of malate was due to an inhibition of isocitrate dehydrogenase. Measurement of mitochondrial nicotinamide adenine nucleotides demonstrated that NAD in particular was more reduced in the presence of malate, citrate and ammonia than in the presence of isomalate, citrate and ammonia (results not shown). We suspect, therefore, that the inhibition of isocitrate oxidation was due to this accumulation of NADH, which leads to inhibition of NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) (see ref. 19).

Citrulline synthesis and intramitochondrial p H It has been reported that the activity of carbamoyl-phosphate synthase is pH

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?~.:

((

Fig. 7. E f f e c t of t h e p H o n c i t r u l l i n e s y n t h e s i s in s o n i c a t e d r a t - l i v e r m i t o c h o n d r i a . S o n i c a t e d m i t o c h o n d r i a (3.2 m g p r o t e i n / m l ) w e r e i n c u b a t e d f o r 10 m i n w i t h 75 m M Tris • HC1, 10 m M NH4C1, 10 m M ornit h i n e , 10 m M N - a c e t y l g l u t a m a t e , 10 m M A T P , 15 m M MgC12, 10 p g / m l o l i g o m y c i n , 1% e t h a n o l a n d 25 m M m a n n i t o l . T h e K H C O 3 c o n c e n t r a t i o n a d d e d w a s a d j u s t e d to give t h e p H r e q u i r e d , u s i n g t h e H e n d e r s o n - H a s s e l b a l c h e q u a t i o n . T h e f o l l o w i n g K H C O 3 c o n c e n t r a t i o n s w e r e u s e d : 3.4, 6, 9 . 4 , 15, 2 3 , 30, 3 7 , 4 6 , 59 a n d 74 m M a t t h e p H v a l u e s o f 6 . 8 , 7 . 0 , 7.2, 7.4, 7 . 6 , 7.7, 7.8, 7.9, 8.0 a n d 8 . 1 , r e s p e c t i v e l y . T h e K ÷ c o n c e n t r a t i o n at t h e v a r i o u s p H v a l u e s w a s k e p t c o n s t a n t at 74 m M w i t h KCl. T h e gas p h a s e w a s 9 5 % 0 2 a n d 5% CO 2.

dependent, the activity increasing as the pH is increased between pH 6 and 7.8 [20]. This is also shown in Fig. 7 where citrulline synthesis was followed in sonicated mitochondria at various pH values. Since different bicarbonate concentrations are in equilibrium with 5% CO2 at different pH values, the concentration of bicarbonate was adapted to the pH, using the Henderson-Hasselbalch equation. At pH 8.1 citrulline production was about twice as fast as at pH 6.8. The pH curve of Fig. 1 is quite similar to that obtained by GuthShrlein and Knappe [20] with isolated carbamoyl-phosphate synthase. T A B L E III CITRULLINE DRIA

SYNTHESIS AND INTRAMITOCHONDRIAL

p H IN I N T A C T R A T - L I V E R M I T O C H O N -

L i v e r m i t o c h o n d r i a (5.2 m g p r o t e i n / m l ) w e r e i n c u b a t e d f o r 30 m i n in a m e d i u m c o n t a i n i n g t h e s t a n d a r d c o m p o n e n t s plus 1 mM E G T A , 3 m M ATP, [ 3 H | H 2 0 (5 p C i / m l ) a n d e i t h e r t r a c e a m o u n t s of | 1 4 C ] s u c r o s e ( 0 . 2 p C i / m l ) o r , in parallel i n c u b a t i o n , 0 . 2 p C i / m l 5 , 5 ' - d i m e t h y l [ 2 - 1 4 C ] o x a z o l i d i n e - 2 , 4 - d i o n e . T h e c o n c e n t r a t i o n s o f t h e o t h e r c o m p o n e n t s , if p r e s e n t , w e r e : 10 m M g l u t a m a t e , 10 m M m a l a t e , 10 m M p y r u v a t e , 20 m M c i t r a t e , 20 m M p h o s p h a t e (in a d d i t i o n t o t h e 5 m M p h o s p h a t e a l r e a d y p r e s e n t in t h e b a s a l m e d i u m ) , 4 p M f l u o r o c i t r a t e a n d 2 p g / m l A 2 3 1 8 7 . T h e t o t a l w a t e r s p a c e w a s a l m o s t t h e s a m e in all i n c u b a t i o n s a n d v a r i e d b e t w e e n 3.0 a n d 3.3 pl p e r m g m i t o c h o n d r i a l P r o t e i n . Additions

Glutamate Glutamate Glutamate Glutamate Glutamate Pyruvate Pyruvate +

+ + + +

A23187 Pi malate malate + citrate

fluorocitrate

A citrulline (pmol/ml)

PHin

% sucrose space

5.45 0.11 4.36 2.23 0.27 7.63 2.56

7.76 7.67 7.69 7.72 7.73 7.81 7.65

64 70 62 57 61 62 62

23 In order to rule out the possibility t hat the inhibition of citrulline synthesis observed u n d er the various conditions described above was due to intramitochondrial pH effects the intramitochondrial pH was measured, using 5,5'dimethyl[2-~4C]oxazolidine-2,4-dione as the pH indicator [21]. Table III shows t hat the variations in intramitochondrial pH were rather small under the various incubation conditions and also shows a lack of correlation between the intramitochondrial pH and the rate of citrulline formation. Addition of A23187 in the presence of glutamate caused a decrease in the intramitochondrial pH from 7.76 to 7.67 whereas citrulline f o r m a t i o n was almost totally blocked. As a control, the phosphate c o n c e n t r a t i o n was enhanced from the basal level of 5 mM to 25 mM. This caused a similar drop in the mitochondrial pH, presumably as a consequence of the p h o s p h a t e - h y d r o x y l exchange across the mitochondrial membrane. In this case, however, only a slight inhibition of citrulline synthesis was observed, in agreement with previous observations by Charles et al. [13]. Addition of citrate in the presence of malate plus glutamate or addition of fluorocitrate in the presence of pyruvate did n o t result in appreciable mitochondrial pH changes, despite the fact that these manipulations drastically inhibited citrulline pr oduc t i on. The inhibition of citrulline synthesis by malate in the presence of glutamate, which was also observed by Charles et al. [13], was n o t associated with any significant intramitochondrial pH change, either. The mechanism of this effect is at present u n d er investigation. Discussion In intact parenchymal cells isolated from rat liver the c o n c e n t r a t i o n of ADP plus ATP in the mitochondrial fraction is 1--1.2 pmol per g wet weight [22, 23], with ATP slightly higher than or equal to ADP, depending on the conditions [22--24]. Assuming that 1 g wet weight equals 60 mg mitochondrial protein [25] it follows that the mitochondrial matrix contains 16--20 nmol ADP plus ATP per mg mitochondrial protein. The Mg c o n t e n t of the mitochondrial inner m e m b r a n e plus matrix is presumably a b o u t 10 n m o l / m g mitochondrial protein [26]. Considering that ATP is a m o r e p o t e n t chelator of Mg 2÷ than citrate or ADP (Kas s = 13 870 M -1 for ATP 4- [27], Kass = 1318 M -1 for ADP 3[27] and Kass = 4507 M -l for citrate 3- [6]), it is unlikely that mitochondrial citrate will ever be so high as to prevent f o r m a t i o n of MgATP, which is one of the substrates of carbamoyl-phosphate synthase [5]. However, the e n z y m e also requires free Mg 2÷ for its activity [3--5]. According to Bogucka and Wojtczak [26] the a m o u n t of total Mg 2÷ present in the mitochondrial matrix is a b o u t 10 nmol per mg mitochondrial protein. Since the volume of the mitochondrial matrix is a b o u t 1 gl per mg protein [28], it follows that the c o n c e n t r a t i o n of total Mg 2÷ present in the mitochondrial matrix is 10 mM. If the ATP c o n c e n t r a t i o n in the mitochondrial matrix is taken to be 8 mM (see Fig. 2), a c o n c e n t r a t i o n of free Mg 2÷ of a b o u t 2.2 mM can be calculated, using the stability constant of the MgATP com pl ex mentioned above and ignoring the binding of Mg 2÷ to matrix proteins. If 10 mM citrate is also present inside the m i t o c h o n d r i a under these conditions, it can be

24 calculated that the mitochondrial free Mg > c o n c e n t r a t i o n will decrease to 0.16 mM. Kinetic studies p e r f o r m e d with low concentrations of carbamoyl-phosphate synthase isolated from bovine liver have shown a K m of a b o u t 0.2 mM for free Mg 2+ under various conditions [5]. Fr om the e x p e r i m e n t of Fig. 1 it can be easily calculated t hat the Km of carbamoyl-phosphate synthase for free Mg 2÷ in sonicated rat-liver m i t o c h o n d r i a is of the same order of magnitude. For example, let us consider the condition with 10 mM total Mg 2÷ and with an initial ATP c o n c e n t r a t i o n of 14 mM present. During the course of the incubation, the ATP c o n c e n t r a t i o n decreased to 10 mM (data n o t shown). Let us therefore take the average ATP concent r a t i on as 12 mM. With 10 mM total Mg 2÷ and 12 mM total ATP present, the corresponding free Mg 2÷ concent rat i on is 0.3 mM. Since under these conditions the rate of citrulline synthesis approached its m a x i m u m value, it follows that the K m for free Mg 2÷ was 0.1--O.2 mM. It has been shown recently that the c o n c e n t r a t i o n of carbamoyl-phosphate synthase in the mitochondrial matrix is probably very high [29,30], perhaps as high as 0.4 mM [30]. It remains to be seen w het her in the intact mitochondrion, at this high e n z y m e concent r a t i on, the c o n c e n t r a t i o n of free Mg 2÷ required to give half-maximal activation of the e n z y m e is also 0 . 1 - 0 . 2 mM. In any case, in our experiments we observed a 50% decrease in the rate o f citrulline p r o d u c t i o n at citrate levels of a b o u t 10 nmol per mg mitochondrial protein (see Figs. 4, 5 and 6B), which is equal to a c o n c e n t r a t i o n of 10 mM. We therefore believe that this inhibitory effect of mitochondrial citrate on citrulline synthesis is due to a decrease in the mitochondrial free Mg 2÷ concentration. Measurements of intramitochondrial citrate in isolated h e p a t o c y t e s under various experimental conditions have indicated that a mitochondrial concentration of citrate of 10 mM is n o t exceptionally high [23,24,31]. In principle, then, it is possible t hat in the intact h e p a t o c y t e , flux through carbamoyl-phosphate synthase can be controlled, at least in part, by the mitochondrial free Mg > c o n c e n t r a t i o n and thus by the mitochondrial c o n c e n t r a t i o n of citrate. The experiments r e p o r t e d in this paper have been p e r f o r m e d with isolated m i t o c h o n d r i a using high concentrations of ammonia. Although these conditions are highly non-physiological, our experiments do dem onst rat e that the activity of carbamoyl-phosphate synthase can be strongly suppressed by citrate. This effect can be of physiological relevance only if it can be d e m o n s t r a t e d (a) that the rate o f citrulline p r o d u c t i o n in isolated m i t o c h o n d r i a is a reliable measure of the activity of carbamoyl-phosphate synthase, and (b) that the maximal activity of carbamoyl-phosphate synthase is n o t in a very large excess of the rate of urea synthesis in vivo. Maximum rates of citrulline p r o d u c t i o n in our experiments ranged from 30-50 n m o l / m i n per mg mitochondrial protein, at 25°C, with either glutamate or pyruvate as the respiratory substrate (see also ref. 13). This value can be converted to 1.8--3 tlmol/min per g liver, since 1 g liver contains 60 mg mitochondrial protein [25]. These rates are equal to those r e p o r t e d by Charles et al. [13] and are slightly higher than those r e p o r t e d by McGivan et al. [2], who used succinate as the respiratory substrate. The m a x i m u m activity of carbamoyl-phosphate synthase in rat liver is 330 p m o l / h per g liver at 37°C [32], which is 5.6 p m o l / m i n per g liver. Thus our values for the rate of citrulline pro-

25 duction (measured at 25°C) are close to the m a x i m u m activity of carbamoylphosphate synthase. We conclude that citrulline production in isolated mitochondria is a good measure of the activity of carbamoyl-phosphate synthase (see ref. 2). In this context it is important to note that ornithine carbamoyltransferase (EC 2.1.3.3) is about 40 times more active than carbamoyl-phosphate synthase [32] so that it is unlikely that this enzyme is a rate-limiting factor in our test system. The second question to be answered is whether the activity of carbamoylphosphate synthase is in great excess of the rate of urea synthesis in vivo, or not. According to Schimke [32] the daily urea excretion in the rat is 70 mg/ g liver which is equal to 0.9 pmol urea/min per g liver. Thus flux through the urea cycle in vivo is 15% of the maximal activity of carbamoyl-phosphate synthase (see above) and about 20% of the maximal capacity of the urea cycle as determined in isolated hepatocytes with high concentrations of ammonia (see ref. 33). In view of the fact that in the liver the ammonia concentration is 0.7 mM [34,35] (which is not saturating for carbamoyl-phosphate synthetase [3-5]) and in view of the fact that the concentration of N-acetylglutamate inside the mitochondria is in all likelihood also n o t saturating [30,36], it must be inferred that the activity of carbamoyl-pl~osphate synthase is not in great excess of the rate of urea synthesis in vivo and may limit flux through the urea cycle in vivo. We believe, therefore, that the inhibitory effects of citrate on the activity of carbamoyl-phosphate synthase, observed in isolated mitochondria, may be of physiological relevance. Whether control of carbamoyl-phosphate synthase activity by mitochondrial citrate really occurs in vivo of course remains to be proven experimentally. Such a mechanism could provide the liver with a means of keeping the NH3 concentration in the hepatocyte within narrow limits. Firstly, it provides the liver with a mechanism to remove NH3 efficiently after a rise of the NH3 concentration in the portal vein: as soon as the concentration of NH3 in the portal vein increases, mitochondrial citrate can be expected to fall, because of removal of a-oxoglutarate for glutamate synthesis and because of a decrease in the redox potential of NAD(P) (see ref. 24). This fall in citrate will then activate carbamoyl-phosphate synthase. Secondly, and this is perhaps even more important, it provides the liver with a mechanism to prevent a complete drain of all NH3 present in the hepatocyte into the urea cycle, so that the concentration of NH3 in the hepatocyte will never fall to zero. The importance of maintaining a certain a m o u n t of NH3 in the hepatocyte has been stressed by Krebs et al. [34]. In the hepatocyte a certain a m o u n t of NH3 must always be available for synthesis of certain nitrogencontaining compounds that are vital for the functioning of the hepatocyte, for example, glutamine, which is required for the biosynthesis of nucleotides. Furthermore, under certain conditions there is a net o u t p u t of glutamine from the liver (see ref. 37). Experiments are currently in progress in order to see whether in the intact hepatocyte, flux through carbamoyl-phosphate synthase under various incubation conditions can be controlled by the concentration of citrate in the mitochondria.

26

Acknowledgements The authors are very grateful to prof. Dr J.M. Tager for critically reviewing the manuscript. This study was supported by a grant from the Netherlands Foundation for Pure Scientific Research (Z.W.O.} under auspices of the Netherlands Foundation for Chemical Research (S.O.N.). References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

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