- Email: [email protected]
Arginine uptake by isolated rat liver mitochondria
Bioehimica et Biophysiea Acta, 802 (1984) 407-412
407
Elsevier
BBA 21933
ARGININE UPTAKE BY ISOLATED RAT LIVER MITOCHONDRIA R.A. F R E E D L A N D a,. G.L. CROZIER a, B.L. HICKS a and A.J. MEIJER b
" Department of Physiological Sciences, School of Veterinary Medicine, University of California Davis, Davis, CA 95616 (U.S.A.), and h Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, P.O. Box 20151, 1000 HD Amsterdam (The Netherlands) (Received June 27th, 1984)
Key words: Arginine uptake," Amino acid transport; Urea synthesis; N-Acetylglutamate; (Rat liver mitochondria)
The question of arginine uptake by mitochondria is important in that arginine is an allosteric effector of N-acetylglutamate synthetase. Thus, changes in mitochondrial arginine concentration have the potential for acutely modifying levels of N-acetylglutamate, a compound necessary for maximal activity of carbamyl phosphate synthesis. Mitochondria were isolated from chow-fed rats, incubated with [guanido-14C]arginine and were centrifuged through silicon oil into perchloric acid for determination of intramitochondrial metabolites. Arginine was separated from urea by cation-exchange resin. Mitochondrial water space was determined by [14C]urea arising from arginase activity associated with the mitochondrial preparations. Extramatrix space was determined by parallel incubations with [inulin- 14 C l c a r b o x y l i c acid or [ 14 C]sucrose. There was considerable degradation of arginine by arginase associated with the mitochondrial preparation. This was inhibited by 7 mM ornithine and 7 mM lysine. Arginine was concentrated intramitochondrially to 4-times the extramitochondrial levels. The concentration ratio was decreased in the presence of ornithine and lysine but not with citrulline, NH4CI , glutamate, glutamate or leucine. No uptake was observed when mitochondria were incubated at 0°C. Mitochondria did not concentrate citrulline. Introduction Marshall et al. [1] have shown that N-acetylglutamate is a necessary allosteric effector of carbamyl-phosphate synthetase (EC 6.3.4.16). In rat liver, N-acetylglutamate activates carbamylphosphate synthetase by changing conformation and subunit structure of the enzyme [2] with a K a of approx 0.8 n m o l / m g mitochondrial protein [3]. N-Acetylglutamate levels increased in liver of rats injected with amino acids [3], and in mitochondria isolated from rats fed high protein diets [4], or injected with glucagon [5,6]. In mitochondria, Nacetylglutamate concentrations were shown to increase over a 12 rain incubation [7], and McGivan * To whom correspondence should be addressed. Abbreviation: Mops, 4-morpholinepropanesulfonic acid. 0005-2728/84/$03.00 © 1984 Elsevier Science Publishers B.V.
et al. [8] correlated changes in mitochondrial Nacetylglutamate content with rates of citrulline synthesis from NH4C1. In isolated rat hepatocytes treated with pent-4-enoate, N-acetylglutamate levels were lower compared with controls, as was the synthesis of urea and citrulline [9]. Thus, there is much evidence suggesting that N-acetylglutamate plays an important regulatory role in carbamyl phosphate synthetase activity. McGivan et al. [4] have shown that rates of ureagenesis from ammonia in cells paralleled rates of citrulline synthesis in mitochondria, indicating that under these conditions, the overall rate of urea synthesis is regulated by carbamyl-phosphate synthetase activity. Therefore, factors which control the synthesis of N-acetylglutamate have the potential for the acute regulation of ureagenesis. N-Acetylglutamate is synthesized in liver mitochondria from acetyl-
408 CoA and glutamate by the mitochondrial aminoacid acyltransferase (EC 2.3.1.1) [10]. Arginine is an allosteric modulator of this enzyme and acts by increasing its Vmax,although it has no effect on K m values for the substrates [11]. Arginine is a specific activator for N-acetylglutamate synthetase: activation does not occur by any other amino acid nor intermediate of the urea cycle [12]. The physiological importance of arginine is positively modulating N-acetylglutamate synthetase is strongly suggested by the experiments of Tatibana and Shigesada [13], who injected arginine intraperitoneally into mice and found increased liver levels of N-acetylglutamate, In rat liver, N-acetylglutamate levels are correlated with arginine, ornithine [13] and urea [14]. There is, however, controversy about whether the arginine effect on N-acetylglutamate synthetase has importance in vivo. The enzyme is active even in the absence of arginine [12] and the optimal activity of the enzyme is higher in sonicated mitochondria from rats fed a standard diet than in mitochondria from glucose-fed rats [4]. Although arginine injected in mice resulted in increased liver N-acetylglutamate [15], N-acetylglutamate was also observed to increase if rats were injected with NH4CI [16] or amino acid mixtures containing no arginine [3]. Obviously, variations in the mitochondrial arginine concentration within the range of its K~ of 5-10 /~M for N-acetylglutamate synthetase [11] must occur if arginine were to play an important regulatory role in N-acetylglutamate synthesis. Total arginine levels in liver of 20-50 n m o l / g wet wt. [15,3,17] exceed the K a for N-acetylglutamate synthesis although it is presently unknown how much of this arginine is present in the mitochondria. Finally, arginase (EC 3.5.3.1) in liver is exceedingly active and appears to be associated with the particulate cellular components, including approx. 10% with the mitochondria [18,19]. The extensive degradation of arginine by mitochondrial preparations have complicated studies of arginine uptake [20], and its effect on citrulline synthesis [19] and ornithine uptake [21]. In this paper, we report our findings on the uptake of arginine by isolated rat liver mitochondria and on the effects of certain amino acids on this. The method is unique in that the use of [guanido-]4C]arginine permitted us to
quantify the extent of degradation of arginine as well as ensure that once separated from urea, intramitochondrial label was arginine and not a product of its degradation such as ornithine. Materials and Methods
Male Sprague-Dawley rats weighing 195-300 g were obtained from the University of California, Davis, breeding colony and were fed Purina rat chow (Ralston Purina, St. Louis, MO) ad libitum. Mitochondria were isolated by standard centrifugation techniques as described by Chappell and Hansford [22]. Livers were homogenized in 2.5 vol. of ice-cold medium consisting of 250 mM mannitol, 5 mM Mops, and 0.5 mM E G T A at pH 7.0. Mitochondrial coupling was verified with a Gilson 5 / 6 oxygraph with a Clark electrode. The mean respiratory control index was 4.1 + 1.2 with succinate as a substrate. Mitochondrial protein was determined by the biuret method as described by Mokrasch and McGilvery [23]. For determination of arginine uptake, 21-42 mg mitochondria were suspended in 7 ml medium (pH 7.4) consisting of 75 mM Mops, 20 mM K2HPO4, 4 mM EGTA, 15 mM KCI, 16.6 mM K H C O 3, 2.2 rag/1 rotenone, 0.01 /LCi/ml [guanido-laC]arginine and substrates as noted in the tables. Incubations were carried out at 25 or 0°C as noted. For separation of the mitochondria from the surrounding medium, a 1 ml sample of the mixture was gently layered over 0.35 ml silicon oil (density, 1.035) which had been layered over 0.15 ml 14% HC104 in a 1.5 ml microcentrifuge tube (Walter Sarstedt, Princeton, N J). Tubes were immediately centrifuged for 1 rain in an Eppendorf 5412 microcentrifuge (12000 × g). In order to obtain accurate estimates of arginine breakdown, synchronous samples of the incubation mixture were deproteinized with HCIO 4. These tubes were also centrifuged. Samples were taken as close to zero time as possible, usually within 30 s, and again at 5, 10, 20 and 30 rain. The top layer of the first set of tubes and a portion of the silicon oil were aspirated, and 0.5 ml of water was added to the acid/mitochondria layer, and the whole thoroughly mixed to ensure dispersion of metabolites. The tubes were re-
409
centrifuged to bring down the oil and protein fractions. Aliquots of both the mitochondrial fraction and the total deproteinized medium were passed over a 7 cm Dowex 50W-X1 (H + form, 50-100 mesh) cation-exchange resin in a pasteur pipette. Urea was eluted in 6 ml of water, and arginine was eluted with 6 ml of 1 M N a O H . The latter was neutralized by the addition of HC1 and radioactivity in aliquots of both fractions was quantitated by liquid scintillation counting (Packard Tricarb 2660). The fluor used was tritoso124. Total water space was routinely determined with [14C]urea arising from degradation of [guanido-14C]arginine. For several mitochondrial preparations, non-matrix water was determined by incubating mitochondria with [3H]or [14C]inulin, or [14C]sucrose prior to spinning through oil. Of the total water associated with the mitochondria, 70% _+ 4.0 was non-matrix water, and the remainder was assumed to be intra-matrix water. Calculations
Calculations of the matrix arginine concentration and medium arginine concentration were as follows: matrix arginine = ([total mito Arg ( d p m ) - n o n matrix Arg (dpm)]/matrix volume (~l))/spec. act. Arg (dpm/~umol) and medium Arg = total Arg in medium ( d p m / m l ) / spec. act. Arg (dpm//lmol).
100 E
H ~ \
.-=
80
c
60
.c "o
~ \ ~
~- 4 . 0 /~M arginine ~ 4 . 0 / ~ M arginine + 7 m M o r n i t h i n• ~ +• - -
40 2O
0
I
~
io
~o
~o
Time (min) Fig. l. Arginine remaining in the medium throughout a 30 min incubation with mitochondria (3-6 m g / m l ) (n = 2). Arginine concentration was added isotope only, i.e., no unlabelled arginine was added in these incubations.
way were compared with those determined with 3H20. These were identical with the exception of those estimates determined at the initial time. At this point, the urea formed was small, and not fully equilibrated with the total water. The addition of 7 mM ornithine inhibited arginine degradation by arginase (Fig. 1) and 7 m M lysine had a similar effect, although this inhibition was not as strong as that of ornithine (data not shown). This is consistent with the finding that ornithine [19] and lysine are inhibitors of arginase [25]. The results in Fig. 2 show that mitochondria were able to concentrate arginine. After a lag period of about 10 min, the mitochondria achieved a mean concentration ratio near four and main-
Results
The activity of arginase in the mitochondrial preparation was high and resulted in rapid and considerable degradation of arginine in the medium (Fig. 1). By 30 min of incubation, as little as 14% of the added arginine remained. In this experiment, utilization of [guanido-laC]arginine combined with deproteinization of samples of the medium in synchrony with mitochondrial separations enabled us to quantitate accurately the extent of arginine degradation at any given point. In addition, the liberation of [14C]urea and its rapid equilibration with water permitted us a convenient estimation of total mitochondrial water space. In a few trials, water spaces obtained with urea in this
,.o
c
•~ ,
10
o U oo
6
~
4
"6
2
-~
4.0 #M arginine
0
0 I
5
1(3
1'5
;~0
3'0
Time (rain)
Fig. 2. Representative plot of the ratio of mitochondrial to external medium arginine levels throughout a 30 min incubation.
410 t a i n e d this value relatively c o n s t a n t t h r o u g h o u t the next 20 min. T h e c o n c e n t r a t i o n of arginine in isolated m i t o c h o n d r i a is in accord with levels f o u n d in c y t o s o l a n d m i t o c h o n d r i a , w h i c h were d e t e r m i n e d in cells d i s r u p t e d b y d i g i t o n i n before s e p a r a t i o n of the two c o m p o n e n t s b y spinning t h r o u g h oil. In these studies, m i t o c h o n d r i a l arginine levels also exceeded cytosolic levels [26]. A few studies were carried out with mitoc h o n d r i a which were u n c o u p l e d during p r e p a r a tion, as j u d g e d b y low r e s p i r a t o r y control index values. These m i t o c h o n d r i a were u n a b l e to conc e n t r a t e arginine, a n d this p r o v i d e d a negative c o n t r o l s h o w i n g that the i n t e g r i t y of the m i t o c h o n d r i a is necessary for arginine uptake. In a d d i t i o n , these studies showed that our results in c o u p l e d m i t o c h o n d r i a could not be e x p l a i n e d by nonspecific b i n d i n g of arginine to the mitochondrial membrane. Effect of o t h e r a m i n o acids a n d NH4C1 on m i t o c h o n d r i a l c o n c e n t r a t i o n of arginine. The level of external arginine had no effect on its c o n c e n t r a tion b y m i t o c h o n d r i a ( T a b l e I). T h e a d d i t i o n of the basic a m i n o acids, o r n i t h i n e or iysine i n h i b i t e d
TABLE I EFFECT OF VARIOUS CONDITIONS ON MITOCHONDRIAL CONCENTRATION OF ARGININE Ratios, with their standard deviations, are concentrations of intramitochondrial arginine over medium arginine, averaged over 10, 20 and 30 min. Numbers in parentheses are the numbers of determinations. Conditions
Ratio
4.0 ~M arginine 1.0 mM arginine 7.0 mM arginine 4.0/~ M arginine + 7 mM ornithine 4.0 p~M arginine+ 1 mM ornithine 7.0 mM arginine + 7 mM ornithine 4.0/~M arginine+ 7 mM lysine 4.0/~M arginine + 7 mM citrulline 4.0 ~M arginine + 7 mM NH~4.0/~M arginine at 0°C 1.0 mM arginine at 0°C 4.0 p~M arginine+0.1 mM ATP 4.0 ~M arginine + 0.1 mM ADP 4.0/~M arginine + 7 mM glutamine 4.0/LM arginine + 7 mM glutamate 4.0 ~M arginine + 7 mM leucine
4.3 _+0.3 (3) 3.3 _+0.3 (2) 3.7 _+0.3 (2) 1.2 +_0.2 (3) 2.4_+0.3 (4) 2.4 1.8_+0.3 (2) 4.1 4.1 0.4 0.5 3.9+0.2 (2) 4.1 _+0.2 (2) 3.7 3.9 3.6
the u p t a k e of arginine by m i t o c h o n d r i a with o r n i t h i n e having a stronger effect. This is consistent with the findings of H i l d e n and Sacktor [27] in r a b b i t renal brush b o r d e r m e m b r a n e vesicles, a n d of Keller [20] in isolated dog kidney m i t o c h o n d r i a . B r a d f o r d a n d M c G i v a n [28] have shown that o r n i t h i n e and citrulline are exchanged across the m i t o c h o n d r i a l m e m b r a n e b y an o r n i t h i n e / c i t r u l l i n e antiporter, a n d we were curious to see wether citrulline affected arginine uptake. F r o m T a b l e I it can be seen that citruiline h a d no effect on arginine c o n c e n t r a t i o n by the mitochondria. L y s o s o m e s are c o s e d i m e n t e d with m i t o c h o n d r i a , a n d we considered the a r g u m e n t that the conc e n t r a t i o n ratio seen here was due to sequestration of arginine by lysosomes based on their p H differential rather than true m i t o c h o n d r i a l uptake. Thus, we tested the effect of 7 m M NH4CI, since lysosomes would also sequester N H 4 (intralysos o m a l p H 5.5 or less). If lysosomal sequestration were an i m p o r t a n t factor, we would see a decrease in arginine u p t a k e in the presence of a m m o n i a . T a b l e I shows that 7 m M N H 4 C I had no effect on arginine uptake, a n d this is an i n d i c a t i o n that the u p t a k e p h e n o m e n o n is not p r i m a r i l y lysosomal. F u r t h e r evidence for this derives from studies of W a r d and M o r t i m o r e [29], who were u n a b l e to show a c c u m u l a t i o n of arginine in lysosomes from rat liver. W e further tested glutamate, an acidic a m i n o acid, and glutamine, a neutral a m i n o acid, b o t h of which are k n o w n to be taken up a n d m e t a b o l i z e d by m i t o c h o n d r i a [30,31]. N e i t h e r these a m i n o acids nor leucine, a neutral a m i n o acid which stimulates m i t o c h o n d r i a l g l u t a m a t e synthesis [32], affected arginine uptake. The a d d i t i o n of A D P or A T P d i d not affect the c o n c e n t r a t i o n ratio i n d i c a t i n g that the availability of A T P or A D P was not limiting for arginine uptake. M i t o c h o n d r i a i n c u b a t e d at 0 ° C failed to a c c u m u l a t e arginine indicating that it was a t e m p e r a t u r e - d e p e n d e n t p h e n o m e n o n . Because of the relative c o n s t a n c y of the ratio over m a n y different conditions, we p o s e d the question that p e r h a p s the p h e n o m e n o n observed was not characteristic of arginine, but rather could be generalized over a range of a m i n o acids, or possibly was related to our m e t h o d o l o g y . To test this, we investigated the u p t a k e of citrulline in studies
411 TABLE II MITOCHONDRIAL LINE
CONCENTRATION
OF
CITRUL-
Ratios are intramitochondrial citrulline concentrations divided by medium citrulline concentrations, averaged over 10, 20 and 30 min. [ureido-14C]Citrulline was used in lieu of [guanido14C]arginine. Conditions
Ratio
5.0 `aM citrulline 5.0 ,aM citrulline + 7 mM ornithine 7.0 mM citrulline 7.0 mM citrulline+ 7 mM ornithine
0.74 0.74 0.69 0.69
using [ureido-14C]citrulline. Citrulline is synthesized in the mitochondria and readily exits this subcellular compartment [21,28,33]. Table II shows that in mitochondria incubated with trace amounts or 7 mM citrulline with or without the addition of ornithine, there was no concentration of citrulline, thus the phenomenon is characteristic of, though not necessarily limited to, arginine. In studies with heart mitochondria, we observed that the tritiated water to [14C]arginine ratio was similar in the surrounding medium and the mitochondria. Tritiated water was used in these experiments to give total water space, since in the absence of arginase there would be no urea produced nor arginine destroyed. The value for arginine concentration per unit of water in the mitochondria compared to the surrounding medium was 0.97 _+ 0.05 for three separate determinations. Discussion
The results of this investigation clearly demonstrate the inhibitory effect of lysine and ornithine on arginine uptake by isolated rat liver mitochondria. This effect has also been noted in renal brush-border membrane vesicles from rabbit [27], isolated renal cortex mitochondria from dogs [20], and isolated kidney mitochondria from chickens [34]. In addition, Bradford and McGiven [28] showed that lysine and 6-aminocaproate, a lysine analogue, as well as ornithine stimulated citrulline efflux from preloaded mitochondria. These studies suggest that a single translocator may be involved in the transport of arginine, lysine and ornithine.
The fact that heart mitochondria failed to concentrate arginine against a gradient indicates that nonspecific binding by inner or outer membrane of the mitochondria could not be the explanation for the observed phenomena with the liver mitochondria. Furthermore, these studies with the heart mitochondria indicate that a proton pumping could not be the sole explanation for this concentration, since both heart and liver mitochondria show this phenomenon. The present investigation also confirms the association of arginase with isolated rat liver mitochondria [19,18], and its inhibition by lysine [25] and ornithine [19]. The metabolic significance of this remains to be established, but one possibility is that arginase provides a means of generating ornithine from arginine near the mitochondrial membrane for anaplerosis of the urea cycle [19]. For the production of urea, ornithine leaves the mitochondria as citrulline and, for the continuation of the cycle, it must return. Raijman and co-workers [17] have shown that ornithine may be a limiting substrate for ornithine transcarbamylase activity. Thus, the functional advantage of the proximity of arginase to the mitochondrial membrane may lie in an important role it may play in regulating the urea cycle by providing ornithine. We have shown that arginine is concentrated by isolated rat liver mitochondria despite the high activity of arginase in mitochondrial preparations. Liver levels of arginine in normal rats range from 20-50 n m o l / g wet wt. [3,15,17]. Assuming cells to be 2 / 3 water, this would mean that the arginine concentration would be 30-75/~M. If the ratio in vivo between the cytosol and mitochondrial arginine levels is similar to the 1 : 4 ratio we have observed in these studies in isolated mitochondria, the mitochondrial concentration of arginine would be 120-300/~M, many times greater than its K, of 5 10 /~M [11] for N-acetylglutamate synthetase. Thus, if the kinetic properties of N-acetylglutamate synthetase as it occurs in the mitochondrial matrix are similar to those of the purified enzyme, it is unlikely that arginine plays an important role in the regulation of N-acetylglutamate synthetase. Cheung and Raijman [19] have shown that the stimulation of citrulline synthesis in isolated mitochondria incubated with substrates for Nacetylglutamate and arginine was only slightly
412 greater than in mitochondria incubated with a r g i n i n e a l o n e . A l t h o u g h i n i t i a l levels o f N - a c e t y l glutamate were not measured, this data, in concert with ours showing substantial mitochondrial uptake of arginine regardless of initial arginine conc e n t r a t i o n i n t h e m e d i u m , give f u r t h e r w e i g h t to the hypothesis that arginine stimulation of citrull i n e s y n t h e s i s in v i v o is o f little i m p o r t a n c e in t h e regulation of urea synthesis. Thus, the metabolic importance of the m i t o c h o n d r i a l c o n c e n t r a t i o n o f a r g i n i n e is u n l i k e l y to be related to an influence of N-acetylglutamate synthetase. Mitochondria can synthesize protein [35], t h u s it is p o s s i b l e t h a t t h e u p t a k e o f t h i s amino a c i d m a y s e r v e to c o m p a r t m e n t a l i z e i n t r a c e l l u l a r a r g i n i n e at a site o f e s s e n t i a l p r o t e i n s y n t h e s i s . S i n c e t h e r e is l i t t l e e v i d e n c e f o r i n t r a m i tochondrial arginase, mitochondrial arginine may a l s o s e r v e as a p r o t e c t e d p o o l o f a n o r n i t h i n e p r e c u r s o r f o r t h e u r e a cycle.
Acknowledgements We thank the following undergraduate students w h o p a r t i c i p a t e d i n v a r i o u s a s p e c t s o f t h i s res e a r c h : D. Byerly, P. C h c u n g , S. D o n o v a n , M . D w i g h t , C. G i l l e s p i e , M . M a l l e r , B. S e e m a n , J. S h r a g e r a n d K. S p r a y b e r r y .
References 1 Marshall, M., Metzenberg, R.L. and Cohen, P.P, (1961) J. Biol. Chem. 236, 2229-2237 2 Guthorlein, G. and Knappe, J. (1968) Eur. J. Biochem. 7, 119-127 3 Stewart, P.M. and Walser, M. (1980) J. Biol. Chem. 255, 5270-5280 4 McGivan, J.D., Bradford, N.M. and Mendes-Mourao, J. (1976) Biochem. J. 154, 415-421 5 Cathelineau, L., Rabier, D., Petit, F. and Kamoun, P. (1980) CR Acad. Sci. Ser. D291, 464-466 6 Verhoeven, A.J., Hensgens, H.E.S.J., Meijer, A.J. and Tager, J.M. (1982) FEBS Lett. 140, 270-272 7 Meijer, A.J. and Van Woerkom, G.M. (1978) FEBS Lett. 86, 117-121 8 McGivan, J.D., Bradford, N.M. and Chappell, J.B. (1974) Biochem. J. 142, 359-364 9 Aoyagi, K., Mori, M. and Tatibana, M. (1979) Biochim. Biophys. Acta 587, 515-521
10 Shigesada, K. and Tatibana, M. (1971) J. Biol. Chem. 246, 5588-5595 11 Shigesada, K. and Tatibana, M. (1978) Eur. J. Biochem. 84, 285-291 12 Shigesada, K. and Tatibana, M. (1971) Biochem. Biophys. Res. Commun. 44, 1117 1124 13 Tatibana, M. and Shigesada, K. (1976) in The Urea Cycle (Grisolia, S., Baguena, R. and Mayor, F., eds.), pp. 301 314, Wiley, New York 14 Saheki, T. and Katsunuma, T. (1977) J. Biochem. 82, 551-558 15 Shigesada, K., Aoyagi, K. and Tatibana, M. (1978) Eur. J. Biochem. 85, 385-391 16 Saheki, T., Ohkubo, T. and Katsunuma, T. (1978) J. Biochem. 84, 1423 1430 17 Raijman, L. (1976) in The Urea Cycle (Grisolia, S., Baguena, R. and Mayor, F., eds.), pp. 243-254, Wiley, New York 18 Mora, J., Mortuscelli, J., Ortiz-Pineda, J. and Soberon, G. (1965) Biochem. J. 96, 28-35 19 Cheung, C.W. and Raijman, L. (1981) Arch. Biochem. Biophys. 200, 643-649 20 Keller, D.M. (1968) Biochim. Biophys. Acta 153, 113-123 21 Bryla, J. and Harris, E.J. (1976) FEBS Lett. 72, 331-336 22 Chappell, J.B. and Hansford, R.G. (1979) in Subcellular Components; Preparation and Fractionation (Birnie, G.D., ed.), 2nd Edn, pp. 77-91, Butterworths, London 23 Mokrasch, C.C. and McGilvery, R.W. (1956) J. Biol. Chem. 221,909 917 24 Frick, U. (1975) Anal. Biochem. 63, 555-558 25 Hunter, L.A. and Downs, C.E. (1945) J. Biol. Chem. 157, 427 445 26 Freedland, R.A., Meijer, A.J. and Tager, J.M. Fed. Proc., in the press 27 Hilden, S.A. and Sacktor, B. (1981) Arch. Biochem. Biophys. 210, 289 297 28 Bradford, N.M. and McGivan, J.D. (1980) FEBS Lett. 113, 294-298 29 Ward, W.F. and Mortimore, G.E. (1978)J. Biol. Chem. 253, 3581 3587 30 Azzi, A., Chappell, J.B. and Robinson, B.H. (1967) Biochem. Biophys. Res. Commun. 29, 148-152 31 Joseph, S.K. and McGivan, J.D. (1978) Biochim. Biophys. Acta 543, 16 28 32 McGivan, J.D., Bradford, N.M., Crompton, M. and Chappell, J.B. (1973) Biochem. J. 134, 209 215 33 Crozier, G.L. and Freedland, R.A. (1983) Fed. Proc. 42, 1311 34 Kwong, E., Tsai, S. and Nesheim, M.C. (1982) J. Nutr. 112, 2081-2090 35 Kroon, A.M. (1966) in The Regulation of Metabolic Processes in Mitochondria (Tager, J.M., Papa, S., Quagliarello, E. and Slater, E.C., eds.), Vol. 7, pp. 397 414, BBA Library, Elsevier Amsterdam
407
Elsevier
BBA 21933
ARGININE UPTAKE BY ISOLATED RAT LIVER MITOCHONDRIA R.A. F R E E D L A N D a,. G.L. CROZIER a, B.L. HICKS a and A.J. MEIJER b
" Department of Physiological Sciences, School of Veterinary Medicine, University of California Davis, Davis, CA 95616 (U.S.A.), and h Laboratory of Biochemistry, B.C.P. Jansen Institute, University of Amsterdam, P.O. Box 20151, 1000 HD Amsterdam (The Netherlands) (Received June 27th, 1984)
Key words: Arginine uptake," Amino acid transport; Urea synthesis; N-Acetylglutamate; (Rat liver mitochondria)
The question of arginine uptake by mitochondria is important in that arginine is an allosteric effector of N-acetylglutamate synthetase. Thus, changes in mitochondrial arginine concentration have the potential for acutely modifying levels of N-acetylglutamate, a compound necessary for maximal activity of carbamyl phosphate synthesis. Mitochondria were isolated from chow-fed rats, incubated with [guanido-14C]arginine and were centrifuged through silicon oil into perchloric acid for determination of intramitochondrial metabolites. Arginine was separated from urea by cation-exchange resin. Mitochondrial water space was determined by [14C]urea arising from arginase activity associated with the mitochondrial preparations. Extramatrix space was determined by parallel incubations with [inulin- 14 C l c a r b o x y l i c acid or [ 14 C]sucrose. There was considerable degradation of arginine by arginase associated with the mitochondrial preparation. This was inhibited by 7 mM ornithine and 7 mM lysine. Arginine was concentrated intramitochondrially to 4-times the extramitochondrial levels. The concentration ratio was decreased in the presence of ornithine and lysine but not with citrulline, NH4CI , glutamate, glutamate or leucine. No uptake was observed when mitochondria were incubated at 0°C. Mitochondria did not concentrate citrulline. Introduction Marshall et al. [1] have shown that N-acetylglutamate is a necessary allosteric effector of carbamyl-phosphate synthetase (EC 6.3.4.16). In rat liver, N-acetylglutamate activates carbamylphosphate synthetase by changing conformation and subunit structure of the enzyme [2] with a K a of approx 0.8 n m o l / m g mitochondrial protein [3]. N-Acetylglutamate levels increased in liver of rats injected with amino acids [3], and in mitochondria isolated from rats fed high protein diets [4], or injected with glucagon [5,6]. In mitochondria, Nacetylglutamate concentrations were shown to increase over a 12 rain incubation [7], and McGivan * To whom correspondence should be addressed. Abbreviation: Mops, 4-morpholinepropanesulfonic acid. 0005-2728/84/$03.00 © 1984 Elsevier Science Publishers B.V.
et al. [8] correlated changes in mitochondrial Nacetylglutamate content with rates of citrulline synthesis from NH4C1. In isolated rat hepatocytes treated with pent-4-enoate, N-acetylglutamate levels were lower compared with controls, as was the synthesis of urea and citrulline [9]. Thus, there is much evidence suggesting that N-acetylglutamate plays an important regulatory role in carbamyl phosphate synthetase activity. McGivan et al. [4] have shown that rates of ureagenesis from ammonia in cells paralleled rates of citrulline synthesis in mitochondria, indicating that under these conditions, the overall rate of urea synthesis is regulated by carbamyl-phosphate synthetase activity. Therefore, factors which control the synthesis of N-acetylglutamate have the potential for the acute regulation of ureagenesis. N-Acetylglutamate is synthesized in liver mitochondria from acetyl-
408 CoA and glutamate by the mitochondrial aminoacid acyltransferase (EC 2.3.1.1) [10]. Arginine is an allosteric modulator of this enzyme and acts by increasing its Vmax,although it has no effect on K m values for the substrates [11]. Arginine is a specific activator for N-acetylglutamate synthetase: activation does not occur by any other amino acid nor intermediate of the urea cycle [12]. The physiological importance of arginine is positively modulating N-acetylglutamate synthetase is strongly suggested by the experiments of Tatibana and Shigesada [13], who injected arginine intraperitoneally into mice and found increased liver levels of N-acetylglutamate, In rat liver, N-acetylglutamate levels are correlated with arginine, ornithine [13] and urea [14]. There is, however, controversy about whether the arginine effect on N-acetylglutamate synthetase has importance in vivo. The enzyme is active even in the absence of arginine [12] and the optimal activity of the enzyme is higher in sonicated mitochondria from rats fed a standard diet than in mitochondria from glucose-fed rats [4]. Although arginine injected in mice resulted in increased liver N-acetylglutamate [15], N-acetylglutamate was also observed to increase if rats were injected with NH4CI [16] or amino acid mixtures containing no arginine [3]. Obviously, variations in the mitochondrial arginine concentration within the range of its K~ of 5-10 /~M for N-acetylglutamate synthetase [11] must occur if arginine were to play an important regulatory role in N-acetylglutamate synthesis. Total arginine levels in liver of 20-50 n m o l / g wet wt. [15,3,17] exceed the K a for N-acetylglutamate synthesis although it is presently unknown how much of this arginine is present in the mitochondria. Finally, arginase (EC 3.5.3.1) in liver is exceedingly active and appears to be associated with the particulate cellular components, including approx. 10% with the mitochondria [18,19]. The extensive degradation of arginine by mitochondrial preparations have complicated studies of arginine uptake [20], and its effect on citrulline synthesis [19] and ornithine uptake [21]. In this paper, we report our findings on the uptake of arginine by isolated rat liver mitochondria and on the effects of certain amino acids on this. The method is unique in that the use of [guanido-]4C]arginine permitted us to
quantify the extent of degradation of arginine as well as ensure that once separated from urea, intramitochondrial label was arginine and not a product of its degradation such as ornithine. Materials and Methods
Male Sprague-Dawley rats weighing 195-300 g were obtained from the University of California, Davis, breeding colony and were fed Purina rat chow (Ralston Purina, St. Louis, MO) ad libitum. Mitochondria were isolated by standard centrifugation techniques as described by Chappell and Hansford [22]. Livers were homogenized in 2.5 vol. of ice-cold medium consisting of 250 mM mannitol, 5 mM Mops, and 0.5 mM E G T A at pH 7.0. Mitochondrial coupling was verified with a Gilson 5 / 6 oxygraph with a Clark electrode. The mean respiratory control index was 4.1 + 1.2 with succinate as a substrate. Mitochondrial protein was determined by the biuret method as described by Mokrasch and McGilvery [23]. For determination of arginine uptake, 21-42 mg mitochondria were suspended in 7 ml medium (pH 7.4) consisting of 75 mM Mops, 20 mM K2HPO4, 4 mM EGTA, 15 mM KCI, 16.6 mM K H C O 3, 2.2 rag/1 rotenone, 0.01 /LCi/ml [guanido-laC]arginine and substrates as noted in the tables. Incubations were carried out at 25 or 0°C as noted. For separation of the mitochondria from the surrounding medium, a 1 ml sample of the mixture was gently layered over 0.35 ml silicon oil (density, 1.035) which had been layered over 0.15 ml 14% HC104 in a 1.5 ml microcentrifuge tube (Walter Sarstedt, Princeton, N J). Tubes were immediately centrifuged for 1 rain in an Eppendorf 5412 microcentrifuge (12000 × g). In order to obtain accurate estimates of arginine breakdown, synchronous samples of the incubation mixture were deproteinized with HCIO 4. These tubes were also centrifuged. Samples were taken as close to zero time as possible, usually within 30 s, and again at 5, 10, 20 and 30 rain. The top layer of the first set of tubes and a portion of the silicon oil were aspirated, and 0.5 ml of water was added to the acid/mitochondria layer, and the whole thoroughly mixed to ensure dispersion of metabolites. The tubes were re-
409
centrifuged to bring down the oil and protein fractions. Aliquots of both the mitochondrial fraction and the total deproteinized medium were passed over a 7 cm Dowex 50W-X1 (H + form, 50-100 mesh) cation-exchange resin in a pasteur pipette. Urea was eluted in 6 ml of water, and arginine was eluted with 6 ml of 1 M N a O H . The latter was neutralized by the addition of HC1 and radioactivity in aliquots of both fractions was quantitated by liquid scintillation counting (Packard Tricarb 2660). The fluor used was tritoso124. Total water space was routinely determined with [14C]urea arising from degradation of [guanido-14C]arginine. For several mitochondrial preparations, non-matrix water was determined by incubating mitochondria with [3H]or [14C]inulin, or [14C]sucrose prior to spinning through oil. Of the total water associated with the mitochondria, 70% _+ 4.0 was non-matrix water, and the remainder was assumed to be intra-matrix water. Calculations
Calculations of the matrix arginine concentration and medium arginine concentration were as follows: matrix arginine = ([total mito Arg ( d p m ) - n o n matrix Arg (dpm)]/matrix volume (~l))/spec. act. Arg (dpm/~umol) and medium Arg = total Arg in medium ( d p m / m l ) / spec. act. Arg (dpm//lmol).
100 E
H ~ \
.-=
80
c
60
.c "o
~ \ ~
~- 4 . 0 /~M arginine ~ 4 . 0 / ~ M arginine + 7 m M o r n i t h i n• ~ +• - -
40 2O
0
I
~
io
~o
~o
Time (min) Fig. l. Arginine remaining in the medium throughout a 30 min incubation with mitochondria (3-6 m g / m l ) (n = 2). Arginine concentration was added isotope only, i.e., no unlabelled arginine was added in these incubations.
way were compared with those determined with 3H20. These were identical with the exception of those estimates determined at the initial time. At this point, the urea formed was small, and not fully equilibrated with the total water. The addition of 7 mM ornithine inhibited arginine degradation by arginase (Fig. 1) and 7 m M lysine had a similar effect, although this inhibition was not as strong as that of ornithine (data not shown). This is consistent with the finding that ornithine [19] and lysine are inhibitors of arginase [25]. The results in Fig. 2 show that mitochondria were able to concentrate arginine. After a lag period of about 10 min, the mitochondria achieved a mean concentration ratio near four and main-
Results
The activity of arginase in the mitochondrial preparation was high and resulted in rapid and considerable degradation of arginine in the medium (Fig. 1). By 30 min of incubation, as little as 14% of the added arginine remained. In this experiment, utilization of [guanido-laC]arginine combined with deproteinization of samples of the medium in synchrony with mitochondrial separations enabled us to quantitate accurately the extent of arginine degradation at any given point. In addition, the liberation of [14C]urea and its rapid equilibration with water permitted us a convenient estimation of total mitochondrial water space. In a few trials, water spaces obtained with urea in this
,.o
c
•~ ,
10
o U oo
6
~
4
"6
2
-~
4.0 #M arginine
0
0 I
5
1(3
1'5
;~0
3'0
Time (rain)
Fig. 2. Representative plot of the ratio of mitochondrial to external medium arginine levels throughout a 30 min incubation.
410 t a i n e d this value relatively c o n s t a n t t h r o u g h o u t the next 20 min. T h e c o n c e n t r a t i o n of arginine in isolated m i t o c h o n d r i a is in accord with levels f o u n d in c y t o s o l a n d m i t o c h o n d r i a , w h i c h were d e t e r m i n e d in cells d i s r u p t e d b y d i g i t o n i n before s e p a r a t i o n of the two c o m p o n e n t s b y spinning t h r o u g h oil. In these studies, m i t o c h o n d r i a l arginine levels also exceeded cytosolic levels [26]. A few studies were carried out with mitoc h o n d r i a which were u n c o u p l e d during p r e p a r a tion, as j u d g e d b y low r e s p i r a t o r y control index values. These m i t o c h o n d r i a were u n a b l e to conc e n t r a t e arginine, a n d this p r o v i d e d a negative c o n t r o l s h o w i n g that the i n t e g r i t y of the m i t o c h o n d r i a is necessary for arginine uptake. In a d d i t i o n , these studies showed that our results in c o u p l e d m i t o c h o n d r i a could not be e x p l a i n e d by nonspecific b i n d i n g of arginine to the mitochondrial membrane. Effect of o t h e r a m i n o acids a n d NH4C1 on m i t o c h o n d r i a l c o n c e n t r a t i o n of arginine. The level of external arginine had no effect on its c o n c e n t r a tion b y m i t o c h o n d r i a ( T a b l e I). T h e a d d i t i o n of the basic a m i n o acids, o r n i t h i n e or iysine i n h i b i t e d
TABLE I EFFECT OF VARIOUS CONDITIONS ON MITOCHONDRIAL CONCENTRATION OF ARGININE Ratios, with their standard deviations, are concentrations of intramitochondrial arginine over medium arginine, averaged over 10, 20 and 30 min. Numbers in parentheses are the numbers of determinations. Conditions
Ratio
4.0 ~M arginine 1.0 mM arginine 7.0 mM arginine 4.0/~ M arginine + 7 mM ornithine 4.0 p~M arginine+ 1 mM ornithine 7.0 mM arginine + 7 mM ornithine 4.0/~M arginine+ 7 mM lysine 4.0/~M arginine + 7 mM citrulline 4.0 ~M arginine + 7 mM NH~4.0/~M arginine at 0°C 1.0 mM arginine at 0°C 4.0 p~M arginine+0.1 mM ATP 4.0 ~M arginine + 0.1 mM ADP 4.0/~M arginine + 7 mM glutamine 4.0/LM arginine + 7 mM glutamate 4.0 ~M arginine + 7 mM leucine
4.3 _+0.3 (3) 3.3 _+0.3 (2) 3.7 _+0.3 (2) 1.2 +_0.2 (3) 2.4_+0.3 (4) 2.4 1.8_+0.3 (2) 4.1 4.1 0.4 0.5 3.9+0.2 (2) 4.1 _+0.2 (2) 3.7 3.9 3.6
the u p t a k e of arginine by m i t o c h o n d r i a with o r n i t h i n e having a stronger effect. This is consistent with the findings of H i l d e n and Sacktor [27] in r a b b i t renal brush b o r d e r m e m b r a n e vesicles, a n d of Keller [20] in isolated dog kidney m i t o c h o n d r i a . B r a d f o r d a n d M c G i v a n [28] have shown that o r n i t h i n e and citrulline are exchanged across the m i t o c h o n d r i a l m e m b r a n e b y an o r n i t h i n e / c i t r u l l i n e antiporter, a n d we were curious to see wether citrulline affected arginine uptake. F r o m T a b l e I it can be seen that citruiline h a d no effect on arginine c o n c e n t r a t i o n by the mitochondria. L y s o s o m e s are c o s e d i m e n t e d with m i t o c h o n d r i a , a n d we considered the a r g u m e n t that the conc e n t r a t i o n ratio seen here was due to sequestration of arginine by lysosomes based on their p H differential rather than true m i t o c h o n d r i a l uptake. Thus, we tested the effect of 7 m M NH4CI, since lysosomes would also sequester N H 4 (intralysos o m a l p H 5.5 or less). If lysosomal sequestration were an i m p o r t a n t factor, we would see a decrease in arginine u p t a k e in the presence of a m m o n i a . T a b l e I shows that 7 m M N H 4 C I had no effect on arginine uptake, a n d this is an i n d i c a t i o n that the u p t a k e p h e n o m e n o n is not p r i m a r i l y lysosomal. F u r t h e r evidence for this derives from studies of W a r d and M o r t i m o r e [29], who were u n a b l e to show a c c u m u l a t i o n of arginine in lysosomes from rat liver. W e further tested glutamate, an acidic a m i n o acid, and glutamine, a neutral a m i n o acid, b o t h of which are k n o w n to be taken up a n d m e t a b o l i z e d by m i t o c h o n d r i a [30,31]. N e i t h e r these a m i n o acids nor leucine, a neutral a m i n o acid which stimulates m i t o c h o n d r i a l g l u t a m a t e synthesis [32], affected arginine uptake. The a d d i t i o n of A D P or A T P d i d not affect the c o n c e n t r a t i o n ratio i n d i c a t i n g that the availability of A T P or A D P was not limiting for arginine uptake. M i t o c h o n d r i a i n c u b a t e d at 0 ° C failed to a c c u m u l a t e arginine indicating that it was a t e m p e r a t u r e - d e p e n d e n t p h e n o m e n o n . Because of the relative c o n s t a n c y of the ratio over m a n y different conditions, we p o s e d the question that p e r h a p s the p h e n o m e n o n observed was not characteristic of arginine, but rather could be generalized over a range of a m i n o acids, or possibly was related to our m e t h o d o l o g y . To test this, we investigated the u p t a k e of citrulline in studies
411 TABLE II MITOCHONDRIAL LINE
CONCENTRATION
OF
CITRUL-
Ratios are intramitochondrial citrulline concentrations divided by medium citrulline concentrations, averaged over 10, 20 and 30 min. [ureido-14C]Citrulline was used in lieu of [guanido14C]arginine. Conditions
Ratio
5.0 `aM citrulline 5.0 ,aM citrulline + 7 mM ornithine 7.0 mM citrulline 7.0 mM citrulline+ 7 mM ornithine
0.74 0.74 0.69 0.69
using [ureido-14C]citrulline. Citrulline is synthesized in the mitochondria and readily exits this subcellular compartment [21,28,33]. Table II shows that in mitochondria incubated with trace amounts or 7 mM citrulline with or without the addition of ornithine, there was no concentration of citrulline, thus the phenomenon is characteristic of, though not necessarily limited to, arginine. In studies with heart mitochondria, we observed that the tritiated water to [14C]arginine ratio was similar in the surrounding medium and the mitochondria. Tritiated water was used in these experiments to give total water space, since in the absence of arginase there would be no urea produced nor arginine destroyed. The value for arginine concentration per unit of water in the mitochondria compared to the surrounding medium was 0.97 _+ 0.05 for three separate determinations. Discussion
The results of this investigation clearly demonstrate the inhibitory effect of lysine and ornithine on arginine uptake by isolated rat liver mitochondria. This effect has also been noted in renal brush-border membrane vesicles from rabbit [27], isolated renal cortex mitochondria from dogs [20], and isolated kidney mitochondria from chickens [34]. In addition, Bradford and McGiven [28] showed that lysine and 6-aminocaproate, a lysine analogue, as well as ornithine stimulated citrulline efflux from preloaded mitochondria. These studies suggest that a single translocator may be involved in the transport of arginine, lysine and ornithine.
The fact that heart mitochondria failed to concentrate arginine against a gradient indicates that nonspecific binding by inner or outer membrane of the mitochondria could not be the explanation for the observed phenomena with the liver mitochondria. Furthermore, these studies with the heart mitochondria indicate that a proton pumping could not be the sole explanation for this concentration, since both heart and liver mitochondria show this phenomenon. The present investigation also confirms the association of arginase with isolated rat liver mitochondria [19,18], and its inhibition by lysine [25] and ornithine [19]. The metabolic significance of this remains to be established, but one possibility is that arginase provides a means of generating ornithine from arginine near the mitochondrial membrane for anaplerosis of the urea cycle [19]. For the production of urea, ornithine leaves the mitochondria as citrulline and, for the continuation of the cycle, it must return. Raijman and co-workers [17] have shown that ornithine may be a limiting substrate for ornithine transcarbamylase activity. Thus, the functional advantage of the proximity of arginase to the mitochondrial membrane may lie in an important role it may play in regulating the urea cycle by providing ornithine. We have shown that arginine is concentrated by isolated rat liver mitochondria despite the high activity of arginase in mitochondrial preparations. Liver levels of arginine in normal rats range from 20-50 n m o l / g wet wt. [3,15,17]. Assuming cells to be 2 / 3 water, this would mean that the arginine concentration would be 30-75/~M. If the ratio in vivo between the cytosol and mitochondrial arginine levels is similar to the 1 : 4 ratio we have observed in these studies in isolated mitochondria, the mitochondrial concentration of arginine would be 120-300/~M, many times greater than its K, of 5 10 /~M [11] for N-acetylglutamate synthetase. Thus, if the kinetic properties of N-acetylglutamate synthetase as it occurs in the mitochondrial matrix are similar to those of the purified enzyme, it is unlikely that arginine plays an important role in the regulation of N-acetylglutamate synthetase. Cheung and Raijman [19] have shown that the stimulation of citrulline synthesis in isolated mitochondria incubated with substrates for Nacetylglutamate and arginine was only slightly
412 greater than in mitochondria incubated with a r g i n i n e a l o n e . A l t h o u g h i n i t i a l levels o f N - a c e t y l glutamate were not measured, this data, in concert with ours showing substantial mitochondrial uptake of arginine regardless of initial arginine conc e n t r a t i o n i n t h e m e d i u m , give f u r t h e r w e i g h t to the hypothesis that arginine stimulation of citrull i n e s y n t h e s i s in v i v o is o f little i m p o r t a n c e in t h e regulation of urea synthesis. Thus, the metabolic importance of the m i t o c h o n d r i a l c o n c e n t r a t i o n o f a r g i n i n e is u n l i k e l y to be related to an influence of N-acetylglutamate synthetase. Mitochondria can synthesize protein [35], t h u s it is p o s s i b l e t h a t t h e u p t a k e o f t h i s amino a c i d m a y s e r v e to c o m p a r t m e n t a l i z e i n t r a c e l l u l a r a r g i n i n e at a site o f e s s e n t i a l p r o t e i n s y n t h e s i s . S i n c e t h e r e is l i t t l e e v i d e n c e f o r i n t r a m i tochondrial arginase, mitochondrial arginine may a l s o s e r v e as a p r o t e c t e d p o o l o f a n o r n i t h i n e p r e c u r s o r f o r t h e u r e a cycle.
Acknowledgements We thank the following undergraduate students w h o p a r t i c i p a t e d i n v a r i o u s a s p e c t s o f t h i s res e a r c h : D. Byerly, P. C h c u n g , S. D o n o v a n , M . D w i g h t , C. G i l l e s p i e , M . M a l l e r , B. S e e m a n , J. S h r a g e r a n d K. S p r a y b e r r y .
References 1 Marshall, M., Metzenberg, R.L. and Cohen, P.P, (1961) J. Biol. Chem. 236, 2229-2237 2 Guthorlein, G. and Knappe, J. (1968) Eur. J. Biochem. 7, 119-127 3 Stewart, P.M. and Walser, M. (1980) J. Biol. Chem. 255, 5270-5280 4 McGivan, J.D., Bradford, N.M. and Mendes-Mourao, J. (1976) Biochem. J. 154, 415-421 5 Cathelineau, L., Rabier, D., Petit, F. and Kamoun, P. (1980) CR Acad. Sci. Ser. D291, 464-466 6 Verhoeven, A.J., Hensgens, H.E.S.J., Meijer, A.J. and Tager, J.M. (1982) FEBS Lett. 140, 270-272 7 Meijer, A.J. and Van Woerkom, G.M. (1978) FEBS Lett. 86, 117-121 8 McGivan, J.D., Bradford, N.M. and Chappell, J.B. (1974) Biochem. J. 142, 359-364 9 Aoyagi, K., Mori, M. and Tatibana, M. (1979) Biochim. Biophys. Acta 587, 515-521
10 Shigesada, K. and Tatibana, M. (1971) J. Biol. Chem. 246, 5588-5595 11 Shigesada, K. and Tatibana, M. (1978) Eur. J. Biochem. 84, 285-291 12 Shigesada, K. and Tatibana, M. (1971) Biochem. Biophys. Res. Commun. 44, 1117 1124 13 Tatibana, M. and Shigesada, K. (1976) in The Urea Cycle (Grisolia, S., Baguena, R. and Mayor, F., eds.), pp. 301 314, Wiley, New York 14 Saheki, T. and Katsunuma, T. (1977) J. Biochem. 82, 551-558 15 Shigesada, K., Aoyagi, K. and Tatibana, M. (1978) Eur. J. Biochem. 85, 385-391 16 Saheki, T., Ohkubo, T. and Katsunuma, T. (1978) J. Biochem. 84, 1423 1430 17 Raijman, L. (1976) in The Urea Cycle (Grisolia, S., Baguena, R. and Mayor, F., eds.), pp. 243-254, Wiley, New York 18 Mora, J., Mortuscelli, J., Ortiz-Pineda, J. and Soberon, G. (1965) Biochem. J. 96, 28-35 19 Cheung, C.W. and Raijman, L. (1981) Arch. Biochem. Biophys. 200, 643-649 20 Keller, D.M. (1968) Biochim. Biophys. Acta 153, 113-123 21 Bryla, J. and Harris, E.J. (1976) FEBS Lett. 72, 331-336 22 Chappell, J.B. and Hansford, R.G. (1979) in Subcellular Components; Preparation and Fractionation (Birnie, G.D., ed.), 2nd Edn, pp. 77-91, Butterworths, London 23 Mokrasch, C.C. and McGilvery, R.W. (1956) J. Biol. Chem. 221,909 917 24 Frick, U. (1975) Anal. Biochem. 63, 555-558 25 Hunter, L.A. and Downs, C.E. (1945) J. Biol. Chem. 157, 427 445 26 Freedland, R.A., Meijer, A.J. and Tager, J.M. Fed. Proc., in the press 27 Hilden, S.A. and Sacktor, B. (1981) Arch. Biochem. Biophys. 210, 289 297 28 Bradford, N.M. and McGivan, J.D. (1980) FEBS Lett. 113, 294-298 29 Ward, W.F. and Mortimore, G.E. (1978)J. Biol. Chem. 253, 3581 3587 30 Azzi, A., Chappell, J.B. and Robinson, B.H. (1967) Biochem. Biophys. Res. Commun. 29, 148-152 31 Joseph, S.K. and McGivan, J.D. (1978) Biochim. Biophys. Acta 543, 16 28 32 McGivan, J.D., Bradford, N.M., Crompton, M. and Chappell, J.B. (1973) Biochem. J. 134, 209 215 33 Crozier, G.L. and Freedland, R.A. (1983) Fed. Proc. 42, 1311 34 Kwong, E., Tsai, S. and Nesheim, M.C. (1982) J. Nutr. 112, 2081-2090 35 Kroon, A.M. (1966) in The Regulation of Metabolic Processes in Mitochondria (Tager, J.M., Papa, S., Quagliarello, E. and Slater, E.C., eds.), Vol. 7, pp. 397 414, BBA Library, Elsevier Amsterdam