- Email: [email protected]
Amino acid incorporation into rat liver mitochondria
628
BIOCHIMICAET BIOPHYSICAACTA
BBA 97317
AMINO ACID I N C O R P O R A T I O N INTO RAT L I V E R MITOCHONDRIA" j. MOCKEL Laboratory of Nuclear Medicine, School of Medicine, University qf Brussels, Brussels (Belgium)
(Received February I7th, i972) (Revised manuscript received May 3rd, i972)
SUMMARY Many discrepancies in the literature on amino acid incorporation into rat liver mitochondria can result from methodological differences. Some of these are evidenced by the present work: incubation temperature, leucine concentration in the medium, properties of the preparation and incubation media. Substrates are required in every system, while additional phosphate is required only in the ADP-substrate-supported system. Atractylate additions and adenine nucleotide omissions definitely show that addition of adenine nucleotides to the incubation medium is not an absolute requirement. The endogenous adenine nucleotide pool would thus be selfsufficient to allow normal protein synthesis and the real function of the ATP-phosphoenolpyruvatepyruvate kinase system would be to provide mitochondria with actively metabolized pyruvate.
INTRODUCTION Autonomous protein synthesis in vitro by isolated rat liver mitochondria is a function which has been clearly established after many controversies 1-1s. However, when one compares the qualitative requirements of the different incubation media as well as the incorporation of amino acids into mitochondrial proteins on a quantitative basis, the results of various authors show major discrepancies 1'3-5' 0,13,14,16,19. If mitochondrial protein synthesis with tissues giving much lower mitochondrial protein yield than liver (thyroid, skeletal muscle) is to be studied, it is of the utmost importance to apply a method giving optimal quantitative and qualitative results. The aim of the present work was thus: (I) To ascertain which of the available methods gives maximal amino acid incorporation. (2) To try to determine the various factors responsible for the important variations in mitochondfial protein synthesis as reported by various authors. As control of the obtained results, the action of compounds known for their effects on mitochondrial protein synthesis was checked. Tile best methodology was then applied to a study of the relations between protein synthesis and substrate, phosphate and adenine nucleotides requirements. " This work was carried out under Contract of the Minist~re de la Politique Scientifique within the framework of the Association Euratom-University of Brussels-University of Pisa. Abbreviation: ATP-PEP-pyruvate kinase system, sytem enriched with ATP, phosphoenolpyruvate and pyruvate kinase. Biochim. Biophys. Acta, 277 (1972) 628-638
AMINO ACID INCORPORATION INTO MITOCHONDRIA
629
MATERIAL AND METHODS
R a t liver mitochondria were prepared from animals weighing lOO-15o g and fasted overnight. They were killed by decapitation and the livers quickly removed and treated in cold 0.25 M sucrose-2 mM EDTA (pH 7.4). Two preparation methods were used: (A) that of Kroon 11 (preincubation with RNAase being omitted) with successive centrifugations of the homogenate at 900 ×g • 5 min, supernatant 4500 × g • IO min, resuspended mitochondrial pellet 12 500 ×g • IO rain, and (B) the method of Schneider and Hogeboom ~° as used b y Wheeldon and Lehninger 8,9 and Beattie and associates 5'6 with successive centrifugations of the homogenate at 600 × g - IO min, supernatant 8 5 o o × g " IO rain, and resuspended mitochondrial pellet washed three or four times at 8500 × g • IO rain. The solutions were freshly prepared with deionized water and filtered on millipore filters before use. All instruments and glassware were sterilized b y autoclaving. The mitochondria were incubated in open round-bottom tubes or beakers containing 1-3 ml of incubation medium at temperatures varying from 20 to 37 °C for I h. Three different media were used: incubation Medium A as described b y Kroon and de Vries 15 containing 5o mM sucrose, 50 mM Tricine buffer (pH 7.4), 20 mM KC1, 30 mM NH4C1, 5 mM MgCI~, I mM EDTA, 20 mM potassium phosphate buffer, 2 mM ADP, 30 mM sodium succinate, IOO/~g/ml cycloheximide and 50/ag/ml of a synthetic amino acid mixture m i n u s leucine; incubation Medium B of Wheeldon and Lehninger 9 containing IOO mM sucrose, IOO mM KC1, IO mM KH2PO 4, IO mM succinate, 15 mM MgCI~, 5 mM proline, 33 mM Tris-HC1 buffer (pH 7.4) and 2 mM ATP; incubation Medium C is the one used b y Beattie et al. ~ containing 5 ° mM Tris-HC1 buffer (pH 7-4), 15 mM MgC12, 5 mM phosphate buffer (pH 7.4), 2 mM ATP, 2 mM EDTA, r5o mM KC1, IO mM succinate and 22.5/~g/ml of a mixture of amino acids. At the end of the I-h incubation, the reaction was stopped b y the addition of ice-cold trichloroacetic acid at a final concentration of 5 % to a total volume of IO ml. The protein pellet was then successively washed once more with cold 5 % trichloroacetic acid, with 5 % trichloroacetic acid at 7 ° °C for 15 min and with ethanol-ether (i : I, v/v) at 35 °C. The dried pellet was then dissolved in i ml soluene (Packard Cy) (45 °C, 45 min), diluted with 14 ml of scintillating fluid (NEN omnifluor 4 g per I 1 toluene) and counted in a liquid scintillation counter. The radioactive precursor was E3H]- or E14C]leucine, or a mixture of 3H- or 14C-labeled amino acids. Precise values relating to labeled precursors quantities, specific activities and modifications to incubation conditions are reported in the legends to tables and figures. The results of each experiment always represent means of three closely agreeing duplicates. Bacterial contamination was evaluated by plating I ml of incubation medium at the end of incubation oil blood agar plates e. After 72 h at 37 °C, growing bacterial colonies were counted: their number never exceeded 80 ml of incubation medium. Protein determination was made according to the method of Lowry e~ al. ~1. Mitochondrial protein yield varied between 17 and 32 mg per g of wet liver tissue. The activity of the mitochondrial inhibitors used was checked regularly by polarographic study of rat liver mitochondrial samples. Atractylate was a kind gift of Prof. Bruni (Padova, Italy). Actinomycin D, RNAase, rifampicin, puromycin, chloramphenicol, ethidium bromide, dinitrophenol, valinomycin, gramicidin and rotenone were products from Calbiochem (Los Angeles, Biochim. Biophys. Acta, 277 (1972) 628-638
63o
J. MOCKEL
Calif., U.S.A.); antimycin, oligomycin and mersalyl were purchased from Sigma Chemical Co. (St. Louis, Mo., U.S.A.); cycloheximide was an Upjohn product (Kalamazoo, Mich., U.S.A.), pyiuvate kinase was from Boehringer (Mannheim, Germany); ~SHlleucine (38o mCi/mM), I14C!leucine (25o mCi/mM) and the 14C-labeled amino acid mixture was purchased from the Radiochemical Centre (Amersham, England), and the 3H-labeled amino acid mixture from NEN-Chenficals (Frankfurt, Germany). All other products were of the purest grade commercially available.
RESULTS
Fig. I shows the effect of temperature on amino acid incorporation into mitochondria with two different systems. In the system of Wheeldon and Lehninger 9, the temperature used by the authors (37 °C) gives a poor protein synthesis, close to the one reported in the original results, and lowering the incubation temperature to 2o °C induces a rise of nearly IOO % in amino acid incorporation. Oil the contrary, the system of Kroon is much less sensitive to temperature variations, protein synthesis is enhanced by increased temperatures and at 3 ° °C (as used by the authors) amino acid incorporation is nearly optimal. C
Leucine i n c o r p o r a t i o n
6
% of
a 25 .~
OI intoproteins
c o n t r o l (37 ~)
20
• 150I-
~ot
.~
/
b
""*, ",
.~ 20
/ Y ~,,,
~~~ ~o 15
._c e ~ 5
50
j...?___ff-'",
o 30@37*40 °
C
t
(*C)
lO
20 i
3oc t
.,b' 6b' "6o '14o o L:~ ' ~3 ~ 100' pln leucine ioo~c 4Ol Ib ; 4SAmCi/mM
I
I
I
I
J i
I
1 15 201a£21025 5 0
20
Fig. I. Effect of temperature on rat liver mitochondrial protein synthesis. / - × , preparation Method A, i ml incubation medium. Labeled precursor: lSH]leucine (o.4/~Ci, io mCi/mM). O - - - O , preparation Method B, 3 ml incubation Medium B. Labeled precursor: E3H~leucine (2/,Ci, 380 mCi/mM), i-h incubation. Mitochondria, 2- 3 mg/ml. Fig. 2. Variations of leucine incorporation rates into mitochondrial proteins for different leucine concentrations. (AI Preparation Method B. Incubation Medium B: 3 ml, 60 min, 37°C. (B) As (A) except temperature: 2o°C. (A and B) [3H]Leucine, constant specific activity of 25 ° mCi/mM. (C) Preparation Method A. Incubation Medium A: i ml, 6o rain, 3o°C, E*Hlleucine (0. 4/,Ci). SA = specific activity.
The results of Fig. 2 show that the concentration of leucine in the incubation medium is a major limiting factor of protein synthesis in both incubation Media A and B. Fig. 2 also shows that amino acid incorporation on a quantitative basis is nearly equal for both systems if "Wheeldon and Lehninger" mitochondria are incubated at 20 °C instead of 37 °C. In the experiments of Table I, mitochondria were all prepared following Method Biochim. Biophys. Acta, 277 (1972) 628-638
AMINO ACID INCORPORATION" INTO MITOCHONDRIA
631
TABLE I MITOCHONDRIAL PROTEIN SYNTHESIS IN D I F F E R E N T INCUBATION MEDIA
All i n c u b a t i o n s were carried o u t for 6o min, a t a t e m p e r a t u r e of 3o°C, in a total v o l u m e of i m l w i t h 2 - 3 m g of m i t o c h o n d r i a . Conditions in (4) are t h e s a m e as in (i) e x c e p t t h a t t h e livers were t r e a t e d a n d t h e m i t o e h o n d r i a p r e p a r e d in t h e ionic " T r i s - K C I " m e d i u m of Chappell a n d P e r r y .2 c o n t a i n i n g i o o m M KC1, 5 ° m M Tris-HC1 buffer (pH 7.4), I m M A T P , 5 m M MgSO 4 a n d I m M E D T A . T h e figures are t h e m e a n v a l u e s r e s u l t i n g f r o m a t least 3 different e x p e r i m e n t s , t h e r e s u l t s of each e x p e r i m e n t b e i n g t h e m e a n of closely agreeing triplicates. W h e n t h e a m i n o acid m i x t u r e w a s t h e tracer, u n l a b e l e d a m i n o acids were o m i t t e d f r o m i n c u b a t i o n Media A a n d C, as well as u n l a b e l e d proline f r o m i n c u b a t i o n M e d i u m B.
Preparation method
Incubation medium
(z ml) (I)
(Kroon) B (Wheeldon) C (Beattie) A
A
A
{2) A (3) A (4) A in "Tris-KCl" m e d i u m
ESH]Leucine (o.4 I~Ci20 mCi/mM)
[14C]Amino acids (0. 4 mCi)
pmoles/mg Percent
dpm/mg Percent
22.9
IOO
7.o5 7.13 6.64
3o.7(28-34) 31.5(25-38) 29(20-38 )
824 331 215 313
IOO
4o.1(38-51) 2 6 . 8 ( I 5 - 4 o) 38(33-41 )
A and incubated at 3o °C for I h in the three different Media A, B and C with the same leucine or amino acids concentration. The results obtained in the Kroon medium are in accordance with those reported b y the author 15. I t appears that, independently of other experimental conditions, the composition of the incubation medium itself is also an important variation factor, since protein synthesis is much lower in Media B and C than in Medium A. The preparation of liver mitochondria in the "Chappell and Perry" medium z2 generally used for skeletal muscle mitochondria preparations also induces an important drop in protein synthesis. These low incorporation values are not modified b y the combined or separate additions to the incubation medium of i mM NAD + or 20/zM cytochrome c. Fig. 3 shows that, in the Kroon system, the kinetics of amino acid incorporation were nearly linear and that the concentrations of KC1 (20 mM) and ADP (2 mM) used by the author/5 were optimal in the particular incubation medium. Addition of bovine serum albumin in the incubation medium did not induce any significant modification.
,°°F If
*°
1¢0~
f. I
I
I
I
100~- x ~ X ~ x ~ x l/ ~x
x
I
|
0 10 20 30 40 5060rain
0 20 40 60 80100KCt
012
4 6 8 10
15ram ADP
Fig. 3. A m i n o acid i n c o r p o r a t i o n i n t o r a t liver m i t o c h o n d r i a as a f unct i on of time (A), K C l •c o n c e n t r a t i o n (13) a n d A D P c o n c e n t r a t i o n (C). P r e p a r a t i o n M e t h o d A. I - h i n c u b a t i o n a t 3o°C ~ f 2 - 3 m g m i t o c h o n d r i a l p r o t e i n in i ml of M e d i u m A w i t h o u t ( × - × ) or w i t h ( O - G ) a d d e d b o v i n e s e r u m a l b u m i n (i m g / m l ) . L a b e l e d precursors: [aH]leucine, (0. 4/~Ci, IO m C i / m M ) (ioo % m e a n = 22.9 p m o l e s l e u c i n e / m g protein) or t4C-labeled a m i n o acid m i x t u r e (o. 4 t, Ci) (ioo % :mean = 824 d p m / m g protein). T h e figures r e p r e s e n t t h e m e a n r e s u l t s f r o m five d i f f e r e n t experixnents.
Biochim. Biophys. Acta, 277 (1972) 6 2 8 - 6 3 8
632
J. MOCKEL
The characteristics of mitochondrial protein synthesis were further defined by the action of various compounds as shown in Table II, mainly in the respirationsupported system. TABLE I I ACTION OF V A R I O U S C O M P O U N D S ON M I T O C H O N D R I A L
PROTEIN SYNTHESIS
Mitochondria were p r e p a r e d following Method A, and 2- 3 mg incubated for 60 rain at 3o'C in I ml of Medium A ( A D P - s u c c i n a t e ) . I n the A T P - P E P - p y r u v a t e kinase system, A D P and succinate were o m i t t e d and replaced b y 2 mM ATP, 5 mM P E P , IOO/~g/ml p y r u v a t e kinase. T r a c e r [3H]- or [l~C]leucine (0. 4 # C i - i o mCi/mM), or 3H- or l~C-labeled a m i n o acids m i x t u r e (0. 4 #Ci). E a c h figure is the m e a n value resulting from at least 3 e x p e r i m e n t s (with range), the figures from each e x p e r i m e n t r e p r e s e n t i n g the m e a n of closely agreeing triplicates.
14dditions to the incubation medium
. 4 D P succinate
.4TP-PEP-pyruvate kinase system
No additions (control) Actinomycin D RNAase Cycloheximide Rifanlpicine 4 or Puromycin Chloramphenicol E t h i d i u m b r o m i d e 0.25 or Antimycin Oligomycin Dinitrophenol Valinomycin Gramicidin P o t a s s i u m arsenate
ioo 95 97 109 lOO-126) 95 94-96 19 13-25 6 O--18 32 27-40 19 3-26 37 7-49 36 9-54 5° 43-55 59 58-60 7° 56 47 35-37
I O0
5o/~g/ml 20/zg/ml i oo #tg/ml 16 ktg/ml ioo/,g/ml I mg/ml 0. 5/~g/ml 2 #g/ml I o #g/ml I oo #M o. 5 # g / m l o. 5 # g / m l 2 mM 4 mM 8 mM lO-2O m i
29 (28-32) 35 (3°-37) 32 (21-36)
Actinomycin D, RNAase, cycloheximide and iifampicin have no effect on mitochondrial protein synthesis, while puromycin, chloramphenicol and, to a lesser extent, ethidium bromide strongly inhibit amino acid incorporation. Antimycin block of electron transport also strongly inhibits, while inhibition of ADP phosphorylation by oligomycin or uncoupling oxidative phosphorylation by dinitrophenol gives a comparable but only partial inhibition of mitochondrial protein synthesis. For the last three compounds, the observed inhibitions are identical in respiration-supported systems or in ATP-generating systems. Antibiotics inducing active cation uptake and uncoupling of oxidative phosphorylation like valinomycin and gramicidin give a 50 60 % inhibition of protein synthesis in a respiration-supported system. Arsenate additions exert increasing inhibitions from 2 to IO mM in the presence of 20 mM phosphate buffer. The action of arsenate in the absence of phosphate was not studied, since the maximal inhibition by arsenate phts phosphate is equivalent to that obtained by the omission of phosphate (see furthei results). Table III shows the quantitative relationship of amino acid incorporation into mitochondria in ADP-substrate- versus A T P - P E P - p y r u v a t e kinase-supported systems. The figures clearly demonstrate that under our experimental conditions, there is no important increase in mitochondfial protein synthesis when an ATP-generating system is used as energy source. Biochim. Biophys. dcta, 277 (1972) 628 638
633
AMINO ACID INCORPORATION INTO MITOCHONDRIA TABLE III MITOCHONDRIAL PROTEIN S Y N T H E S I S IN A T P - P E P - P Y R U V A T E versus A T P - AND ADP--sUBSTRATE-SUPPOETED SYSTEMS
KINASE-SUPPORTED
SYSTEM
M i t o c h o n d r i a were p r e p a r e d following M e t h o d A, a n d 2-3 m g of m i t o c h o n d r i a l p r o t e i n s i n c u b a t e d a t 3o°C for 6o m i n in I m l of M e d i u m A, a d e n i n e n u c l e o t i d e s a n d s u b s t r a t e s b e i n g a d d e d as i n d i c a t e d in t h e table. T r a c e r : [aH]- or [t4C]leucine (0.4/zCi-Io m C i / m M ) , or sH- or 14C-labeled a m i n o acid m i x t u r e (o. 4/zCi). A dditions to the incubation medium Plus
Plus Plus Plus Plus
2 mM ATP 5 mM PEP IOO p g / m l p y r u v a t e 2 mM ATP 3o m M s u c c i n a t e 2 m M A D P - 3 o mM 2 mM ADP-3 o mM 2 mM ADP-3 o mM
°/o of incorporation
I oo kinase
succinate pyruvate glutamate
83 (81-89) 87 (79-99) lO 5 lO8
T A B L E IV E F F E C T S ON M I T O C H O N D R I A L P R O T E I N S Y N T H E S I S O F S U B S T R A T E S , P H O S P H A T E A N D A D E N I N E N U C L E O T I D E O M I S S I O N , A N D O F A D D I T I O N S OF C O M P O U N D S B L O C K I N G T H E I R N O R M A L M E T A B O L I S M OR T R A N S P O R T
M i t o c h o n d r i a were p r e p a r e d following M e t h o d A, a n d 2 - 3 m g of m i t o c h o n d r i a l p r o t e i n s i n c u b a t e d a t 3o°C for 60 m i n in: (i) Column A : I m l M e d i u m A w i t h 2 m M A D P a n d s u b s t r a t e s as i n d i c a t e d in t h e table. (2) Column B: I m l M e d i u m A w i t h no A D P or s u b s t r a t e , b u t 2 m M A T P , 5 m M P E P , i o o / z g / m l p y r u v a t e kinase. Label: [SH]- or [I4C]leucine (o. 4 p C i - i o m C i / m M ) , or 8H- or x*C-labeled a m i n o acid m i x t u r e (o. 4 #Ci). W h e n (ranges) are indicated, t h e r e s u l t s are m e a n s of a t least 3 different e x p e r i m e n t s , each e x p e r i m e n t i n v o l v i n g closely agreeing triplicates. Additions or omissions to the incubation medium
-- s u c c i n a t e - - s u c c i n a t e + 12 pM r o t e n o n e +3 ° mM succinate+ I2/,M rotenone + 3° m M s u c c i n a t e + 3 ° m M s u c c i n a t e + IO m M m a l o n a t e (in C o l u m n A only) + IO m M m a l o n a t e + 12/~M r o t e n o n e --Pt
+ 3 5 pM m e r s a l y l +3 ° mM pyruvate +3 ° mM glutamate + 5° p M a t r a c t y l a t e + 5°/*M atractylate + 3° mM succinate - - / A D P (Column A) | o r A T P (Column B) +30 mM succinate + 3° m M s u c c i n a t e +50 pM atractylate +3 ° mM pyruvate + 3° m M p y r u v a t e +50/,M atractylate + 30 m M g l u t a m a t e +3 ° mM glutamate + 5o p M a t r a e t y l a t e + 5° pM atractylate
A A DP
3 ° (27- 32)
B A TP-PEP-pyruvate kinase IOO 59 76 98
5 8 - 60) 7 3 - 78 )
78 98
7 3 - 85) 9 3 - 99)
132
lO5-175)
93 (73-112)
24 48
2 2 - 27) 4 2 - 49)
86 (63-11o) i28
68 54
6 3 - 72) 5 I - 56)
128 77
75
64- 8I)
47
4 3 - 52)
86 (8o- 92) ioo 45 (38- 47) 13 ( s - ~6) 28 ( 2 2 - 3 ° ) 2 6 (19- 32 ) lO 5 133 7 2 (62-1o4)
67
Biochim. Biophys. Acta, 277 (1972) 628-638
634
J. MOCKEL
The results of Table IV show the effects in both systems of substrate, phosphate and adenine nucleotides omission and of various added compounds affecting their normal metabolism oI transport. In a respiration-supported system, Initochondrial protein synthesis is strongly dependent on the presence of exogenous substrate and phosphate as demonstrated by the addition of malonate (or malonate plus rotenone) ol mersalyl, or by the omission of succinate or phosphate from the incubation medium. Other substrates, like pyruvate and glutamate also provide efficient and even better means than succinate to ensure normal amino acid incorporation. On the other hand, protein synthesis proceeds nearly nolmally in the absence of ADP for each of these substrates. Atraetylate addition to the complete system sometimes induces a slight inhibition, which becomes even less significant if ADP is omitted from the medium. In the A T P - P E P - p y r u v a t e kinase system, no additional substrate is required, as shown by the lack of effect of suceinate. However, there is an important rotenoneinduced drop in protein synthesis, partially compensated by further suecinate addition. Phosphate omission and mersalyl addition clearly show that the external ATPgenerating system is not as dependent on external phosphate as the ADP-substratesupported system. Here, atractylate not only does not inhibit protein synthesis, but frequently induces a stimulation exceeding 30 °/'o. However, when ATP is omitted, amino acid incorporation dlops to 20 o/ /o of the control. In these conditions, succinate, pyruvate and atractylate, each separately, exert a IOO °/o stinmlation effect. Furthelmore, addition of substrates and atractylate together in the absence of ATP provide additive stimulations, since protein synthesis then goes up from 24 to 68--75 o, of the control.
DISCUSSION
Increased temperatures induce a rise in state 4 respiration and a reduction of respiratory control rates with liver, heart and skeletal muscle mitochondria 2a'24. This mechanism probably explains the decreased amino acid incorporation by increased incubation temperatures in Medium B. The fact that these variations are not parallel in the two Media A and B might be related to differences in sensitivity to temperature of mitoehondrial coupling due to the different composition of both media. The importance of leucine concentration in the incubation medium as a major rate-limiting factor in mitoehondrial amino acid incorporation into proteins is emphasized by the results shown in Fig. 2. This fact, already demonstrated by Kroon and de Vries 15 for their own system holds true at various temperatures in the other systems. Adding high leueine concentrations and cold amino acids to the medium when leucine is the tracer certainly minimizes the effects of variations most probably occurring from one experiment to another in the small endogenous mitochondrial amino acids pool. As to the importance of the incubation medium composition, Table I clearly shows that independently of preparation methods, incubation temperature and leucine concentration, mitochondrial protein synthesis m a y be very diffelent depending on the incubation medium used. Many parameters m a y be involved, but osmolarity of the medium is probably an important factor, as showed b y Haldar and Freeman 4. Biochim. Biophys. Acta, 277 (1972) 628-638
AMINO ACID INCORPORATION INTO MITOCHONDRIA
635
In relation to this work, it must be noted t h a t the respective calculated osmolarities of Media A, B and C are approximately 300, 39 ° and 410 mosmoles/1. Properties of the preparation medium ale also important since mitochondria from livers treated in the Chappell and Perry ionic medium 22 and incubated in Medium A also give a particularly low yield in mitochondrial protein synthesis. It has been reported by Hedman 25 that treatment of tissues in such media without sucrose depleted the mitochondria of soluble coenzymes such as NAD ÷ and cytochrome c. Since addition of these two compounds to the incubation medium has no effect, it m a y be hypothesized that other soluble substances important for normal protein synthesis are also lost during the preparation of mitocbondria. It thus seems that a correct study of skeletal muscle mitochondrial protein synthesis should be done with "sucrose"-prepared mitochondria, even ii these procedures give lower mitochondrial protein yield 25. From the results of Fig. I and Fig. 2 and from Table I, it is evident that despite temperature and leucine concentiation colrections for the other two media, the Kroon medium 15 gives much better quantitative results. Fig. 3 further shows that under these experimental conditions kinetics of amino acid incorporation were satisfactory, and there was no advantage in increasing ADP or KC1 concentration or adding bovine serum albumin to the incubation medium, as has been recently reported 3°. The quality of our mitochondrial preparations were tested in the Kroon medium 15, as shown in Table II, b y addition of various compounds. Their action has generally been studied by different authors and our results agree with some of them (refs 5, 6, 9, 11-15)- Only a few particular points and differences with the literature will thus be discussed. The complete lack of inhibition of mitochondrial protein synthesis by rifampicin additions at high concentrations deserves some comment. Some experiments tend to demonstrate that mitochondrial DNA-dependent RNA polymerase is insensitive to rifampicin 27-29, while other authors clearly show an inhibition of mitochondlial RNA synthesis by rifampicin 31-33. Its lack of effect on amino acid incorporation in the present experiments could be explained by: (I) Protection in vivo of RNA polymerase b y a cofactor x, as proposed by Kfintzel and Schafer 33. (2) Impermeability of intact mitochondria to the antibiotic. (3) Effective inhibition of RNA polymerase during the experiments, protein synthesis being unaffected by this inhibition for a short-time incubation (60 rain). Ethidium bromide, a known inhibitor of mitochondrial DNA replication a4-37 also inhibits mitochondlial protein synthesis to a large extent (ref. 15, this work). This could imply that mitochondlial protein synthesis is continuously dependent on DNA replication as proposed b y Kroon and de Vries 15. However, the half life of mitochondrial DNA should allow protein synthesis to proceed normally for a I-h incubation in the presence of ethidium bromide. Its effect could thus also result from the block of some other mitochondrial metabolic process which could affect amino acid incorporation more directly than DNA replication suppression. The action of antibiotics like valinomycin and gramicidin which induce an energy-dependent active cation uptake s8 has never been tested to our knowledge. Their inhibitory action on mitochondrial protein synthesis in a respiration-supported system is not surprising if related to their uncoupling properties. Biochim. Biophys. Acta, 277 (1972) 628-638
636
j. MOCKEL
The results of Table 1V show the effects of substrate, phosphate and adenine nucleotide deprivation in both ADP--substrate and A T P - P E P - p y r u v a t e kinase systems. Additional substrates were an absolute requirement with ADP, but succinate could be successfully replaced by NAD-linked substrates like pyruvate and glutamate (Tables I I I and IV). No supplementary substrates were necessary in the A T P - P E P pyruvate kinase system. However, in contradiction with previous reports by Wheeldon and Lehninger 9 and Williams and Birt ag, oligomycin and antimycin inhibited mitochondrial protein synthesis exactly to the same extent as shown in Table II. Furthermore there was an important rotenone-induced inhibition partially relieved by succinate addition. This rather suggests that even with external ATP generation, normal substrate metabolism remains necessary for amino acid incorporation, the substrate possibly being pyruvate in such a system. The plesence of phosphate in the medium has always been found necessary in ADP-substrate-supported system and our results agree with these previous data 1' s,9. The similar inhibition induced by mersalyl which blocks phosphate uptake 4° gives indirect confirmation of this requirement. However, with the ATP-generating system, this phosphate requirement nearly disappears, most probably as tim result of continuous phosphate generation induced by phosphoenolpyruvate and ATP breakdown. Atractylate additions at high concentrations have a slight and variable inhibitory effect in the A D P - s u b s t r a t e system, while with A T P - P E P - p y r u v a t e kinase it does not inhibit protein synthesis at all. Atractylate has been shown to be a specific inhibitor of adenine nucleotide translocation 41-45. These results, further confirmed by the fact that omission of ADP leaves protein synthesis intact with the different substrates, would thus imply that the endogenous pool of mitochondrial adenine nucleotides is selfsufficient to ensure normal protein synthesis. However, in the A T P - P E P pyruvate kinase system, A T P omission induces a major drop in protein synthesis. If A T P is useless b y itself, the system could have beneficial side effects by providing the mitochondria with pyruvate coming from P E P breakdown and by preventing leakage in the external medium of endogenous adenine nucleotides. If such is the case, addition to a P E P - p y r u v a t e kinase system of substrates or atractylate should tend to restore amino acid incorporation to control levels. This is indeed the fact since pyruvate, or succinate, or atractylate, each separately, effectively doubles mitochondrial protein synthesis in such conditions (Table IV). Substrate and atractylate effects are additive, and their simultaneous addition restored amino acid incorporation to 68-75 3/0 of the control. Adenine nucleotide requirements for mitochondrial protein synthesis have been a matter of important controversy throughout the literature. Our results are in accordance with some data 4s-4s and contradictory to m a n y others s'9,a~,49-Sa. The discrepancies, once more found in the literature, could finally simply result from variable endogenous adenine nucleotide pools in the mitochondrial samples studied, since preparation methods, number of final washes of the mitochondrial pellet and characteristics of the incubation media, which could all affect the size of this pool as well as the mitochondrial ATPase activity, show a huge range of variations. Atractylate, on the other hand, has already been shown to stimulate ATP-requiring reactions such as citrulline synthesis 55'56. What our results definitely demonstrate is that addition of adenine nucleotide is no absolute requirement for normal mitochondrial protein synthesis. It remains, however, possible that in the Biochim. Biophys. Acta, 277 (1972) 628-638
AMINO ACID INCORPORATION INTO MITOCHONDRIA
637
p r e s e n c e of a v e r y l o w e n d o g e n o u s a d e n i n e n u c l e o t i d e p o o l o r of a v e r y h i g h m i t o c h o n d r i a l A T P a s e activity, a d e n i n e n u c l e o t i d e omission in the m e d i u m or a t r a c t y l a t e a d d i t i o n s could e x h i b i t entirely different effects.
ACKNOWLE DGEMENTS W e w i s h t o t h a n k D r D. S. B e a t t i e , D r A. M. K r o o n a n d D r J . E . D u m o n t f o r h e l p f u l d i s c u s s i o n s a n d c r i t i c i s m s , Mrs L. C o l l y n - L e g a l l a i s f o r h e r e x c e l l e n t t e c h n i c a l a s s i s t a n c e , Miss J . H e n n a u x a n d Miss Ch. B o r r e y f o r t h e d r a w i n g of t h e f i g u r e s a n d t y p i n g of t h e m a n u s c r i p t .
REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 2o 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38 39 4° 41 42 43
D. B. Roodyn, R. J. Reiss and T. S. Work, Biochem. J., 8o (1961) 9. D. 13. Roodyn, J. W. Suttie and T. S. Work, Biochem. J., 83 (I962) 29. S. Sandell, H. L6w and A. yon der Decken, Biochem. J . , IO4 (1967) 575. D. Haldar and K. B. Freeman, Biochem. J., I I I (1969) 653. D. S. Beattie, R. E. Basford and S. B. Koritz, Biochemistry, 6 (I967) 3099 . D. S. Beattie, R. E. Basford and S. B. Koritz, J. Biol. Chem., 242 (1967) 3366. D. S. Beattie, G. M. Patton and R. N. Stuchell, J. Biol. Chem., 245 (197 o) 2177. L. Wheeldon, Biochem. Biophys. Res. Commun., 24 (1966) 4o7 . L. W. Wheeldon and A. L. Lehninger, Biochemistry, 5 (1967) 3533. A. M. Kroon, Biochim. Biophys. Acta, 72 (1963) 391. A. M. Kroon, Biochim. Biophys. Acta, 91 (1964) 145. A. M. Kroon, Biochim. Biophys. Acta, io8 (I965) 275. A. M. Kroon, C. Saccone and M. J. 13otman, Biochim. Biophys. Acta, 142 (1967) 552. A. M. Kroon and H. de Vries, Syrup. Soc. Exp. Biol., 24 (197 o) 181. A. M. Kroon and H. de Vries, in Boardmann, Linnane and Smillie, Autonomy and Biogenesis of Mitochondria and Chloroplasts, North-Holland, i97 I, p. 318. F. C. Firkin and A. W. Linnane, F E B S Lett., 2 (1969) 33 o. M. W. Gordon and G. G. Deanin, J. Biol. Chem., 243 (I968) 4222. D. Halder, Biochem. Biophys. Res. Commun., 42 (1971) 899. D. E. S. Truman and A. Korner, Biochem..l., 83 (I962) 588. W. C. Schneider and G. H. Hogeboom, J. Biol. Chem., I83 (1956) I23. O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Chem., 193 (1951) 265. J. B. Chappell and S. V. Perry, Nature, 173 (1954) lO94. J. Mockel and J. E. Dumont, Eur. J. Clin. Invest., I (197 o) 32. G. A. Brooks, K. J. Hittelman, J. A. Faulkner and R. E. Beyer, Am. J. Physiol., 22o (1971) lO53. I¢. Hedman, Exp. Cell Res., 38 (1965) i. D, Neubert and H. Helge, Biochem. Biophys. Res. Commun., 18 (1965) 600. E. Wintersberger and V. Wintersberger, F E B S Lett., 6 (197 o) 58. Ming-Jer Tsai, G. Michaelis and R. S. Criddle, Proc. Natl. ,4cad. Sci. U.S., 68 (1971) 473. E. Wintersberger, Biochem. Biophys. Res. Commun., 4 ° (197 o) 1179. J. L. Coote and T. S. Work, Eur. J. Biochem., 23 (1971) 564. S. J. Surzycki, Proc. Natl. Acad. Sci. U.S., 63 (1969) 1327. L. G. Shmerling, Biochem. Biophys. Res. Commun., 37 (1969) ooo. H. Kiintzel and K. P. Schafer, Nature New Biol., 231 (1971) 265. E. S. Goldring, L. I. Grossman, D. Krupnick, D. R. Cryer and J. Marmur, J. Mol. Biol., 52 (197 ° ) 323. H. ]3. Horwitz and C. E. Holt, J. Cell Biol., 49 (1971) 546. K. Radsak, K. Kato, N. Sato and H. Koprowski, Exp. Cell Res., 66 (1971) 41o. P- S. Perlman and H. R. Mahler, Nature New Biol., 231 (I97I), 12. J. B. Chappell and A. R. Crofts, Biochem. J., 95 (1965) 393. 14. L. Williams and L. M. Birt, Eur. J. Biochem., 22 (1971) 87. D. D. Tyler, Biochem. J., I I I (1969) 665. E. D. Duee and P. V. Vignais, Biochim. Biophys. Aaa, lO 7 (1964) 184. G. Brierley and R. L. O'Brien, J. Biol. Chem., 24 ° (1965) 4532. H. H. Winkler, F. L. Bygrane and A. L. Lehninger, J. Biol. Chem., 243 (1968) 2o.
Biochim. Biophys. Acta, 277 (1972) 628-638
638 44 45 46 47 48 49 50 51 52 53 54 55 56
j.
MOCKEL
E. P f a f f a n d M. K l i n g e n b e r g , Euv. J . Biochem., 6 (1968) 66. M. J. W e i d e m a n n , H. E r d e l t a n d M. K l i n g e n b e r g , Eur..]. Biochem., I6 (197 o) 313. A. M. K r o o n , Biochim. Biophys. Acta, 72 (1963) 39t. E. ~Vintersberger, Biochemistry., 341 (I965) 409. S. V. G a n g a l a n d S. P. B e s s m a n , Biochem. Biophys. Res. Commun., 33 (1968) 675. S. 1~. C h a n a n d L. R i c h a r d s o n , J. Biol. Chem., 244 (I969) lO39. U. B r o n s e r t a n d W. N e u p e r t , in J. M. Tager, S. P a p a , E. Quagliariello a n d E. C. Slater, Regulation of Metabolic Processes in Mitochondria, B . B . A . L i b r a r y , Vol. 7, Elsevier, A m s t e r d a m , 1966, p. 426. W . Sebald, Th. B u c h e r , t3. Olbrich a n d F . K a u d e w i t z , F E B S Lett., I (1963) 236. :~-. J. L a m b , G. D. C l a r k - W a l k e r a n d A. "W. L i n n a n e , Biochim. Biophys. Acta, 161 (1968) 415. G. E. Kalf, Arch. Biochem. Biophys., IOi (1963) 35 o. M. L e d e r m a n a n d G. A t t a r d i , Biochem. Biophys. Res. Commun., 4 ° (197 o) 1492. ~V. D. J. G r a a f m a n s , R. Charles a n d J. M. Tager, Biochim. Biophys. Acta, 153 (1968) 916. R. Charles a n d S. G. v a n d e n Bergh, Bioehim. Biophys..4eta, 731 (I967) 393-
Biochim. Biophys. .4cta, 277 (1972) 6 2 8 - 6 3 8
BIOCHIMICAET BIOPHYSICAACTA
BBA 97317
AMINO ACID I N C O R P O R A T I O N INTO RAT L I V E R MITOCHONDRIA" j. MOCKEL Laboratory of Nuclear Medicine, School of Medicine, University qf Brussels, Brussels (Belgium)
(Received February I7th, i972) (Revised manuscript received May 3rd, i972)
SUMMARY Many discrepancies in the literature on amino acid incorporation into rat liver mitochondria can result from methodological differences. Some of these are evidenced by the present work: incubation temperature, leucine concentration in the medium, properties of the preparation and incubation media. Substrates are required in every system, while additional phosphate is required only in the ADP-substrate-supported system. Atractylate additions and adenine nucleotide omissions definitely show that addition of adenine nucleotides to the incubation medium is not an absolute requirement. The endogenous adenine nucleotide pool would thus be selfsufficient to allow normal protein synthesis and the real function of the ATP-phosphoenolpyruvatepyruvate kinase system would be to provide mitochondria with actively metabolized pyruvate.
INTRODUCTION Autonomous protein synthesis in vitro by isolated rat liver mitochondria is a function which has been clearly established after many controversies 1-1s. However, when one compares the qualitative requirements of the different incubation media as well as the incorporation of amino acids into mitochondrial proteins on a quantitative basis, the results of various authors show major discrepancies 1'3-5' 0,13,14,16,19. If mitochondrial protein synthesis with tissues giving much lower mitochondrial protein yield than liver (thyroid, skeletal muscle) is to be studied, it is of the utmost importance to apply a method giving optimal quantitative and qualitative results. The aim of the present work was thus: (I) To ascertain which of the available methods gives maximal amino acid incorporation. (2) To try to determine the various factors responsible for the important variations in mitochondfial protein synthesis as reported by various authors. As control of the obtained results, the action of compounds known for their effects on mitochondrial protein synthesis was checked. Tile best methodology was then applied to a study of the relations between protein synthesis and substrate, phosphate and adenine nucleotides requirements. " This work was carried out under Contract of the Minist~re de la Politique Scientifique within the framework of the Association Euratom-University of Brussels-University of Pisa. Abbreviation: ATP-PEP-pyruvate kinase system, sytem enriched with ATP, phosphoenolpyruvate and pyruvate kinase. Biochim. Biophys. Acta, 277 (1972) 628-638
AMINO ACID INCORPORATION INTO MITOCHONDRIA
629
MATERIAL AND METHODS
R a t liver mitochondria were prepared from animals weighing lOO-15o g and fasted overnight. They were killed by decapitation and the livers quickly removed and treated in cold 0.25 M sucrose-2 mM EDTA (pH 7.4). Two preparation methods were used: (A) that of Kroon 11 (preincubation with RNAase being omitted) with successive centrifugations of the homogenate at 900 ×g • 5 min, supernatant 4500 × g • IO min, resuspended mitochondrial pellet 12 500 ×g • IO rain, and (B) the method of Schneider and Hogeboom ~° as used b y Wheeldon and Lehninger 8,9 and Beattie and associates 5'6 with successive centrifugations of the homogenate at 600 × g - IO min, supernatant 8 5 o o × g " IO rain, and resuspended mitochondrial pellet washed three or four times at 8500 × g • IO rain. The solutions were freshly prepared with deionized water and filtered on millipore filters before use. All instruments and glassware were sterilized b y autoclaving. The mitochondria were incubated in open round-bottom tubes or beakers containing 1-3 ml of incubation medium at temperatures varying from 20 to 37 °C for I h. Three different media were used: incubation Medium A as described b y Kroon and de Vries 15 containing 5o mM sucrose, 50 mM Tricine buffer (pH 7.4), 20 mM KC1, 30 mM NH4C1, 5 mM MgCI~, I mM EDTA, 20 mM potassium phosphate buffer, 2 mM ADP, 30 mM sodium succinate, IOO/~g/ml cycloheximide and 50/ag/ml of a synthetic amino acid mixture m i n u s leucine; incubation Medium B of Wheeldon and Lehninger 9 containing IOO mM sucrose, IOO mM KC1, IO mM KH2PO 4, IO mM succinate, 15 mM MgCI~, 5 mM proline, 33 mM Tris-HC1 buffer (pH 7.4) and 2 mM ATP; incubation Medium C is the one used b y Beattie et al. ~ containing 5 ° mM Tris-HC1 buffer (pH 7-4), 15 mM MgC12, 5 mM phosphate buffer (pH 7.4), 2 mM ATP, 2 mM EDTA, r5o mM KC1, IO mM succinate and 22.5/~g/ml of a mixture of amino acids. At the end of the I-h incubation, the reaction was stopped b y the addition of ice-cold trichloroacetic acid at a final concentration of 5 % to a total volume of IO ml. The protein pellet was then successively washed once more with cold 5 % trichloroacetic acid, with 5 % trichloroacetic acid at 7 ° °C for 15 min and with ethanol-ether (i : I, v/v) at 35 °C. The dried pellet was then dissolved in i ml soluene (Packard Cy) (45 °C, 45 min), diluted with 14 ml of scintillating fluid (NEN omnifluor 4 g per I 1 toluene) and counted in a liquid scintillation counter. The radioactive precursor was E3H]- or E14C]leucine, or a mixture of 3H- or 14C-labeled amino acids. Precise values relating to labeled precursors quantities, specific activities and modifications to incubation conditions are reported in the legends to tables and figures. The results of each experiment always represent means of three closely agreeing duplicates. Bacterial contamination was evaluated by plating I ml of incubation medium at the end of incubation oil blood agar plates e. After 72 h at 37 °C, growing bacterial colonies were counted: their number never exceeded 80 ml of incubation medium. Protein determination was made according to the method of Lowry e~ al. ~1. Mitochondrial protein yield varied between 17 and 32 mg per g of wet liver tissue. The activity of the mitochondrial inhibitors used was checked regularly by polarographic study of rat liver mitochondrial samples. Atractylate was a kind gift of Prof. Bruni (Padova, Italy). Actinomycin D, RNAase, rifampicin, puromycin, chloramphenicol, ethidium bromide, dinitrophenol, valinomycin, gramicidin and rotenone were products from Calbiochem (Los Angeles, Biochim. Biophys. Acta, 277 (1972) 628-638
63o
J. MOCKEL
Calif., U.S.A.); antimycin, oligomycin and mersalyl were purchased from Sigma Chemical Co. (St. Louis, Mo., U.S.A.); cycloheximide was an Upjohn product (Kalamazoo, Mich., U.S.A.), pyiuvate kinase was from Boehringer (Mannheim, Germany); ~SHlleucine (38o mCi/mM), I14C!leucine (25o mCi/mM) and the 14C-labeled amino acid mixture was purchased from the Radiochemical Centre (Amersham, England), and the 3H-labeled amino acid mixture from NEN-Chenficals (Frankfurt, Germany). All other products were of the purest grade commercially available.
RESULTS
Fig. I shows the effect of temperature on amino acid incorporation into mitochondria with two different systems. In the system of Wheeldon and Lehninger 9, the temperature used by the authors (37 °C) gives a poor protein synthesis, close to the one reported in the original results, and lowering the incubation temperature to 2o °C induces a rise of nearly IOO % in amino acid incorporation. Oil the contrary, the system of Kroon is much less sensitive to temperature variations, protein synthesis is enhanced by increased temperatures and at 3 ° °C (as used by the authors) amino acid incorporation is nearly optimal. C
Leucine i n c o r p o r a t i o n
6
% of
a 25 .~
OI intoproteins
c o n t r o l (37 ~)
20
• 150I-
~ot
.~
/
b
""*, ",
.~ 20
/ Y ~,,,
~~~ ~o 15
._c e ~ 5
50
j...?___ff-'",
o 30@37*40 °
C
t
(*C)
lO
20 i
3oc t
.,b' 6b' "6o '14o o L:~ ' ~3 ~ 100' pln leucine ioo~c 4Ol Ib ; 4SAmCi/mM
I
I
I
I
J i
I
1 15 201a£21025 5 0
20
Fig. I. Effect of temperature on rat liver mitochondrial protein synthesis. / - × , preparation Method A, i ml incubation medium. Labeled precursor: lSH]leucine (o.4/~Ci, io mCi/mM). O - - - O , preparation Method B, 3 ml incubation Medium B. Labeled precursor: E3H~leucine (2/,Ci, 380 mCi/mM), i-h incubation. Mitochondria, 2- 3 mg/ml. Fig. 2. Variations of leucine incorporation rates into mitochondrial proteins for different leucine concentrations. (AI Preparation Method B. Incubation Medium B: 3 ml, 60 min, 37°C. (B) As (A) except temperature: 2o°C. (A and B) [3H]Leucine, constant specific activity of 25 ° mCi/mM. (C) Preparation Method A. Incubation Medium A: i ml, 6o rain, 3o°C, E*Hlleucine (0. 4/,Ci). SA = specific activity.
The results of Fig. 2 show that the concentration of leucine in the incubation medium is a major limiting factor of protein synthesis in both incubation Media A and B. Fig. 2 also shows that amino acid incorporation on a quantitative basis is nearly equal for both systems if "Wheeldon and Lehninger" mitochondria are incubated at 20 °C instead of 37 °C. In the experiments of Table I, mitochondria were all prepared following Method Biochim. Biophys. Acta, 277 (1972) 628-638
AMINO ACID INCORPORATION" INTO MITOCHONDRIA
631
TABLE I MITOCHONDRIAL PROTEIN SYNTHESIS IN D I F F E R E N T INCUBATION MEDIA
All i n c u b a t i o n s were carried o u t for 6o min, a t a t e m p e r a t u r e of 3o°C, in a total v o l u m e of i m l w i t h 2 - 3 m g of m i t o c h o n d r i a . Conditions in (4) are t h e s a m e as in (i) e x c e p t t h a t t h e livers were t r e a t e d a n d t h e m i t o e h o n d r i a p r e p a r e d in t h e ionic " T r i s - K C I " m e d i u m of Chappell a n d P e r r y .2 c o n t a i n i n g i o o m M KC1, 5 ° m M Tris-HC1 buffer (pH 7.4), I m M A T P , 5 m M MgSO 4 a n d I m M E D T A . T h e figures are t h e m e a n v a l u e s r e s u l t i n g f r o m a t least 3 different e x p e r i m e n t s , t h e r e s u l t s of each e x p e r i m e n t b e i n g t h e m e a n of closely agreeing triplicates. W h e n t h e a m i n o acid m i x t u r e w a s t h e tracer, u n l a b e l e d a m i n o acids were o m i t t e d f r o m i n c u b a t i o n Media A a n d C, as well as u n l a b e l e d proline f r o m i n c u b a t i o n M e d i u m B.
Preparation method
Incubation medium
(z ml) (I)
(Kroon) B (Wheeldon) C (Beattie) A
A
A
{2) A (3) A (4) A in "Tris-KCl" m e d i u m
ESH]Leucine (o.4 I~Ci20 mCi/mM)
[14C]Amino acids (0. 4 mCi)
pmoles/mg Percent
dpm/mg Percent
22.9
IOO
7.o5 7.13 6.64
3o.7(28-34) 31.5(25-38) 29(20-38 )
824 331 215 313
IOO
4o.1(38-51) 2 6 . 8 ( I 5 - 4 o) 38(33-41 )
A and incubated at 3o °C for I h in the three different Media A, B and C with the same leucine or amino acids concentration. The results obtained in the Kroon medium are in accordance with those reported b y the author 15. I t appears that, independently of other experimental conditions, the composition of the incubation medium itself is also an important variation factor, since protein synthesis is much lower in Media B and C than in Medium A. The preparation of liver mitochondria in the "Chappell and Perry" medium z2 generally used for skeletal muscle mitochondria preparations also induces an important drop in protein synthesis. These low incorporation values are not modified b y the combined or separate additions to the incubation medium of i mM NAD + or 20/zM cytochrome c. Fig. 3 shows that, in the Kroon system, the kinetics of amino acid incorporation were nearly linear and that the concentrations of KC1 (20 mM) and ADP (2 mM) used by the author/5 were optimal in the particular incubation medium. Addition of bovine serum albumin in the incubation medium did not induce any significant modification.
,°°F If
*°
1¢0~
f. I
I
I
I
100~- x ~ X ~ x ~ x l/ ~x
x
I
|
0 10 20 30 40 5060rain
0 20 40 60 80100KCt
012
4 6 8 10
15ram ADP
Fig. 3. A m i n o acid i n c o r p o r a t i o n i n t o r a t liver m i t o c h o n d r i a as a f unct i on of time (A), K C l •c o n c e n t r a t i o n (13) a n d A D P c o n c e n t r a t i o n (C). P r e p a r a t i o n M e t h o d A. I - h i n c u b a t i o n a t 3o°C ~ f 2 - 3 m g m i t o c h o n d r i a l p r o t e i n in i ml of M e d i u m A w i t h o u t ( × - × ) or w i t h ( O - G ) a d d e d b o v i n e s e r u m a l b u m i n (i m g / m l ) . L a b e l e d precursors: [aH]leucine, (0. 4/~Ci, IO m C i / m M ) (ioo % m e a n = 22.9 p m o l e s l e u c i n e / m g protein) or t4C-labeled a m i n o acid m i x t u r e (o. 4 t, Ci) (ioo % :mean = 824 d p m / m g protein). T h e figures r e p r e s e n t t h e m e a n r e s u l t s f r o m five d i f f e r e n t experixnents.
Biochim. Biophys. Acta, 277 (1972) 6 2 8 - 6 3 8
632
J. MOCKEL
The characteristics of mitochondrial protein synthesis were further defined by the action of various compounds as shown in Table II, mainly in the respirationsupported system. TABLE I I ACTION OF V A R I O U S C O M P O U N D S ON M I T O C H O N D R I A L
PROTEIN SYNTHESIS
Mitochondria were p r e p a r e d following Method A, and 2- 3 mg incubated for 60 rain at 3o'C in I ml of Medium A ( A D P - s u c c i n a t e ) . I n the A T P - P E P - p y r u v a t e kinase system, A D P and succinate were o m i t t e d and replaced b y 2 mM ATP, 5 mM P E P , IOO/~g/ml p y r u v a t e kinase. T r a c e r [3H]- or [l~C]leucine (0. 4 # C i - i o mCi/mM), or 3H- or l~C-labeled a m i n o acids m i x t u r e (0. 4 #Ci). E a c h figure is the m e a n value resulting from at least 3 e x p e r i m e n t s (with range), the figures from each e x p e r i m e n t r e p r e s e n t i n g the m e a n of closely agreeing triplicates.
14dditions to the incubation medium
. 4 D P succinate
.4TP-PEP-pyruvate kinase system
No additions (control) Actinomycin D RNAase Cycloheximide Rifanlpicine 4 or Puromycin Chloramphenicol E t h i d i u m b r o m i d e 0.25 or Antimycin Oligomycin Dinitrophenol Valinomycin Gramicidin P o t a s s i u m arsenate
ioo 95 97 109 lOO-126) 95 94-96 19 13-25 6 O--18 32 27-40 19 3-26 37 7-49 36 9-54 5° 43-55 59 58-60 7° 56 47 35-37
I O0
5o/~g/ml 20/zg/ml i oo #tg/ml 16 ktg/ml ioo/,g/ml I mg/ml 0. 5/~g/ml 2 #g/ml I o #g/ml I oo #M o. 5 # g / m l o. 5 # g / m l 2 mM 4 mM 8 mM lO-2O m i
29 (28-32) 35 (3°-37) 32 (21-36)
Actinomycin D, RNAase, cycloheximide and iifampicin have no effect on mitochondrial protein synthesis, while puromycin, chloramphenicol and, to a lesser extent, ethidium bromide strongly inhibit amino acid incorporation. Antimycin block of electron transport also strongly inhibits, while inhibition of ADP phosphorylation by oligomycin or uncoupling oxidative phosphorylation by dinitrophenol gives a comparable but only partial inhibition of mitochondrial protein synthesis. For the last three compounds, the observed inhibitions are identical in respiration-supported systems or in ATP-generating systems. Antibiotics inducing active cation uptake and uncoupling of oxidative phosphorylation like valinomycin and gramicidin give a 50 60 % inhibition of protein synthesis in a respiration-supported system. Arsenate additions exert increasing inhibitions from 2 to IO mM in the presence of 20 mM phosphate buffer. The action of arsenate in the absence of phosphate was not studied, since the maximal inhibition by arsenate phts phosphate is equivalent to that obtained by the omission of phosphate (see furthei results). Table III shows the quantitative relationship of amino acid incorporation into mitochondria in ADP-substrate- versus A T P - P E P - p y r u v a t e kinase-supported systems. The figures clearly demonstrate that under our experimental conditions, there is no important increase in mitochondfial protein synthesis when an ATP-generating system is used as energy source. Biochim. Biophys. dcta, 277 (1972) 628 638
633
AMINO ACID INCORPORATION INTO MITOCHONDRIA TABLE III MITOCHONDRIAL PROTEIN S Y N T H E S I S IN A T P - P E P - P Y R U V A T E versus A T P - AND ADP--sUBSTRATE-SUPPOETED SYSTEMS
KINASE-SUPPORTED
SYSTEM
M i t o c h o n d r i a were p r e p a r e d following M e t h o d A, a n d 2-3 m g of m i t o c h o n d r i a l p r o t e i n s i n c u b a t e d a t 3o°C for 6o m i n in I m l of M e d i u m A, a d e n i n e n u c l e o t i d e s a n d s u b s t r a t e s b e i n g a d d e d as i n d i c a t e d in t h e table. T r a c e r : [aH]- or [t4C]leucine (0.4/zCi-Io m C i / m M ) , or sH- or 14C-labeled a m i n o acid m i x t u r e (o. 4/zCi). A dditions to the incubation medium Plus
Plus Plus Plus Plus
2 mM ATP 5 mM PEP IOO p g / m l p y r u v a t e 2 mM ATP 3o m M s u c c i n a t e 2 m M A D P - 3 o mM 2 mM ADP-3 o mM 2 mM ADP-3 o mM
°/o of incorporation
I oo kinase
succinate pyruvate glutamate
83 (81-89) 87 (79-99) lO 5 lO8
T A B L E IV E F F E C T S ON M I T O C H O N D R I A L P R O T E I N S Y N T H E S I S O F S U B S T R A T E S , P H O S P H A T E A N D A D E N I N E N U C L E O T I D E O M I S S I O N , A N D O F A D D I T I O N S OF C O M P O U N D S B L O C K I N G T H E I R N O R M A L M E T A B O L I S M OR T R A N S P O R T
M i t o c h o n d r i a were p r e p a r e d following M e t h o d A, a n d 2 - 3 m g of m i t o c h o n d r i a l p r o t e i n s i n c u b a t e d a t 3o°C for 60 m i n in: (i) Column A : I m l M e d i u m A w i t h 2 m M A D P a n d s u b s t r a t e s as i n d i c a t e d in t h e table. (2) Column B: I m l M e d i u m A w i t h no A D P or s u b s t r a t e , b u t 2 m M A T P , 5 m M P E P , i o o / z g / m l p y r u v a t e kinase. Label: [SH]- or [I4C]leucine (o. 4 p C i - i o m C i / m M ) , or 8H- or x*C-labeled a m i n o acid m i x t u r e (o. 4 #Ci). W h e n (ranges) are indicated, t h e r e s u l t s are m e a n s of a t least 3 different e x p e r i m e n t s , each e x p e r i m e n t i n v o l v i n g closely agreeing triplicates. Additions or omissions to the incubation medium
-- s u c c i n a t e - - s u c c i n a t e + 12 pM r o t e n o n e +3 ° mM succinate+ I2/,M rotenone + 3° m M s u c c i n a t e + 3 ° m M s u c c i n a t e + IO m M m a l o n a t e (in C o l u m n A only) + IO m M m a l o n a t e + 12/~M r o t e n o n e --Pt
+ 3 5 pM m e r s a l y l +3 ° mM pyruvate +3 ° mM glutamate + 5° p M a t r a c t y l a t e + 5°/*M atractylate + 3° mM succinate - - / A D P (Column A) | o r A T P (Column B) +30 mM succinate + 3° m M s u c c i n a t e +50 pM atractylate +3 ° mM pyruvate + 3° m M p y r u v a t e +50/,M atractylate + 30 m M g l u t a m a t e +3 ° mM glutamate + 5o p M a t r a e t y l a t e + 5° pM atractylate
A A DP
3 ° (27- 32)
B A TP-PEP-pyruvate kinase IOO 59 76 98
5 8 - 60) 7 3 - 78 )
78 98
7 3 - 85) 9 3 - 99)
132
lO5-175)
93 (73-112)
24 48
2 2 - 27) 4 2 - 49)
86 (63-11o) i28
68 54
6 3 - 72) 5 I - 56)
128 77
75
64- 8I)
47
4 3 - 52)
86 (8o- 92) ioo 45 (38- 47) 13 ( s - ~6) 28 ( 2 2 - 3 ° ) 2 6 (19- 32 ) lO 5 133 7 2 (62-1o4)
67
Biochim. Biophys. Acta, 277 (1972) 628-638
634
J. MOCKEL
The results of Table IV show the effects in both systems of substrate, phosphate and adenine nucleotides omission and of various added compounds affecting their normal metabolism oI transport. In a respiration-supported system, Initochondrial protein synthesis is strongly dependent on the presence of exogenous substrate and phosphate as demonstrated by the addition of malonate (or malonate plus rotenone) ol mersalyl, or by the omission of succinate or phosphate from the incubation medium. Other substrates, like pyruvate and glutamate also provide efficient and even better means than succinate to ensure normal amino acid incorporation. On the other hand, protein synthesis proceeds nearly nolmally in the absence of ADP for each of these substrates. Atraetylate addition to the complete system sometimes induces a slight inhibition, which becomes even less significant if ADP is omitted from the medium. In the A T P - P E P - p y r u v a t e kinase system, no additional substrate is required, as shown by the lack of effect of suceinate. However, there is an important rotenoneinduced drop in protein synthesis, partially compensated by further suecinate addition. Phosphate omission and mersalyl addition clearly show that the external ATPgenerating system is not as dependent on external phosphate as the ADP-substratesupported system. Here, atractylate not only does not inhibit protein synthesis, but frequently induces a stimulation exceeding 30 °/'o. However, when ATP is omitted, amino acid incorporation dlops to 20 o/ /o of the control. In these conditions, succinate, pyruvate and atractylate, each separately, exert a IOO °/o stinmlation effect. Furthelmore, addition of substrates and atractylate together in the absence of ATP provide additive stimulations, since protein synthesis then goes up from 24 to 68--75 o, of the control.
DISCUSSION
Increased temperatures induce a rise in state 4 respiration and a reduction of respiratory control rates with liver, heart and skeletal muscle mitochondria 2a'24. This mechanism probably explains the decreased amino acid incorporation by increased incubation temperatures in Medium B. The fact that these variations are not parallel in the two Media A and B might be related to differences in sensitivity to temperature of mitoehondrial coupling due to the different composition of both media. The importance of leucine concentration in the incubation medium as a major rate-limiting factor in mitoehondrial amino acid incorporation into proteins is emphasized by the results shown in Fig. 2. This fact, already demonstrated by Kroon and de Vries 15 for their own system holds true at various temperatures in the other systems. Adding high leueine concentrations and cold amino acids to the medium when leucine is the tracer certainly minimizes the effects of variations most probably occurring from one experiment to another in the small endogenous mitochondrial amino acids pool. As to the importance of the incubation medium composition, Table I clearly shows that independently of preparation methods, incubation temperature and leucine concentration, mitochondrial protein synthesis m a y be very diffelent depending on the incubation medium used. Many parameters m a y be involved, but osmolarity of the medium is probably an important factor, as showed b y Haldar and Freeman 4. Biochim. Biophys. Acta, 277 (1972) 628-638
AMINO ACID INCORPORATION INTO MITOCHONDRIA
635
In relation to this work, it must be noted t h a t the respective calculated osmolarities of Media A, B and C are approximately 300, 39 ° and 410 mosmoles/1. Properties of the preparation medium ale also important since mitochondria from livers treated in the Chappell and Perry ionic medium 22 and incubated in Medium A also give a particularly low yield in mitochondrial protein synthesis. It has been reported by Hedman 25 that treatment of tissues in such media without sucrose depleted the mitochondria of soluble coenzymes such as NAD ÷ and cytochrome c. Since addition of these two compounds to the incubation medium has no effect, it m a y be hypothesized that other soluble substances important for normal protein synthesis are also lost during the preparation of mitocbondria. It thus seems that a correct study of skeletal muscle mitochondrial protein synthesis should be done with "sucrose"-prepared mitochondria, even ii these procedures give lower mitochondrial protein yield 25. From the results of Fig. I and Fig. 2 and from Table I, it is evident that despite temperature and leucine concentiation colrections for the other two media, the Kroon medium 15 gives much better quantitative results. Fig. 3 further shows that under these experimental conditions kinetics of amino acid incorporation were satisfactory, and there was no advantage in increasing ADP or KC1 concentration or adding bovine serum albumin to the incubation medium, as has been recently reported 3°. The quality of our mitochondrial preparations were tested in the Kroon medium 15, as shown in Table II, b y addition of various compounds. Their action has generally been studied by different authors and our results agree with some of them (refs 5, 6, 9, 11-15)- Only a few particular points and differences with the literature will thus be discussed. The complete lack of inhibition of mitochondrial protein synthesis by rifampicin additions at high concentrations deserves some comment. Some experiments tend to demonstrate that mitochondrial DNA-dependent RNA polymerase is insensitive to rifampicin 27-29, while other authors clearly show an inhibition of mitochondlial RNA synthesis by rifampicin 31-33. Its lack of effect on amino acid incorporation in the present experiments could be explained by: (I) Protection in vivo of RNA polymerase b y a cofactor x, as proposed by Kfintzel and Schafer 33. (2) Impermeability of intact mitochondria to the antibiotic. (3) Effective inhibition of RNA polymerase during the experiments, protein synthesis being unaffected by this inhibition for a short-time incubation (60 rain). Ethidium bromide, a known inhibitor of mitochondrial DNA replication a4-37 also inhibits mitochondlial protein synthesis to a large extent (ref. 15, this work). This could imply that mitochondlial protein synthesis is continuously dependent on DNA replication as proposed b y Kroon and de Vries 15. However, the half life of mitochondrial DNA should allow protein synthesis to proceed normally for a I-h incubation in the presence of ethidium bromide. Its effect could thus also result from the block of some other mitochondrial metabolic process which could affect amino acid incorporation more directly than DNA replication suppression. The action of antibiotics like valinomycin and gramicidin which induce an energy-dependent active cation uptake s8 has never been tested to our knowledge. Their inhibitory action on mitochondrial protein synthesis in a respiration-supported system is not surprising if related to their uncoupling properties. Biochim. Biophys. Acta, 277 (1972) 628-638
636
j. MOCKEL
The results of Table 1V show the effects of substrate, phosphate and adenine nucleotide deprivation in both ADP--substrate and A T P - P E P - p y r u v a t e kinase systems. Additional substrates were an absolute requirement with ADP, but succinate could be successfully replaced by NAD-linked substrates like pyruvate and glutamate (Tables I I I and IV). No supplementary substrates were necessary in the A T P - P E P pyruvate kinase system. However, in contradiction with previous reports by Wheeldon and Lehninger 9 and Williams and Birt ag, oligomycin and antimycin inhibited mitochondrial protein synthesis exactly to the same extent as shown in Table II. Furthermore there was an important rotenone-induced inhibition partially relieved by succinate addition. This rather suggests that even with external ATP generation, normal substrate metabolism remains necessary for amino acid incorporation, the substrate possibly being pyruvate in such a system. The plesence of phosphate in the medium has always been found necessary in ADP-substrate-supported system and our results agree with these previous data 1' s,9. The similar inhibition induced by mersalyl which blocks phosphate uptake 4° gives indirect confirmation of this requirement. However, with the ATP-generating system, this phosphate requirement nearly disappears, most probably as tim result of continuous phosphate generation induced by phosphoenolpyruvate and ATP breakdown. Atractylate additions at high concentrations have a slight and variable inhibitory effect in the A D P - s u b s t r a t e system, while with A T P - P E P - p y r u v a t e kinase it does not inhibit protein synthesis at all. Atractylate has been shown to be a specific inhibitor of adenine nucleotide translocation 41-45. These results, further confirmed by the fact that omission of ADP leaves protein synthesis intact with the different substrates, would thus imply that the endogenous pool of mitochondrial adenine nucleotides is selfsufficient to ensure normal protein synthesis. However, in the A T P - P E P pyruvate kinase system, A T P omission induces a major drop in protein synthesis. If A T P is useless b y itself, the system could have beneficial side effects by providing the mitochondria with pyruvate coming from P E P breakdown and by preventing leakage in the external medium of endogenous adenine nucleotides. If such is the case, addition to a P E P - p y r u v a t e kinase system of substrates or atractylate should tend to restore amino acid incorporation to control levels. This is indeed the fact since pyruvate, or succinate, or atractylate, each separately, effectively doubles mitochondrial protein synthesis in such conditions (Table IV). Substrate and atractylate effects are additive, and their simultaneous addition restored amino acid incorporation to 68-75 3/0 of the control. Adenine nucleotide requirements for mitochondrial protein synthesis have been a matter of important controversy throughout the literature. Our results are in accordance with some data 4s-4s and contradictory to m a n y others s'9,a~,49-Sa. The discrepancies, once more found in the literature, could finally simply result from variable endogenous adenine nucleotide pools in the mitochondrial samples studied, since preparation methods, number of final washes of the mitochondrial pellet and characteristics of the incubation media, which could all affect the size of this pool as well as the mitochondrial ATPase activity, show a huge range of variations. Atractylate, on the other hand, has already been shown to stimulate ATP-requiring reactions such as citrulline synthesis 55'56. What our results definitely demonstrate is that addition of adenine nucleotide is no absolute requirement for normal mitochondrial protein synthesis. It remains, however, possible that in the Biochim. Biophys. Acta, 277 (1972) 628-638
AMINO ACID INCORPORATION INTO MITOCHONDRIA
637
p r e s e n c e of a v e r y l o w e n d o g e n o u s a d e n i n e n u c l e o t i d e p o o l o r of a v e r y h i g h m i t o c h o n d r i a l A T P a s e activity, a d e n i n e n u c l e o t i d e omission in the m e d i u m or a t r a c t y l a t e a d d i t i o n s could e x h i b i t entirely different effects.
ACKNOWLE DGEMENTS W e w i s h t o t h a n k D r D. S. B e a t t i e , D r A. M. K r o o n a n d D r J . E . D u m o n t f o r h e l p f u l d i s c u s s i o n s a n d c r i t i c i s m s , Mrs L. C o l l y n - L e g a l l a i s f o r h e r e x c e l l e n t t e c h n i c a l a s s i s t a n c e , Miss J . H e n n a u x a n d Miss Ch. B o r r e y f o r t h e d r a w i n g of t h e f i g u r e s a n d t y p i n g of t h e m a n u s c r i p t .
REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 18 19 2o 21 22 23 24 25 26 27 28 29 3° 31 32 33 34 35 36 37 38 39 4° 41 42 43
D. B. Roodyn, R. J. Reiss and T. S. Work, Biochem. J., 8o (1961) 9. D. 13. Roodyn, J. W. Suttie and T. S. Work, Biochem. J., 83 (I962) 29. S. Sandell, H. L6w and A. yon der Decken, Biochem. J . , IO4 (1967) 575. D. Haldar and K. B. Freeman, Biochem. J., I I I (1969) 653. D. S. Beattie, R. E. Basford and S. B. Koritz, Biochemistry, 6 (I967) 3099 . D. S. Beattie, R. E. Basford and S. B. Koritz, J. Biol. Chem., 242 (1967) 3366. D. S. Beattie, G. M. Patton and R. N. Stuchell, J. Biol. Chem., 245 (197 o) 2177. L. Wheeldon, Biochem. Biophys. Res. Commun., 24 (1966) 4o7 . L. W. Wheeldon and A. L. Lehninger, Biochemistry, 5 (1967) 3533. A. M. Kroon, Biochim. Biophys. Acta, 72 (1963) 391. A. M. Kroon, Biochim. Biophys. Acta, 91 (1964) 145. A. M. Kroon, Biochim. Biophys. Acta, io8 (I965) 275. A. M. Kroon, C. Saccone and M. J. 13otman, Biochim. Biophys. Acta, 142 (1967) 552. A. M. Kroon and H. de Vries, Syrup. Soc. Exp. Biol., 24 (197 o) 181. A. M. Kroon and H. de Vries, in Boardmann, Linnane and Smillie, Autonomy and Biogenesis of Mitochondria and Chloroplasts, North-Holland, i97 I, p. 318. F. C. Firkin and A. W. Linnane, F E B S Lett., 2 (1969) 33 o. M. W. Gordon and G. G. Deanin, J. Biol. Chem., 243 (I968) 4222. D. Halder, Biochem. Biophys. Res. Commun., 42 (1971) 899. D. E. S. Truman and A. Korner, Biochem..l., 83 (I962) 588. W. C. Schneider and G. H. Hogeboom, J. Biol. Chem., I83 (1956) I23. O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Chem., 193 (1951) 265. J. B. Chappell and S. V. Perry, Nature, 173 (1954) lO94. J. Mockel and J. E. Dumont, Eur. J. Clin. Invest., I (197 o) 32. G. A. Brooks, K. J. Hittelman, J. A. Faulkner and R. E. Beyer, Am. J. Physiol., 22o (1971) lO53. I¢. Hedman, Exp. Cell Res., 38 (1965) i. D, Neubert and H. Helge, Biochem. Biophys. Res. Commun., 18 (1965) 600. E. Wintersberger and V. Wintersberger, F E B S Lett., 6 (197 o) 58. Ming-Jer Tsai, G. Michaelis and R. S. Criddle, Proc. Natl. ,4cad. Sci. U.S., 68 (1971) 473. E. Wintersberger, Biochem. Biophys. Res. Commun., 4 ° (197 o) 1179. J. L. Coote and T. S. Work, Eur. J. Biochem., 23 (1971) 564. S. J. Surzycki, Proc. Natl. Acad. Sci. U.S., 63 (1969) 1327. L. G. Shmerling, Biochem. Biophys. Res. Commun., 37 (1969) ooo. H. Kiintzel and K. P. Schafer, Nature New Biol., 231 (1971) 265. E. S. Goldring, L. I. Grossman, D. Krupnick, D. R. Cryer and J. Marmur, J. Mol. Biol., 52 (197 ° ) 323. H. ]3. Horwitz and C. E. Holt, J. Cell Biol., 49 (1971) 546. K. Radsak, K. Kato, N. Sato and H. Koprowski, Exp. Cell Res., 66 (1971) 41o. P- S. Perlman and H. R. Mahler, Nature New Biol., 231 (I97I), 12. J. B. Chappell and A. R. Crofts, Biochem. J., 95 (1965) 393. 14. L. Williams and L. M. Birt, Eur. J. Biochem., 22 (1971) 87. D. D. Tyler, Biochem. J., I I I (1969) 665. E. D. Duee and P. V. Vignais, Biochim. Biophys. Aaa, lO 7 (1964) 184. G. Brierley and R. L. O'Brien, J. Biol. Chem., 24 ° (1965) 4532. H. H. Winkler, F. L. Bygrane and A. L. Lehninger, J. Biol. Chem., 243 (1968) 2o.
Biochim. Biophys. Acta, 277 (1972) 628-638
638 44 45 46 47 48 49 50 51 52 53 54 55 56
j.
MOCKEL
E. P f a f f a n d M. K l i n g e n b e r g , Euv. J . Biochem., 6 (1968) 66. M. J. W e i d e m a n n , H. E r d e l t a n d M. K l i n g e n b e r g , Eur..]. Biochem., I6 (197 o) 313. A. M. K r o o n , Biochim. Biophys. Acta, 72 (1963) 39t. E. ~Vintersberger, Biochemistry., 341 (I965) 409. S. V. G a n g a l a n d S. P. B e s s m a n , Biochem. Biophys. Res. Commun., 33 (1968) 675. S. 1~. C h a n a n d L. R i c h a r d s o n , J. Biol. Chem., 244 (I969) lO39. U. B r o n s e r t a n d W. N e u p e r t , in J. M. Tager, S. P a p a , E. Quagliariello a n d E. C. Slater, Regulation of Metabolic Processes in Mitochondria, B . B . A . L i b r a r y , Vol. 7, Elsevier, A m s t e r d a m , 1966, p. 426. W . Sebald, Th. B u c h e r , t3. Olbrich a n d F . K a u d e w i t z , F E B S Lett., I (1963) 236. :~-. J. L a m b , G. D. C l a r k - W a l k e r a n d A. "W. L i n n a n e , Biochim. Biophys. Acta, 161 (1968) 415. G. E. Kalf, Arch. Biochem. Biophys., IOi (1963) 35 o. M. L e d e r m a n a n d G. A t t a r d i , Biochem. Biophys. Res. Commun., 4 ° (197 o) 1492. ~V. D. J. G r a a f m a n s , R. Charles a n d J. M. Tager, Biochim. Biophys. Acta, 153 (1968) 916. R. Charles a n d S. G. v a n d e n Bergh, Bioehim. Biophys..4eta, 731 (I967) 393-
Biochim. Biophys. .4cta, 277 (1972) 6 2 8 - 6 3 8