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The influence of fatty acid composition on the restoration of succinate-cytochrome c reductase activity by phospholipids in extracted mitochondria
Chem. Phys. Lipids 1 (1966) 20-32 © North-Holland Publ. Co., Amsterdam
THE INFLUENCE
OF FATTY ACID COMPOSITION
ON THE RESTORATION REDUCTASE
OF SUCCINATE-CYTOCHROME
A C T I V I T Y BY P H O S P H O L I P I D S
EXTRACTED
c
IN
MITOCHONDRIA
G. G. DE PURY and F. D. COLLINS Russell Grimwade School of Biochemistry, University of Melbourne, Parkville N. 2, Victoria, Australia
1. The requirement of the electron transport chain for phospholipids, previously shown for beef heart mitochondria, has been confirmed for rat liver mitochondria. The succinatecytochrome c reductase activity of mitochondria whose lipids had been extracted with acetone has been restored by incubating with micelles of phospholipids. 2. Phospholipids that differ only in their fatty acid composition have been prepared from the livers of normal rats and rats deficient in essential fatty acids. 3. Restoration of enzyme activity was, within limits, proportional to the amount of phospholipid bound by mitochondrial protein and, once bound, pbospholipids from normal and deficient rats were equally effective. However, the rate of binding could be influenced by the fatty acid composition of the phospholipids. 4. It is suggested that enzymes of the electron transport chain do not have any special requirement for phospbotipids that contain essential fatty acids.
Introduction G r e e n and Fleischer 1) have shown that the enzyme complexes o f the electron t r a n s p o r t chain in beef heart m i t o c h o n d r i a have a r e q u i r e m e n t for p h o s p h o l i p i d . P h o s p h o l i p i d is needed for several steps in electron t r a n s p o r t ; succinate to u b i q u i n o n e , reduced u b i q u i n o n e to c y t o c h r o m e c a n d reduced c y t o c h r o m e c to oxygen2). It has been d e m o n s t r a t e d t h a t the a m o u n t o f enzyme activity restored is p r o p o r t i o n a l to the a m o u n t o f p h o s p h o l i p i d b o u n d by m i t o c h o n d r i a l p r o t e i n I 3). Different types o f p h o s p h o l i p i d s have been used to reactivate enzyme activity: lecithin, p h o s p h a t i d y l e t h a n o l a m i n e a n d c a r d i o l i p i n , as well as mixed m i t o c h o n d r i a l phospholipidsZ,3). It has been shown that, a l t h o u g h these p h o s p h o l i p i d s differ in the readiness with which they are b o u n d by m i t o c h o n d r i a l protein, they are all equally effective in restoring enzyme activity once they have been b o u n d 3). However, c o m p a r i s o n s have n o t been m a d e previously between p h o s p h o l i p i d s t h a t differ only in their fatty acid composition. The fatty acid c o m p o s i t i o n o f p h o s p h o l i p i d s in rats t h a t have been fed a
PHOSPHOLIPID FATTY ACIDS IN ELECTRON TRANSFER
21
diet deficient in essential fatty acids is markedly different from that of normal rats; the linoleic series of fatty acids (the essential fatty acids) are replaced by fatty acids of the oleic and palmitoleic series4-9). There have been reports that the activity of mitochondrial enzyme systems are altered in rats deficient in essential fatty acids 1°,11) and that oxidative phosphorylation is affected12.a3), which suggests that fatty acid composition may influence the activity of these enzymes. Materials ANIMALS
The rats were an inbred strain of Sprague-Dawley obtained from Holtzman's Rat Farm, Madison, Wis., U.S.A. Male animals at weaning were fed either a stock or a fat-flee diet 14) and after 12 weeks the animals fed the fat-flee diet were showing all the characteristic symptoms of a deficiency of essential fatty acids15). MITOCHONDRIA TREATED WITH AQUEOUS ACETONE*
Mitochondria from livers of normal rats were extracted with 90 per cent aqueous acetone by the method of Lester and Fleischer 16) and the extracted mitochondria were then resuspended in 0.88 M sucrose, buffered with 10 mM tris chloride, p H 7.5. This stock suspension was stored at - 1 8 ° until required. The mitochondria were exposed to acetone for only 12 to 13 min instead of the 20 min recommended for beef heart mitochondria16), because it was found that with rat liver mitochondria the longer extraction lowered the amount of enzyme activity that could subsequently be restored by phospholipid. Analysis of the extracted mitochondria showed that this treatment with acetone had extracted approximately 80 per cent of the phospholipids (table 1). PHOSPHOLIPID MICELLES
Phospholipids were obtained from liver of normal and deficient rats. Liver lipids were extracted and purified as described by Wheeldon and Collins 19) and the phospholipids isolated by the method of Galanos and Kapoulas2°). Phospholipid micelles were prepared by ultrasonic irradiation of an emulsion of phospholipids in 20 m M tris acetate buffer, p H 8.0, containing 1 mM EDTA. From 12 to 15 ml of emulsion, containing 10 to 20/tM of phospholipids per ml, were ultrasonically irradiated for 30 rain in a 60 watt ultrasonic disintegrator (Measuring and Scientific Equipment Ltd., London). In order * The abbreviation M T A A is used for mitochondria which have been treated with aqueous acetone.
22
G. G, DE PURY AND F. D. COLLINS TABLE 1
Total phosphorus and lipid phosphorus content of rat liver mitochondria and MTAA. Total phosphorus was determined by digesting samples of mitochondria with perchloric acid and estimating the phosphorus content of the digest17), Lipid phosphorus was determined by precipitating samples of mitochondria with 10 per cent trichloracetic acid,
washing tbe precipitate twice with 5 per cent trichloracetic acid and then extracting the lipid phosphorus with chloroform-ethanol (2:1 v/v) in three successive half-hour extractions at room temperature. The combined extracts were taken to dryness and analysed for phosphoruslS). /tM of phosphorus/mg of protein Total phosphorus Lipid phosphorus Mitochondria Mean MTAA
Mean
0.33
0.19
0.33 0.33
0.21 0.20
0.13
0.02
--
0.04
0.13 O. 13
0.05 0.04
to prevent oxidation the buffer was saturated with nitrogen and emulification and irradiation were carried out at 0 °. The resulting opalescent solutions of micelles were centrifuged at 80 000 x g for 30 rain to remove any aggregations and were then stored at 5° under nitrogen. The ultrasonic irradiation resulted in at least 80 per cent of the phospholipid remaining in micellar solution even after centrifuging. Care was taken that micelles of phospholipids from normal and deficient rats were always prepared in an identical manner, so that the only difference between them would be in their fatty acid composition. Analyses confirmed that their fatty acid compositions were markedly different (table 2), as has been reported previously8,9). OTHER MATERIALS Cytochrome c prepared from horse heart by the method of Keilin and Hartree 2~) and containing 0.30 per cent iron was the gift of Dr. R. W. Henderson of this department. Ubiquinon%o was purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. Other chemicals were of analytical grade quality and all solutions were made with distilled and de-ionized water. Solvents were dried and redistilled before use. Methods
The procedures of Fleischer et al)) were followed in measuring the restoration of succinate-cytochrome c reductase activity in extracted mitochondria and in re-isolating mitochondrial protein after incubation with phospholipid micelles. Phosphorus was determined by the method of Berenblum and
PHOSPHOLIPID FATTY ACIDS IN ELECTRONTRANSFER
23
TABLE 2 Fatty acid composition of phospholipid micelles. The methyl esters of phospholipid fatty acids were prepared and analysed by gas-liquid chromatography on poly(ethylene g l y c o l ) adipate a t 173 ° as described by Collins°~l). Values are the mean of six preparations and the standard errors are shown. Amount ( m o l e s / 1 0 0 m o l e s ) Normal Myristic a c i d n-Pentadecanoic a c i d Palmitic acid Palmitoleic acid
n-Heptadecanoic a c i d Stearic a c i d Oleic a c i d Unidentified Linoleic acid
6,9-Octadecadienoic a c i d 5,8,11-Eicosatrienoic acid Arachidonic a c i d
0.l 0.1 19.3 1.3 0.7 29.4 9.8
: 0.04 - : 0.04 v: 1.8 - 0.3 :_ 0.08 ! 1.2 ~ 0.7 -17.5 ~ 1.2 21.6 - ' 0.4
Deficient 0.2 ! 0.08 17.3 = 0.8 9.2 - 0 . 7 29.9 0.6 27.2 - 0.7 1.7 ! 0.02 0.8 ~ 0.3 18.0 :[ 1.0 3.3 _J: 0.3
These micelles also contained no more than 3 % of fatty acids with retention times longer than that of arachidonic acid.
Chain 17) or as described by Collins18). Protein was determined by the method of Cleland and Slater23), using bovine serum albumin as a standard. Results RESTORATION OF SUCCINATE-CYTOCHROME C REDUCTASE ACTIVITY IN EXTRACTED MITOCHONDRIA BY INCUBATION WITH PHOSPHOLIPID MICELLES
M T A A were unable to reduce cytochrome c in the presence of succinate and cyanide. However some succinate-cytochrome c reductase activity could be restored by incubating the M T A A with ubiquinoneso and micelles of mixed phospholipids for 20 min before assaying enzyme activity (table 3). Ubiquinoneso by itself did not restore any enzyme activity, but when it was not included in an incubation mixture less enzyme activity was restored by phospholipid, showing that both ubiquinoneso and pbospholipid were needed for maximum activity (cf. Redfearn and Burgose4)). In the experiment described in table 3 phospholipids from normal rats restored less activity than did phospholipids from deficient rats. This difference was not due to an insufficient concentration of ubiquinoneso, because doubling the concentration of ubiquinoneso in the incubation medium containing "normal" phospholipids did not increase the restored activity.
24
¢;. G. DE PURY AND F. D. COLLINS TABLE 3
Effect of ubiquinones0 and phospholipids on the restoration of succinate-cytochrome c reductase activity in MTAA. Before assaying enzyme activity, 0.1 ml of MTAA suspension, containing approximately 25/tg of mitochondria protein, were added to cuvettes containing 2.8 ml of a mixture of ubiquinones0, mixed phospholipid micelles, 2 nag of cytochronae c and 60 mM potassium phosphate buffer, pH 7.5. After incubating for 20 rain at 30 °, the enzyme assay was started by adding 0.05 nal of 60 naM potassium cyanide and 0.05 ml of 300 mM potassium succinate. Ubiquinoneso was in an ethanolic solution, containing 2 mg/mI. Mixed phospholipids (/~M/cuvette) 0.00 0.00 0.32 0.35 0.35 0.32
Type
Ubiquinones0 (ld/cuvette)
Restored enzyme activity (ttM/min/mg of protein)
0 20 20 20 0 40
0.00 0.00 0.29 0.44 0.13 0.27
Normal Deficient Deficient Normal
TABLE 4
Effect of storage at -- 18~ on the restoration of succinate-cytochrome c reductase activity in MTAA. MTAA were stored, as a suspension in buffered 0.88 M sucrose, at a concentration of either 0.21 mg of protein/nal or 2.4 nag of protein/ml. All suspensions of MTAA were diluted to 0.21 mg of protein/nal before adding to cuvettes for the incubation with phospbolipid micelles. All incubations contained 20/A of an ethanolic solution of ubiquinone (2 nag/ml) and were otherwise the same as those described in table 3. Mixed phospholipids (pM/cuvette)
Type
MTAA
0.19 0.19
Deficient Deficient
0.19
Deficient
0.16 0.16
Normal Normal
Freshly prepared Frozen for 12 days in concentrated suspension Frozen for 14 days in dilute suspension Freshly prepared Frozen for 12 days in concentrated suspension
T h e d a t a o f t a b l e 4 s h o w t h a t if M T A A
Restored enzyme activity (/~M/cuvette) of protein) 0.46 0.49
0.24 0.13 0.18
were frozen in a relatively concen-
t r a t e d s u s p e n s i o n i n b u f f e r e d 0.88 M s u c r o s e , t h e y c o u l d b e s t o r e d a t - 18 ° f o r a t l e a s t 12 d a y s , w i t h o u t a f f e c t i n g t h e s u b s e q u e n t r e s t o r a t i o n o f t h e i r enzyme activity. However, if the suspension was diluted about ten-fold, with buffered sucrose, before freezing and storing, restoration of enzyme activity
PHOSPHOLIPID
FATTY ACIDS IN ELECTRON TRANSFER
25
was impaired. Hence all stock suspensions of M T A A were frozen as concentrated suspensions for storage and were thawed and diluted just before use. Comparisons were made of the effectiveness of phospholipids from normal and deficient rats in restoring succinate-cytochrome c reductase activity in extracted mitochondria. Fig. 1 shows the results of a typical experiment. As the concentrations of phospholipid micelles in the incubation were increased the amount of enzyme activity restored in the M T A A increased towards a maximum value. However, "deficient" phospholipid micelles restored more activity than "normal" phospholipid micelles at all concentrations, and the maximum activity obtained with "deficient" phospholipids was higher than that with "normal" phospholipids. This experiment was repeated six times and the results of these seven experiments are summarized in table 5. In every experiment enzyme activity was increased by incubating with progressively higher concentrations of phospholipid micelles until a maximum activity was reached, but the activity produced by equal concentrations of phospholipid and the maximum activity obtained varied between different preparations of M T A A and phospholipid micelles. In five experiments phospholipids from deficient rats were more effective than "normal" phospholipids of the same concentration in restoring enzyme activity and they pro-
"2
0.4
0
h "V
~
0.3
E ]E ::k 02 >
~ /
• Normal V Deficient
u 01 E N klJ
I
I
01 02 Concentrat;~n of phospholipid (~l.M/ml)
I
03
Fig. 1. Restoration ofsuccinate-cytochrome c reductase activity in MTAA by incubation with micelles of phospholipids from normal and deficient rats. Incubations and enzyme assays were carried out as described in table 3. All incubations contained 20/tl of an ethanolic solution of ubiquinones0 (2 mg/ml) and the final concentration of MTAA was 8.7/~g of protein/ml. V, "normal" phospholipids; A, "deficient" phospholipids.
26
G.G.
D E P U R Y A N D F. D . C O L L I N S
TABLE 5 Comparison of the succinate-cytochrome c reductase activity restored by micelles of mixed phospholipids from normal and deficient rats. This table summarizes the results of experiments similar to that described in fig. 1. The data have been obtained by interpolation between experimental points. Each experiment used different preparations of MTAA and micelles, except that the same preparation of MTAA was used in experiments (4), (5) and (6) and the same paired preparations of micelles were used in experiments (3) and (4).
Experiment
Concentration of phospholipids (~M/ml)
(1)
Enzyme activity (/~M/min/mg protein) Normal Deficient
Ratio deficient/normal
0.08 0.12 0.20
0.20 0.29 0.31
0.46 0.43
2.25
(2)
0.08 0.11
0.09 0.10
0.17 0.19
1.95 1.82
(3)*
0.08 0.12 0.20
0.19 0.29 0.32
0.32 0.36 0.40
1.64 1.27 1.24
(4)
0.08 0.12 0.20
0.11 0.18 0.32
0.18 0.27 0.39
1.66 1.49 1.23
(5)
0.08 0.12 0.20
0.ll 0.17 0.23
0.11 0.17 0.24
1.00 1.01 1.03
(6)
0.08 0.12 0.20 0.28
0.11 0.16 0.16 0.15
0.1 I 0.18 0.19 0.18
0.98 1.12 1.16
0.08 0.12 0.20 0.28
0.07 0.14 0.16 0.17
0.05 0.11 0.17 0.18
0.53 0.77
(7)
1.50
1.22
1.06 1.06
* Data from fig. 1. duced greater maximum activity than did "normal"
p h o s p h o l i p i d s . I n ex-
p e r i m e n t (5), h o w e v e r , these differences w e r e n o t a p p a r e n t , a n d in e x p e r i m e n t (7) " d e f i c i e n t " p h o s p h o l i p i d s p r o d u c e d o n l y a slightly g r e a t e r m a x i m u m e n z y m e activity, w h i l e " n o r m a l " p h o s p h o l i p i d s p r o d u c e d m o r e a c t i v i t y w h e n l o w c o n c e n t r a t i o n s o f m i c e l l e s w e r e used. COMPARISON OF RESTORED ENZYME ACTIVITY WITH THAT FOUND IN UNEXTRACTED MITOCHONDRIA T h e s u c c i n a t e - c y t o c h r o m e c r e d u c t a s e a c t i v i t y o f freshly p r e p a r e d m i t o c h o n d r i a t h a t h a d n o t b e e n t r e a t e d w i t h a c e t o n e was q u i t e v a r i a b l e a n d t h e r e
PHOSPHOLIPID FATTY ACIDS IN ELECTRON TRANSFER
27
was no significant difference in activity between mitochondria prepared from the livers of normal or deficient rats (table 6). The maximum enzyme activities restored in M T A A (table 5) were between 38 per cent and I05 per cent of the average activities found in unextracted mitochondria, indicating that incubation with ubiquinon%o and phospholipid micelles restored the greater part of the enzyme activity that had been lost on extracting mitochondria with aqueous acetone. TABLE 6 S u c c i n a t e - c y t o c h r o m e c reductase activity o f unextracted m i t o c h o n d r i a f r o m livers o f n o r m a l and deficient rats. T h e m e t h o d o f assay was the s a m e as that used for M T A A , except that phospholipid micelles a n d ubiquinones0 were n o t added. T h e n u m b e r o f a n i m a l s e x a m i n e d a n d the s t a n d a r d errors are shown. E n z y m e activity ( / z M / m i n / m g o f protein) N o r m a l (13) Deficient (I 1)
0.44 ~0.07 0.43 ! 0.03
CORRELATION OF ENZYME ACTIVITY WITH BINDING OF PHOSPHOLIPID MICELLES
Analysis of particles of MTAA re-isolated after incubation with phospholipid micelles showed that they had bound phospholipid and also that increasing the concentration of phospholipid in the incubation medium increased the amount of phospholipid bound. Fig. 2 gives the results of four experiments in which comparisons were made of the binding by M T A A of phospholipids from normal and deficient rats. In two experiments more of the "deficient" phospholipids were bound than were " n o r m a l " phospholipids, while in the other two experiments the binding of both types of phospholipids was the same. There was also a considerable variation between experiments in the amounts of phospholipid that were bound. However, assaying the succinate-cytochrome c reductase activity of the re-isolated MTAA showed that restored enzyme activity was directly proportional to the amount of phospholipid bound (fig. 3). In each experiment the same relationship was found up to approximately 0.4/lM of phospholipid bound per mg of protein; uptake greater than this did not lead to any further increase in enzyme activity. Fig. 3 thus shows that once bound by M T A A all the preparations of phospholipids were equally effective in restoring enzyme activity, regardless of whether they were from normal or deficient rats. In the one experiment where binding occurred much in excess of 0.4 ItM per mg of protein, restored enzyme activity reached a maximum value and the
28
6 . G . D E P U R Y A N D F. D . C O L L I N S
16 0 L O.
'~ 12 C~
E 0.8 t
~ 0.4 0
a_
C
t
[
I
i
[
I
1.0 2 0 3.0 Concentr-ation of phospholLpid
I
I
J
4.0 (}J.M/ml)
Fig. 2. Comparison of the binding by MTAA of micelles of phospholipids from normal and deficient rats. Micelles of mixed phospholipids in 3 ml of 20 mM tris acetate buffer, pH 8.0, were mixed with 3 ml of a suspension of MTAA in 0.88 M sucrose, buffered with 10 mM tris chloride, pH 7.5. The final concentration of MTAA was approximately 0.3 mg of protein/ml. After incubating the mixture for 20 rain at 30°, the MTAA were re-isolated a) and analysed to determine the amount of phospholipid they had bound. The different symbols show the results of four experiments comparing "normal" phospholipids (closed symbols; O , l , &, ~') with "deficient" phospholipids (open symbols; } , ~ , ~,, V).
m a x i m u m activity o b t a i n e d with "deficient" p h o s p h o l i p i d s in this experim e n t was greater than that o b t a i n e d with " n o r m a l " p h o s p h o l i p i d s .
Discussion T h e experimental results d e m o n s t r a t e that after extraction with a q u e o u s acetone rat liver m i t o c h o n d r i a lose a b o u t 80 per cent o f their p h o s p h o l i p i d s and also all their s u c c i n a t e - c y t o c h r o m e c reductase activity. The enzyme activity can be restored by i n c u b a t i n g with ubiquinone50 and micelles o f mixed p h o s p h o l i p i d s f r o m rat liver, a n d the a m o u n t o f activity restored is p r o p o r t i o n a l to the a m o u n t o f p h o s p h o l i p i d b o u n d by the extracted mitoc h o n d r i a . These results are very similar to those that have been o b t a i n e d with extracted beef heart m i t o c h o n d r i a S ) , a n d this similarity suggests that the i n f o r m a t i o n o b t a i n e d by G r e e n a n d his c o - w o r k e r s l) f r o m experiments on beef h e a r t m i t o c h o n d r i a is also valid for rat liver m i t o c h o n d r i a . W h e n M T A A were i n c u b a t e d with high c o n c e n t r a t i o n s o f p h o s p h o l i p i d micelles, they b o u n d p h o s p h o l i p i d far in excess o f the a m o u n t f o u n d in unextracted m i t o c h o n d r i a . These excessive a m o u n t s o f p h o s p h o l i p i d did not
PHOSPHOLIPID
FATTY
ACIDS
IN ELECTRON
29
TRANSFER
O L Q_
0.24 v
•o
E o
"-- o.16 :::L
%-,
2~ +_~ >
OIIAT N o r m a l ODZ~2 D e f i c i e n t
uc~ 0.©8 ©
E N
0
|
I
I
I
I
04 0.8 Phospholipid bound
I
I
I
I
I
12 16 2.0 ( l ~ M / m g of p r o t e i n )
Fig. 3. Correlation of restored enzyme activity in MTAA with binding of micelles of phospholipids from normal and deficient rats. The particles of MTAA that had been reisolated after incubation with pbospholipid micelles as described in fig. 2 were assayed for succinate-cytochrome c reductase activity and for bound phospholipid. The enzyme assay was carried out as described in table 3, except that instead of being incubated with phospholipid micelles and ubiquinones0 for 20 min, the re-isolated particles were incubated with ubiquinones0 alone for 3 rain (longer incubations being found to lower enzyme activity). The symbols are as described in fig. 2.
result in any increase in enzyme activity, a n d they were p r o b a b l y o f no physiological significance, except to indicate t h a t the particles o f m i t o c h o n d r i a l p r o t e i n c o n t i n u e d to b i n d p h o s p h o l i p i d micelles after the sites which required lipid for enzyme activity had been filled. A similar excessive b i n d i n g occurs when beef heart m i t o c h o n d r i a are i n c u b a t e d with micelles o f c a r d i o l i p i n , but n o t when they are i n c u b a t e d with micelles o f mixed phosp h o l i p i d s f r o m mitochondriaS). The excessive u p t a k e by M T A A m a y have been due to p o l a r b o n d s being f o r m e d between anionic g r o u p s o f the phosp h o l i p i d s a n d free a m i n o g r o u p s in the m i t o c h o n d r i a l protein in a d d i t i o n to h y d r o p h o b i c bonds, which are k n o w n to occur between fatty acids o f phosp h o l i p i d s a n d h y d r o p h o b i c areas o f m i t o c h o n d r i a l structural proteina,2~). A l s o an excessive a m o u n t o f p h o s p h o l i p i d m a y have been b o u n d because m i t o c h o n d r i a f r o m rat liver, being m o r e fragile t h a n those f r o m beef heart, m a y have suffered greater d a m a g e d u r i n g extraction, so that m o r e sites suitable for b o t h h y d r o p h o b i c a n d p o l a r b o n d i n g were exposed. The v a r i a t i o n between experiments in the a m o u n t o f p h o s p h o l i p i d b o u n d
30
G . G , D E P U R Y A N D F. D. C O L L I N S
by M T A A shows that preparations of MTAA, and perhaps also of micelles, differed in the readiness with which they interacted. It is likely that this variation was due to differences in the number of binding sites per mg of protein that were available to micelles, showing that quantitative comparisons cannot be made between results of experiments which used different preparations of MTAA. However in all the experiments the same enzyme activity was restored per/~M of phospholipid bound, indicating that all the preparations of MTAA had the same capacity for electron transport, even if they differed in their readiness to bind phospholipid. The experiments comparing phospholipids from normal and deficient rats demonstrate that differences in fatty acid composition also can affect the binding of phospholipids by MTAA but that, once phospholipids are bound, differences in fatty acid composition do not influence the effectiveness of phospholipids in restoring enzyme activity. This conclusion is an extension of the previous discovery by Green and his co-workers 1) that, while some types of phospholipids such as lecithin, were bound less readily than others, all the phospholipids investigated were equally effective in promoting enzyme activity once they had been bound by mitochondrial protein 3). In one experiment (fig. 3), an excessive binding of phospholipids from a deficient rat restored a greater enzyme activity than did "normal" phospholipids, which may indicate that the "deficient" phospholipids were able to penetrate to more sites that required phospholipid for enzyme activity. However, the results with binding of physiological amounts of phospholipids indicate that the enzymes of the electron transport chain that transfer electrons from succinate through to cytochrome c do not have any special requirement for phospholipids containing essential fatty acids. Moreover, by inference from similar experiments using different types of phospholipids %3), it is likely that this conclusion applies equally to other enzymes in the electron transport chain. Fatty acid composition, however, can influence binding of phospholipid micelles by mitochondrial protein. In some binding experiments "deficient" phospholipids were bound more readily than "normal" phospholipids, while in other experiments no differences were observed (fig. 3). Since it has been shown that restoration of enzyme activity is proportional to the amount of phospholipid bound by MTAA, the results of earlier experiments, in which enzyme activity was measured in the presence of micelles of phospholipids from normal or deficient rats (table 5), also can be used to compare the amounts of "normal" and "deficient" phospholipids that were bound. These comparisons show that, while in a few experiments phospholipids from normal and deficient rats were bound to an equal extent, in most experiments phospholipids from deficient rats were bound more readily. However, ex-
PHOSPHOL1PID FATTY ACIDS IN ELECTRON 'IRANSFER
3]
p e r i m e n t s with lecithins have shown that it is the rate o f b i n d i n g which is affected by fatty acid composition26). Therefore, since these e x p e r i m e n t s with micelles o f mixed p h o s p h o l i p i d s m e a s u r e d enzyme activity or u p t a k e after a c o n s t a n t p e r i o d o f 20 min, close c o m p a r i s o n s between the perform a n c e o f n o r m a l a n d deficient p h o s p h o l i p i d s c a n n o t be m a d e ; it is p r o b a b l e that the p h o s p h o l i p i d micelles were not b o u n d at a c o n s t a n t rate for 20 min, so the total u p t a k e o f p h o s p h o l i p i d was n o t a reliable measure o f rate of binding. M o r e o v e r , since p o l a r b o n d i n g , which w o u l d n o t have been influenced by fatty acid c o m p o s i t i o n , was p r o b a b l y occurring as well as h y d r o p h o b i c b o n d i n g , differences between the rates o f b i n d i n g o f " n o r m a l " and "deficient" p h o s p h o l i p i d s could have been obscured. Nevertheless, the results o f these experiments suggest t h a t fatty acid c o m p o s i t i o n does influence the binding o f p h o s p h o l i p i d s by structural p r o t e i n a n d that p h o s p h o l i p i d s from rats deficient in essential fatty acids are b o u n d m o r e quickly t h a n are "normal" phospholipids.
Acknowledgements Dr. E, L. F r e n c h o f the D i v i s i o n o f A n i m a l Health, C o m m o n w e a l t h Scientific a n d Industrial R e s e a r c h O r g a n i z a t i o n , k i n d l y m a d e available the ultrasonic d i s i n t e g r a t o r . F i n a n c i a l s u p p o r t f r o m the N a t i o n a l H e a l t h a n d M e d i c a l Research C o u n cil o f A u s t r a l i a is gratefully a c k n o w l e d g e d .
References 1) D. E. Green and S. Fleischer, Biochim. Biophys. Acta 70 (1963) 554. 2) G. P. Brierley, A. J. Merola and S. Fleischer, Biochim. Biophys. Acta 64 (1962) 218. 3) S. Fleischer, G. P. Brierley, H. Klouwen and D. B. Slautterback, J. Biol. Chem, 237 (1962) 3264. 4) F. D. Collins, Biochem. Biophys. Res. Commun. 9 (1962) 289. 5) H. Mohrhauer and R. T. Holman, J. Lipid Res. 4 (1963) 151. 6) H. Mohrhauer and R. T. Holman, J. Lipid Res. 4 (1963) 346. 7) J. J. Rahm and R. T. Holman, J. Lipid Res. 5 (1964) 169. 8) L. A. Biran, W. Bartley, C. W. Carter and A. Renshaw, Biochem. J. 93 (1964) 492. 9) R. M. Johnson and T. lto, J. Lipid Res. 6 (1965) 75. 10) H. O. Kunkel and J. N. Williams, J. Biol. Chem. 189 (1951) 755. 11) P. G. Tulpule and V. N. Patwardban, Arch. Biochem. Biophys. 39 (1952) 450. 12) P. D. Klein and R. M. Johnson, J. Biol. Chem. 211 (1954) 103. 13) P. G. Tulpule and J. N. Williams, J. Biol. Chem. 217 (1955) 229. 14) F. D. Collins, Biochem. J. 99 (1966) 117. 15) E. Aaes-Jorgensen, Physiol. Rev. 41 (1961) 1. 16) R. L. Lester and S, Fleischer, Biochim. Biophys. Acta 47 (1961) 358. 17) I. Berenblum and E. Chain, Biochem. J. 32 (1938) 295. 18) F. D. Collins, Biochem. J. 72 (1959) 532. 19) L. W. Wheeldon and F. D. Collins, Biochem. J. 66 (1957) 435. 20) D. S. Galanos and V. M. Kapoulas, J. Lipid Res. 3 (1962) 134.
32 21) 22) 23) 24) 25)
G.G.
D E P U R Y A N D F. D . C O L L I N S
F. D. Collins, Biochem. J. 88 (1963) 319. D. Keilin and E. F. Hartree, Biochem. Prep. 2 (1952) 1. K. W. Cleland and E. C. Slater, Biochem. J. 53 (1953) 547. E. R. Redfearn and J. Burgos, Nature 209 (1966) 711. S. H. Richardson, H. O. Hultin and S. Fleischer, Arch. Biochem. Biophys. 105 (1964) 254. 26) G. G. de Pury and F. D. Collins, Chem. Phys. Lipids, 1 (1966) 1.
THE INFLUENCE
OF FATTY ACID COMPOSITION
ON THE RESTORATION REDUCTASE
OF SUCCINATE-CYTOCHROME
A C T I V I T Y BY P H O S P H O L I P I D S
EXTRACTED
c
IN
MITOCHONDRIA
G. G. DE PURY and F. D. COLLINS Russell Grimwade School of Biochemistry, University of Melbourne, Parkville N. 2, Victoria, Australia
1. The requirement of the electron transport chain for phospholipids, previously shown for beef heart mitochondria, has been confirmed for rat liver mitochondria. The succinatecytochrome c reductase activity of mitochondria whose lipids had been extracted with acetone has been restored by incubating with micelles of phospholipids. 2. Phospholipids that differ only in their fatty acid composition have been prepared from the livers of normal rats and rats deficient in essential fatty acids. 3. Restoration of enzyme activity was, within limits, proportional to the amount of phospholipid bound by mitochondrial protein and, once bound, pbospholipids from normal and deficient rats were equally effective. However, the rate of binding could be influenced by the fatty acid composition of the phospholipids. 4. It is suggested that enzymes of the electron transport chain do not have any special requirement for phospbotipids that contain essential fatty acids.
Introduction G r e e n and Fleischer 1) have shown that the enzyme complexes o f the electron t r a n s p o r t chain in beef heart m i t o c h o n d r i a have a r e q u i r e m e n t for p h o s p h o l i p i d . P h o s p h o l i p i d is needed for several steps in electron t r a n s p o r t ; succinate to u b i q u i n o n e , reduced u b i q u i n o n e to c y t o c h r o m e c a n d reduced c y t o c h r o m e c to oxygen2). It has been d e m o n s t r a t e d t h a t the a m o u n t o f enzyme activity restored is p r o p o r t i o n a l to the a m o u n t o f p h o s p h o l i p i d b o u n d by m i t o c h o n d r i a l p r o t e i n I 3). Different types o f p h o s p h o l i p i d s have been used to reactivate enzyme activity: lecithin, p h o s p h a t i d y l e t h a n o l a m i n e a n d c a r d i o l i p i n , as well as mixed m i t o c h o n d r i a l phospholipidsZ,3). It has been shown that, a l t h o u g h these p h o s p h o l i p i d s differ in the readiness with which they are b o u n d by m i t o c h o n d r i a l protein, they are all equally effective in restoring enzyme activity once they have been b o u n d 3). However, c o m p a r i s o n s have n o t been m a d e previously between p h o s p h o l i p i d s t h a t differ only in their fatty acid composition. The fatty acid c o m p o s i t i o n o f p h o s p h o l i p i d s in rats t h a t have been fed a
PHOSPHOLIPID FATTY ACIDS IN ELECTRON TRANSFER
21
diet deficient in essential fatty acids is markedly different from that of normal rats; the linoleic series of fatty acids (the essential fatty acids) are replaced by fatty acids of the oleic and palmitoleic series4-9). There have been reports that the activity of mitochondrial enzyme systems are altered in rats deficient in essential fatty acids 1°,11) and that oxidative phosphorylation is affected12.a3), which suggests that fatty acid composition may influence the activity of these enzymes. Materials ANIMALS
The rats were an inbred strain of Sprague-Dawley obtained from Holtzman's Rat Farm, Madison, Wis., U.S.A. Male animals at weaning were fed either a stock or a fat-flee diet 14) and after 12 weeks the animals fed the fat-flee diet were showing all the characteristic symptoms of a deficiency of essential fatty acids15). MITOCHONDRIA TREATED WITH AQUEOUS ACETONE*
Mitochondria from livers of normal rats were extracted with 90 per cent aqueous acetone by the method of Lester and Fleischer 16) and the extracted mitochondria were then resuspended in 0.88 M sucrose, buffered with 10 mM tris chloride, p H 7.5. This stock suspension was stored at - 1 8 ° until required. The mitochondria were exposed to acetone for only 12 to 13 min instead of the 20 min recommended for beef heart mitochondria16), because it was found that with rat liver mitochondria the longer extraction lowered the amount of enzyme activity that could subsequently be restored by phospholipid. Analysis of the extracted mitochondria showed that this treatment with acetone had extracted approximately 80 per cent of the phospholipids (table 1). PHOSPHOLIPID MICELLES
Phospholipids were obtained from liver of normal and deficient rats. Liver lipids were extracted and purified as described by Wheeldon and Collins 19) and the phospholipids isolated by the method of Galanos and Kapoulas2°). Phospholipid micelles were prepared by ultrasonic irradiation of an emulsion of phospholipids in 20 m M tris acetate buffer, p H 8.0, containing 1 mM EDTA. From 12 to 15 ml of emulsion, containing 10 to 20/tM of phospholipids per ml, were ultrasonically irradiated for 30 rain in a 60 watt ultrasonic disintegrator (Measuring and Scientific Equipment Ltd., London). In order * The abbreviation M T A A is used for mitochondria which have been treated with aqueous acetone.
22
G. G, DE PURY AND F. D. COLLINS TABLE 1
Total phosphorus and lipid phosphorus content of rat liver mitochondria and MTAA. Total phosphorus was determined by digesting samples of mitochondria with perchloric acid and estimating the phosphorus content of the digest17), Lipid phosphorus was determined by precipitating samples of mitochondria with 10 per cent trichloracetic acid,
washing tbe precipitate twice with 5 per cent trichloracetic acid and then extracting the lipid phosphorus with chloroform-ethanol (2:1 v/v) in three successive half-hour extractions at room temperature. The combined extracts were taken to dryness and analysed for phosphoruslS). /tM of phosphorus/mg of protein Total phosphorus Lipid phosphorus Mitochondria Mean MTAA
Mean
0.33
0.19
0.33 0.33
0.21 0.20
0.13
0.02
--
0.04
0.13 O. 13
0.05 0.04
to prevent oxidation the buffer was saturated with nitrogen and emulification and irradiation were carried out at 0 °. The resulting opalescent solutions of micelles were centrifuged at 80 000 x g for 30 rain to remove any aggregations and were then stored at 5° under nitrogen. The ultrasonic irradiation resulted in at least 80 per cent of the phospholipid remaining in micellar solution even after centrifuging. Care was taken that micelles of phospholipids from normal and deficient rats were always prepared in an identical manner, so that the only difference between them would be in their fatty acid composition. Analyses confirmed that their fatty acid compositions were markedly different (table 2), as has been reported previously8,9). OTHER MATERIALS Cytochrome c prepared from horse heart by the method of Keilin and Hartree 2~) and containing 0.30 per cent iron was the gift of Dr. R. W. Henderson of this department. Ubiquinon%o was purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. Other chemicals were of analytical grade quality and all solutions were made with distilled and de-ionized water. Solvents were dried and redistilled before use. Methods
The procedures of Fleischer et al)) were followed in measuring the restoration of succinate-cytochrome c reductase activity in extracted mitochondria and in re-isolating mitochondrial protein after incubation with phospholipid micelles. Phosphorus was determined by the method of Berenblum and
PHOSPHOLIPID FATTY ACIDS IN ELECTRONTRANSFER
23
TABLE 2 Fatty acid composition of phospholipid micelles. The methyl esters of phospholipid fatty acids were prepared and analysed by gas-liquid chromatography on poly(ethylene g l y c o l ) adipate a t 173 ° as described by Collins°~l). Values are the mean of six preparations and the standard errors are shown. Amount ( m o l e s / 1 0 0 m o l e s ) Normal Myristic a c i d n-Pentadecanoic a c i d Palmitic acid Palmitoleic acid
n-Heptadecanoic a c i d Stearic a c i d Oleic a c i d Unidentified Linoleic acid
6,9-Octadecadienoic a c i d 5,8,11-Eicosatrienoic acid Arachidonic a c i d
0.l 0.1 19.3 1.3 0.7 29.4 9.8
: 0.04 - : 0.04 v: 1.8 - 0.3 :_ 0.08 ! 1.2 ~ 0.7 -17.5 ~ 1.2 21.6 - ' 0.4
Deficient 0.2 ! 0.08 17.3 = 0.8 9.2 - 0 . 7 29.9 0.6 27.2 - 0.7 1.7 ! 0.02 0.8 ~ 0.3 18.0 :[ 1.0 3.3 _J: 0.3
These micelles also contained no more than 3 % of fatty acids with retention times longer than that of arachidonic acid.
Chain 17) or as described by Collins18). Protein was determined by the method of Cleland and Slater23), using bovine serum albumin as a standard. Results RESTORATION OF SUCCINATE-CYTOCHROME C REDUCTASE ACTIVITY IN EXTRACTED MITOCHONDRIA BY INCUBATION WITH PHOSPHOLIPID MICELLES
M T A A were unable to reduce cytochrome c in the presence of succinate and cyanide. However some succinate-cytochrome c reductase activity could be restored by incubating the M T A A with ubiquinoneso and micelles of mixed phospholipids for 20 min before assaying enzyme activity (table 3). Ubiquinoneso by itself did not restore any enzyme activity, but when it was not included in an incubation mixture less enzyme activity was restored by phospholipid, showing that both ubiquinoneso and pbospholipid were needed for maximum activity (cf. Redfearn and Burgose4)). In the experiment described in table 3 phospholipids from normal rats restored less activity than did phospholipids from deficient rats. This difference was not due to an insufficient concentration of ubiquinoneso, because doubling the concentration of ubiquinoneso in the incubation medium containing "normal" phospholipids did not increase the restored activity.
24
¢;. G. DE PURY AND F. D. COLLINS TABLE 3
Effect of ubiquinones0 and phospholipids on the restoration of succinate-cytochrome c reductase activity in MTAA. Before assaying enzyme activity, 0.1 ml of MTAA suspension, containing approximately 25/tg of mitochondria protein, were added to cuvettes containing 2.8 ml of a mixture of ubiquinones0, mixed phospholipid micelles, 2 nag of cytochronae c and 60 mM potassium phosphate buffer, pH 7.5. After incubating for 20 rain at 30 °, the enzyme assay was started by adding 0.05 nal of 60 naM potassium cyanide and 0.05 ml of 300 mM potassium succinate. Ubiquinoneso was in an ethanolic solution, containing 2 mg/mI. Mixed phospholipids (/~M/cuvette) 0.00 0.00 0.32 0.35 0.35 0.32
Type
Ubiquinones0 (ld/cuvette)
Restored enzyme activity (ttM/min/mg of protein)
0 20 20 20 0 40
0.00 0.00 0.29 0.44 0.13 0.27
Normal Deficient Deficient Normal
TABLE 4
Effect of storage at -- 18~ on the restoration of succinate-cytochrome c reductase activity in MTAA. MTAA were stored, as a suspension in buffered 0.88 M sucrose, at a concentration of either 0.21 mg of protein/nal or 2.4 nag of protein/ml. All suspensions of MTAA were diluted to 0.21 mg of protein/nal before adding to cuvettes for the incubation with phospbolipid micelles. All incubations contained 20/A of an ethanolic solution of ubiquinone (2 nag/ml) and were otherwise the same as those described in table 3. Mixed phospholipids (pM/cuvette)
Type
MTAA
0.19 0.19
Deficient Deficient
0.19
Deficient
0.16 0.16
Normal Normal
Freshly prepared Frozen for 12 days in concentrated suspension Frozen for 14 days in dilute suspension Freshly prepared Frozen for 12 days in concentrated suspension
T h e d a t a o f t a b l e 4 s h o w t h a t if M T A A
Restored enzyme activity (/~M/cuvette) of protein) 0.46 0.49
0.24 0.13 0.18
were frozen in a relatively concen-
t r a t e d s u s p e n s i o n i n b u f f e r e d 0.88 M s u c r o s e , t h e y c o u l d b e s t o r e d a t - 18 ° f o r a t l e a s t 12 d a y s , w i t h o u t a f f e c t i n g t h e s u b s e q u e n t r e s t o r a t i o n o f t h e i r enzyme activity. However, if the suspension was diluted about ten-fold, with buffered sucrose, before freezing and storing, restoration of enzyme activity
PHOSPHOLIPID
FATTY ACIDS IN ELECTRON TRANSFER
25
was impaired. Hence all stock suspensions of M T A A were frozen as concentrated suspensions for storage and were thawed and diluted just before use. Comparisons were made of the effectiveness of phospholipids from normal and deficient rats in restoring succinate-cytochrome c reductase activity in extracted mitochondria. Fig. 1 shows the results of a typical experiment. As the concentrations of phospholipid micelles in the incubation were increased the amount of enzyme activity restored in the M T A A increased towards a maximum value. However, "deficient" phospholipid micelles restored more activity than "normal" phospholipid micelles at all concentrations, and the maximum activity obtained with "deficient" phospholipids was higher than that with "normal" phospholipids. This experiment was repeated six times and the results of these seven experiments are summarized in table 5. In every experiment enzyme activity was increased by incubating with progressively higher concentrations of phospholipid micelles until a maximum activity was reached, but the activity produced by equal concentrations of phospholipid and the maximum activity obtained varied between different preparations of M T A A and phospholipid micelles. In five experiments phospholipids from deficient rats were more effective than "normal" phospholipids of the same concentration in restoring enzyme activity and they pro-
"2
0.4
0
h "V
~
0.3
E ]E ::k 02 >
~ /
• Normal V Deficient
u 01 E N klJ
I
I
01 02 Concentrat;~n of phospholipid (~l.M/ml)
I
03
Fig. 1. Restoration ofsuccinate-cytochrome c reductase activity in MTAA by incubation with micelles of phospholipids from normal and deficient rats. Incubations and enzyme assays were carried out as described in table 3. All incubations contained 20/tl of an ethanolic solution of ubiquinones0 (2 mg/ml) and the final concentration of MTAA was 8.7/~g of protein/ml. V, "normal" phospholipids; A, "deficient" phospholipids.
26
G.G.
D E P U R Y A N D F. D . C O L L I N S
TABLE 5 Comparison of the succinate-cytochrome c reductase activity restored by micelles of mixed phospholipids from normal and deficient rats. This table summarizes the results of experiments similar to that described in fig. 1. The data have been obtained by interpolation between experimental points. Each experiment used different preparations of MTAA and micelles, except that the same preparation of MTAA was used in experiments (4), (5) and (6) and the same paired preparations of micelles were used in experiments (3) and (4).
Experiment
Concentration of phospholipids (~M/ml)
(1)
Enzyme activity (/~M/min/mg protein) Normal Deficient
Ratio deficient/normal
0.08 0.12 0.20
0.20 0.29 0.31
0.46 0.43
2.25
(2)
0.08 0.11
0.09 0.10
0.17 0.19
1.95 1.82
(3)*
0.08 0.12 0.20
0.19 0.29 0.32
0.32 0.36 0.40
1.64 1.27 1.24
(4)
0.08 0.12 0.20
0.11 0.18 0.32
0.18 0.27 0.39
1.66 1.49 1.23
(5)
0.08 0.12 0.20
0.ll 0.17 0.23
0.11 0.17 0.24
1.00 1.01 1.03
(6)
0.08 0.12 0.20 0.28
0.11 0.16 0.16 0.15
0.1 I 0.18 0.19 0.18
0.98 1.12 1.16
0.08 0.12 0.20 0.28
0.07 0.14 0.16 0.17
0.05 0.11 0.17 0.18
0.53 0.77
(7)
1.50
1.22
1.06 1.06
* Data from fig. 1. duced greater maximum activity than did "normal"
p h o s p h o l i p i d s . I n ex-
p e r i m e n t (5), h o w e v e r , these differences w e r e n o t a p p a r e n t , a n d in e x p e r i m e n t (7) " d e f i c i e n t " p h o s p h o l i p i d s p r o d u c e d o n l y a slightly g r e a t e r m a x i m u m e n z y m e activity, w h i l e " n o r m a l " p h o s p h o l i p i d s p r o d u c e d m o r e a c t i v i t y w h e n l o w c o n c e n t r a t i o n s o f m i c e l l e s w e r e used. COMPARISON OF RESTORED ENZYME ACTIVITY WITH THAT FOUND IN UNEXTRACTED MITOCHONDRIA T h e s u c c i n a t e - c y t o c h r o m e c r e d u c t a s e a c t i v i t y o f freshly p r e p a r e d m i t o c h o n d r i a t h a t h a d n o t b e e n t r e a t e d w i t h a c e t o n e was q u i t e v a r i a b l e a n d t h e r e
PHOSPHOLIPID FATTY ACIDS IN ELECTRON TRANSFER
27
was no significant difference in activity between mitochondria prepared from the livers of normal or deficient rats (table 6). The maximum enzyme activities restored in M T A A (table 5) were between 38 per cent and I05 per cent of the average activities found in unextracted mitochondria, indicating that incubation with ubiquinon%o and phospholipid micelles restored the greater part of the enzyme activity that had been lost on extracting mitochondria with aqueous acetone. TABLE 6 S u c c i n a t e - c y t o c h r o m e c reductase activity o f unextracted m i t o c h o n d r i a f r o m livers o f n o r m a l and deficient rats. T h e m e t h o d o f assay was the s a m e as that used for M T A A , except that phospholipid micelles a n d ubiquinones0 were n o t added. T h e n u m b e r o f a n i m a l s e x a m i n e d a n d the s t a n d a r d errors are shown. E n z y m e activity ( / z M / m i n / m g o f protein) N o r m a l (13) Deficient (I 1)
0.44 ~0.07 0.43 ! 0.03
CORRELATION OF ENZYME ACTIVITY WITH BINDING OF PHOSPHOLIPID MICELLES
Analysis of particles of MTAA re-isolated after incubation with phospholipid micelles showed that they had bound phospholipid and also that increasing the concentration of phospholipid in the incubation medium increased the amount of phospholipid bound. Fig. 2 gives the results of four experiments in which comparisons were made of the binding by M T A A of phospholipids from normal and deficient rats. In two experiments more of the "deficient" phospholipids were bound than were " n o r m a l " phospholipids, while in the other two experiments the binding of both types of phospholipids was the same. There was also a considerable variation between experiments in the amounts of phospholipid that were bound. However, assaying the succinate-cytochrome c reductase activity of the re-isolated MTAA showed that restored enzyme activity was directly proportional to the amount of phospholipid bound (fig. 3). In each experiment the same relationship was found up to approximately 0.4/lM of phospholipid bound per mg of protein; uptake greater than this did not lead to any further increase in enzyme activity. Fig. 3 thus shows that once bound by M T A A all the preparations of phospholipids were equally effective in restoring enzyme activity, regardless of whether they were from normal or deficient rats. In the one experiment where binding occurred much in excess of 0.4 ItM per mg of protein, restored enzyme activity reached a maximum value and the
28
6 . G . D E P U R Y A N D F. D . C O L L I N S
16 0 L O.
'~ 12 C~
E 0.8 t
~ 0.4 0
a_
C
t
[
I
i
[
I
1.0 2 0 3.0 Concentr-ation of phospholLpid
I
I
J
4.0 (}J.M/ml)
Fig. 2. Comparison of the binding by MTAA of micelles of phospholipids from normal and deficient rats. Micelles of mixed phospholipids in 3 ml of 20 mM tris acetate buffer, pH 8.0, were mixed with 3 ml of a suspension of MTAA in 0.88 M sucrose, buffered with 10 mM tris chloride, pH 7.5. The final concentration of MTAA was approximately 0.3 mg of protein/ml. After incubating the mixture for 20 rain at 30°, the MTAA were re-isolated a) and analysed to determine the amount of phospholipid they had bound. The different symbols show the results of four experiments comparing "normal" phospholipids (closed symbols; O , l , &, ~') with "deficient" phospholipids (open symbols; } , ~ , ~,, V).
m a x i m u m activity o b t a i n e d with "deficient" p h o s p h o l i p i d s in this experim e n t was greater than that o b t a i n e d with " n o r m a l " p h o s p h o l i p i d s .
Discussion T h e experimental results d e m o n s t r a t e that after extraction with a q u e o u s acetone rat liver m i t o c h o n d r i a lose a b o u t 80 per cent o f their p h o s p h o l i p i d s and also all their s u c c i n a t e - c y t o c h r o m e c reductase activity. The enzyme activity can be restored by i n c u b a t i n g with ubiquinone50 and micelles o f mixed p h o s p h o l i p i d s f r o m rat liver, a n d the a m o u n t o f activity restored is p r o p o r t i o n a l to the a m o u n t o f p h o s p h o l i p i d b o u n d by the extracted mitoc h o n d r i a . These results are very similar to those that have been o b t a i n e d with extracted beef heart m i t o c h o n d r i a S ) , a n d this similarity suggests that the i n f o r m a t i o n o b t a i n e d by G r e e n a n d his c o - w o r k e r s l) f r o m experiments on beef h e a r t m i t o c h o n d r i a is also valid for rat liver m i t o c h o n d r i a . W h e n M T A A were i n c u b a t e d with high c o n c e n t r a t i o n s o f p h o s p h o l i p i d micelles, they b o u n d p h o s p h o l i p i d far in excess o f the a m o u n t f o u n d in unextracted m i t o c h o n d r i a . These excessive a m o u n t s o f p h o s p h o l i p i d did not
PHOSPHOLIPID
FATTY
ACIDS
IN ELECTRON
29
TRANSFER
O L Q_
0.24 v
•o
E o
"-- o.16 :::L
%-,
2~ +_~ >
OIIAT N o r m a l ODZ~2 D e f i c i e n t
uc~ 0.©8 ©
E N
0
|
I
I
I
I
04 0.8 Phospholipid bound
I
I
I
I
I
12 16 2.0 ( l ~ M / m g of p r o t e i n )
Fig. 3. Correlation of restored enzyme activity in MTAA with binding of micelles of phospholipids from normal and deficient rats. The particles of MTAA that had been reisolated after incubation with pbospholipid micelles as described in fig. 2 were assayed for succinate-cytochrome c reductase activity and for bound phospholipid. The enzyme assay was carried out as described in table 3, except that instead of being incubated with phospholipid micelles and ubiquinones0 for 20 min, the re-isolated particles were incubated with ubiquinones0 alone for 3 rain (longer incubations being found to lower enzyme activity). The symbols are as described in fig. 2.
result in any increase in enzyme activity, a n d they were p r o b a b l y o f no physiological significance, except to indicate t h a t the particles o f m i t o c h o n d r i a l p r o t e i n c o n t i n u e d to b i n d p h o s p h o l i p i d micelles after the sites which required lipid for enzyme activity had been filled. A similar excessive b i n d i n g occurs when beef heart m i t o c h o n d r i a are i n c u b a t e d with micelles o f c a r d i o l i p i n , but n o t when they are i n c u b a t e d with micelles o f mixed phosp h o l i p i d s f r o m mitochondriaS). The excessive u p t a k e by M T A A m a y have been due to p o l a r b o n d s being f o r m e d between anionic g r o u p s o f the phosp h o l i p i d s a n d free a m i n o g r o u p s in the m i t o c h o n d r i a l protein in a d d i t i o n to h y d r o p h o b i c bonds, which are k n o w n to occur between fatty acids o f phosp h o l i p i d s a n d h y d r o p h o b i c areas o f m i t o c h o n d r i a l structural proteina,2~). A l s o an excessive a m o u n t o f p h o s p h o l i p i d m a y have been b o u n d because m i t o c h o n d r i a f r o m rat liver, being m o r e fragile t h a n those f r o m beef heart, m a y have suffered greater d a m a g e d u r i n g extraction, so that m o r e sites suitable for b o t h h y d r o p h o b i c a n d p o l a r b o n d i n g were exposed. The v a r i a t i o n between experiments in the a m o u n t o f p h o s p h o l i p i d b o u n d
30
G . G , D E P U R Y A N D F. D. C O L L I N S
by M T A A shows that preparations of MTAA, and perhaps also of micelles, differed in the readiness with which they interacted. It is likely that this variation was due to differences in the number of binding sites per mg of protein that were available to micelles, showing that quantitative comparisons cannot be made between results of experiments which used different preparations of MTAA. However in all the experiments the same enzyme activity was restored per/~M of phospholipid bound, indicating that all the preparations of MTAA had the same capacity for electron transport, even if they differed in their readiness to bind phospholipid. The experiments comparing phospholipids from normal and deficient rats demonstrate that differences in fatty acid composition also can affect the binding of phospholipids by MTAA but that, once phospholipids are bound, differences in fatty acid composition do not influence the effectiveness of phospholipids in restoring enzyme activity. This conclusion is an extension of the previous discovery by Green and his co-workers 1) that, while some types of phospholipids such as lecithin, were bound less readily than others, all the phospholipids investigated were equally effective in promoting enzyme activity once they had been bound by mitochondrial protein 3). In one experiment (fig. 3), an excessive binding of phospholipids from a deficient rat restored a greater enzyme activity than did "normal" phospholipids, which may indicate that the "deficient" phospholipids were able to penetrate to more sites that required phospholipid for enzyme activity. However, the results with binding of physiological amounts of phospholipids indicate that the enzymes of the electron transport chain that transfer electrons from succinate through to cytochrome c do not have any special requirement for phospholipids containing essential fatty acids. Moreover, by inference from similar experiments using different types of phospholipids %3), it is likely that this conclusion applies equally to other enzymes in the electron transport chain. Fatty acid composition, however, can influence binding of phospholipid micelles by mitochondrial protein. In some binding experiments "deficient" phospholipids were bound more readily than "normal" phospholipids, while in other experiments no differences were observed (fig. 3). Since it has been shown that restoration of enzyme activity is proportional to the amount of phospholipid bound by MTAA, the results of earlier experiments, in which enzyme activity was measured in the presence of micelles of phospholipids from normal or deficient rats (table 5), also can be used to compare the amounts of "normal" and "deficient" phospholipids that were bound. These comparisons show that, while in a few experiments phospholipids from normal and deficient rats were bound to an equal extent, in most experiments phospholipids from deficient rats were bound more readily. However, ex-
PHOSPHOL1PID FATTY ACIDS IN ELECTRON 'IRANSFER
3]
p e r i m e n t s with lecithins have shown that it is the rate o f b i n d i n g which is affected by fatty acid composition26). Therefore, since these e x p e r i m e n t s with micelles o f mixed p h o s p h o l i p i d s m e a s u r e d enzyme activity or u p t a k e after a c o n s t a n t p e r i o d o f 20 min, close c o m p a r i s o n s between the perform a n c e o f n o r m a l a n d deficient p h o s p h o l i p i d s c a n n o t be m a d e ; it is p r o b a b l e that the p h o s p h o l i p i d micelles were not b o u n d at a c o n s t a n t rate for 20 min, so the total u p t a k e o f p h o s p h o l i p i d was n o t a reliable measure o f rate of binding. M o r e o v e r , since p o l a r b o n d i n g , which w o u l d n o t have been influenced by fatty acid c o m p o s i t i o n , was p r o b a b l y occurring as well as h y d r o p h o b i c b o n d i n g , differences between the rates o f b i n d i n g o f " n o r m a l " and "deficient" p h o s p h o l i p i d s could have been obscured. Nevertheless, the results o f these experiments suggest t h a t fatty acid c o m p o s i t i o n does influence the binding o f p h o s p h o l i p i d s by structural p r o t e i n a n d that p h o s p h o l i p i d s from rats deficient in essential fatty acids are b o u n d m o r e quickly t h a n are "normal" phospholipids.
Acknowledgements Dr. E, L. F r e n c h o f the D i v i s i o n o f A n i m a l Health, C o m m o n w e a l t h Scientific a n d Industrial R e s e a r c h O r g a n i z a t i o n , k i n d l y m a d e available the ultrasonic d i s i n t e g r a t o r . F i n a n c i a l s u p p o r t f r o m the N a t i o n a l H e a l t h a n d M e d i c a l Research C o u n cil o f A u s t r a l i a is gratefully a c k n o w l e d g e d .
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