A proportion of rat liver mitochondrial carnitine palmitoyltransferase can be made activatable by malonyl-CoA

377

Biochimica et BiophysicaActa. 1085(1991)377-380

© 1991 ElsevierScience PublishersB.V. All rights reserved 000Y..2760/91/$03.50 ADONIS 000527601)02671

A proportion of rat liver mitochondrial carnitine palmitoyltransferase can be made activatable by malonyl-CoA Iraj Ghadiminejad and David Saggerson Department of Biochemistry and MolecularBiology. Unil'ersi~"College London. GowerStreet. London (U.K.)

(Received 25 February 1991)

Key words: Carnitine palmitoyltransferase;Enzymeactivation:MalonyI-CoA:(Rat liver) Treatment of rat liver mitochondrial membranes with cholate yields a soluble extract containing carnitine palmitoyltransferase (CPT) activity that is insensitive to malonyI-CoA. As found previously (1. Ghadiminejad and D. Saggerson (1990) FEBS Lett. 269, 406-408), addition of polyethylenen glycol 6000 (PEG 6000) to this extract confered sensitivity to malonyI-CoA on the CPT. It is now shown that a sub-population of the CPT activity which is sedimentable at 7000 × g after addition of PEG 6000 is activated by malonyI-CoA, whereas the remainder is inhibited by malonyI-CoA. The presence of KCI increases the proportion of the activatable form of CPT. Possible physiological significance of this finding is discussed.

Introduction Carnitine palmitoyltransferase participates in translocation of fatty acids into mitochondria. Two forms have been identified; the overt CPT~ Iocalised to the outer membrane, and the latent CPT2 to the inner membrane [1-4]. CPT I is potently inhibited by malonyl-CoA [5] and is therefore distinguished from CPT 2 which, in situ, is insensitive to malonyI-CoA. A high affinity binding site for malonyI-CoA has been recognised in mitochondrial outer membranes [6,7]. Evidence from radiation activation analysis [6], use of anti-CPT sera [2] and separation of components by chromatography [8] or detergent extraction [3,4] leads to the conclusion that the malonyI-CoA binding entity (or malonyI-CoA sensitivity confering factor) is a separate entity from CPT t itself, it is proposed that CPT I and CPT 2 are different polypeptides [9,10] and they differ in physical properties in that C P T z is easily solubilised by detergents whereas CPT~ is not [3,4,11]. However, experiments [3,4] in which cholate extracts

from rat liver mitochondrial outer membranes can be made to confer inhibitability by malonyI-CoA on C P T z derived from the inner membrane suggest that CPT~ and CPT 2 should have some common domain(s) able to interact productively with the malonyI-CoA sensitivity confering factor from the outer membrane. Here we have further explored the experimental approach used in [3,4] in which addition of PEG 6000 to cholatesolubilized extracts of mitochondrial membranes permited conferment of malonyI-CoA sensitivity on CPT 2. We report the novel observation that, in this artificial system, a proportion of the available CPT is made susceptible to activation by malonyI-CoA. Materials and Methods Chemicals

These were as described in [2] with the exception of sodium cholate and polyethylene glycol 6000 which were from Sigma Chemical Co. (Poole, Dorset, U.K.) and Koch-Light (Haverhill, Suffolk, U.K.), respectively. Isolation o f mitochondrial membranes

Abbreviations: CPT~ and Clrf2, The overt and latent forms, respectively, of carnitine palmitoyltransferase; PEG 6000, polyethylene glycol 6000; PMSF, phenylmethylsulphonylfluoride. Enzymes: EC 2.3.1.21. carnitine palmitoyltransferase. Correspondence: E.D. Sagger~n, Dept. Biochemistryand Molecular Biology, UniversityCollege London, Gov,cr Street, London WCIE 6BT, U.K.

Rat liver mitochondria were isolated and washed as described in [2] and then suspended in ' p h o s p h a t e / P M S F / K C I ' (20 mM potassium phosphate buffer [pH 7.2] containing PMSF (0.1 m g / m l ) and 0.3 M KCI) at a protein concentration of 8-10 m g / m l . The mitochondria were sonicated in ice with a probe sonicator (MSE Soniprep 150). This was performed eight times

378 for 15 s bursts at 10 p.m amplitude, w:m 1:~ s cooling periods. After centrifugation at 115 000 × g for 45 min, the supernatant was discarded and the pellet resuspcnded in p h o s p h a t e / P M S F / K C l . The centrifugation and resuspension process was repeated twice, and the final pellet resuspended in p h o s p h a t e / P M S F with or without 0.3 M KCI and stored frozen at - 7 0 ° C until used. Treatment o f membranes with cholate Mitochondrial m e m b r a n e s at approx. 8 - 1 0 m g / m l were treated with 15 mM sodium cholate solution at a ratio of 10:1 (/xl of d e t e r g e n t to mg of protein). After 45 min incubation on ice with frequent mixing, the mixtures were centrifuged at l 1 5 0 0 0 × g for 1 h at 4 ° C . The pellets were resuspended in the appropriate buffer (see individual Tables or Figs) and the supernatants used for t r e a t m e n t with P E G 6000. Treatment with polyethyleneglycol 6000 P E G 6000 (at 80 m g / m l ) was added to 115000 × g supernatants obtained after t r e a t m e n t of m e m b r a n e s with cholate. After s ' a n d i n g on ice for approx. 1 h, aliquots were taken for CPT assay in the presence and absence of 100 tzM maionyl-CoA and the r e m a i n d e r centrifuged at 7000 × g for 15 min at 4 ° C . The supernatants were decanted and the pellets r e s u s p e n d e d in p h o s p h a t e / P M S F / K C I or p h o s p h a t e / P M S F . Both centrifugal fractions were assayed for C P T activity in thc presence and absence of 100 ~ M malonyI-CoA. Assay o f carnitine palmitoyltramferase All assays were in the direction of palmitoylcarnitine formation and were performed at 2 5 ° C in a final volume of 1.0 ml. Reactions were initiated by addition of 0.4 mM L-carnitine or L-[Me-3H]carnitine as appropriate. Both spectrophotometric [2] and radioehemical [12] assays contained 150 mM sucrose, 60 mM KCI, 25 mM Tris-HCl buffer (pH 6.8), 1 mM E D T A , fatty acid-poor albumin (1.3 m g / m l ) and 4 0 / z M palmitoyl-

CoA. The spectrophotometric assay additiona;'y contained 0.1 mM 4,4'-dithiodipyridine to monitor release of CoASH at 324 nm. Protein was m e a s u r e d [2,13] using bovine albumin as a standard. Results

We found previously [3,4] that cholate can solubilize C P T 2 from liver mitochondrial inner m e m b r a n e s and a malonyl-CoA sensitivity confering factor from outer membranes. However, the d e t e r g e n t causes negligible solubilization of the CPT I catalytic entity [4]. As shown in [3], 115 000 × g s u p e r n a t a n t s obtained after cholate t r e a t m e n t of mitochondrial m e m b r a n e s (inner + outer) contain C P T activity (CPT 2) which is insensitive to malonyl-CoA. However, addition of P E G 6000 confers some inhibitability by malonyI-CoA on this C P T 2, presumably by facilitating a productive interaction between the enzyme and the solubilized mal0nyI-CoA sensitivity confering factor. This p h e n o m e n o n was reproduced in E x p e r i m e n t 1 of Table I using m e m b r a n e s resuspended in p h o s p h a t e / P M S F buffer. U n d e r the conditions of E x p e r i m e n t 1 cholate solubilized approx. 30% of the available C P T activity (i.e., that which is represented in row B). In E x p e r i m e n t 2 of T a b l e I these procedures were r e p e a t e d with m e m b r a n e s that had been r e s u s p e n d e d in p h o s p h a t e / P M S F / K C I . Cholate now solubilized approx. 60% of the m e m b r a n e C P T activity and, most surprisingly, addition of P E G 6000 to the 115 000 × g s u p e r n a t a n t s resulted in a C P T activity that showed a small, but significant, activation by 100 t t M maionyI-CoA (row C). After addition of P E G 6000 some of the previously soluble CPT b e c a m e sedimentable. Initially we att e m p t e d fraetionation by eentrifugation at 30000 × g for 30 min. However, this resulted in total loss of sensitivity to malonyI-CoA (both activation or inhibition) in the resulting pellet or s u p e r n a t a n t (results not shown). This suggested that excessive centrifugal forces may disrupt productive interactions between C P T and

TABLE I lzffects of cholute attd PEG 6000 in the presence or absence of KCI to modify the response of CPT to malonyI.CoA

Mitochondrial membranes were resuspended in phosphate/PMSF buffer without (Expt. 1) or with 0. 3M KCI (Expt. 2). As described under Materials and Methods, these were then treated with cholate, centrifuged at 115090x g and the resulting supernatants then treated with PEG 61)0(I. Dithiodipyridine-linked spectrophotometric assays of CPT were performed in the presence and absence of 100/J.M malonyl-CoA. Specific activities are expressed as nmol/min per mg protein. The values are means ± S.E. for six separate membrane preparations. ~ Indicates P < 0.0l for significance of effects of malonyI-CoA (paired t-test). * Indicates P < 0.001 for comparison of malonyI-CoA effects between Expts. I and 2 (unpaired t-test}.

A Membranes B I 1500(IX g supernatant C 1150(~)x g supermaant + PEG 6011O

Experiment I: without KCI

Exp~,lment 2: with KCI

spec. act. 14 +0.8 6.45:0.4

slzec, act. 14 + 1.3 7.9 5:0.6

6.9_+0.3

% effect of malonyI-CoA -45±3 a - 15:1 -38+ I "

6.8_+0.5

% effect of malonyl-CoA - 12::t:2a,* - 1 5:1 + 14_+2"-*

379 TABLE II Emergence of a malonyI-CoA activated CPT actit'ity after treatment of cholate t~rtract~ with PEG 6000

Mitochondrial membranes were resuspended in phosphate/PMSF buffer without or with 0.3 M KCL treated with cholate, centrifuged at 1150110x g and the resulting supernatants then tree:ted with PEG 61)()l)(see Materials and Methods or Table 19. After centrifugation at 7IX)Ilx g. the resulting pellets and supernatants were assayed for CPT ( ± II)(I tzM malonyI-CoA) by i~oth the dithiodipyridinc-liakcd spectrophotometrie assay and the radiochemical assay (dithiodipyridine absent}. Specific activities are expressed as nmol/min per mg protein. The values are means ± S.E. for six separate membrane preparations. All effects of malonyl-CoA were significant (P < 1).(105).

A70{X)Xg B 7000× g C 7000x g D 7(100× g

pelletf+KCI) pellet {nn KCI) super.(+KCI) super. (no KCI)

Spectrophotomctric assay spec. act "; effect of malonyI-CoA

Radiochemlcal assay spec. act r; effect of malonyI-CoA

7.8+11.1 4.9+0.1 5.9_+_0.4 5.4 ± IL2

5.(t+iL2 4 I ±0.4 4.4+{I.3 7.5 + I}.l

-23_+3 +38+_,5 - 12±" - 28 ± I

the m a l o n y I - C o A sensitivity c o n f e r i n g factor. By contrast, less h a r s h c o n d i t i o n s ( 7 0 0 0 x g for 15 min at 4 ° C ) yielded pellet a n d s u p e r n a t a n t fractions both of w h i c h c o n t a i n e d malonyI-CoA-sensitive C P T ( F a b l e ! !). T h e m o s t n o t e w o r t h y finding w a s that, irrespective of the r e s u s p e n s i o n buffer, the pelleted C P T w a s activ a t e d by m a l o n y l - C o A , w h e r e a s t h a t r e m a i n i n g in the s u p e r n a t a n t w a s inhibited. A t first sight (in view o f the finding in row C o f T a b l e !), it m i g h t a p p e a r a n o m a lous t h a t p h o s p h a t e / P M S F r e s u s p e n d e d m e m b r a n e s ultimately yielded a m a l o n y l - C o A - a c t i v a t a b l e pellet C P T a f t e r P E G 6000 a d d i t i o n (row A, T a b l e IlL H o w ever, this is e x p l a i n e d by the fact t h a t only 1 1 - 2 0 % of t h e C P T e x t r a c t e d by ebolate f r o m these m e m b r a n e s is in a P E G 6000-precipitable form. In u n c e n t r i f u g e d extracts this small p r o p o r t i o n o f the m a l o n y I - C o A activatable C P T is likely to be m a s k e d by the far l a r g e r p r o p o r t i o n o f m a l o n y l - C o A - i n h i b i t a b l e enzyme. By c o n t r a s t , 4 4 - 5 ! % o f the C P T e x t r a c t e d by c h o l a t e from

+16+_ 2 +57± 12 - 15+ 4 - II± 1

t r e a t e d m e m b r a n e s was precipitablc by P E G 6000. T h i o l - g r o u p r e a g e n t s affect the d e g r e e of inhibition of m e m b r a n e - b o u n d liver C P T by n m l o n y I - C o A [14]. W e t h e r e f o r e d u p l i c a t e d Experim e n t 1 of T a b l e I1 by also p e r f o r m i n g a r a d i o c h e m i c a l assay for CPT. A l t h o u g h tbc t h i o l - g r o u p r e a g e n t 4.4'dithiodir,yridine w a s a b s e n t from this assay, activation of P E G - p r c c i p i t a t e d C P T by m a l o n y l - C o A w a s broadly similar to that o b s e r v e d in the s p e c t r o p h o t o m e t r i c assay (rows A a n d B, T a b l e II). In thc c x p e r i m e n t s s h o w n in Figs. I a n d 2 the P E G 600l)-precipitated C P T o b t a i n e d f r o m c h o l a t e extracts of p h o s p h a t c / P M S F r e s u s p e n d e d m e m b r a n e s w a s inv e s t i g a t e d with r e g a r d to d e p e n d e n c e of the activation p h e n o m e n o n o n [palmitoyI-CoA] or [malonyI-CoA]. Fig. 1 shows that the fold-activation by 100 p.M malonylC o A w a s a p p r e c i a b l y h i g h e r at low c o n c e n t r a t i o n s o f palmitoyI-CoA. A b o v e 20 ,uM p a l m i t o y I - C o A the de-

phosphatc/PMSF/KCl

i

to~-I

° II

to

,.i p.%LP.lU'U'OYl.('ot- , ' O N t ' N

qll-o

I

100

(°MI

Fig. I. Effect of palmitoyI-C~A concentration on the activation of CPT by malonyl-CoA. Total mitochondrial membram.s were obtained from fed rats, resuspended in phosphate/PMSF, treated with cholate and then centrifuged at 115000× g. PEG 60(H}was added to the resulting supernatants followed by centrifugation at 7(HIOx ~ to obtain peqets which were resuspended in phosphate/PMSF and then assayed for CPT at the indicated eoncentra:ions of palmitoylCoA+ Ill0 p.M malonyI-CoA, e, spectrophotometric assay {4.4'-dithiodipyridine present): Q, radiochemical assay (4,4'-dithiodipyridine absent); i , radiochemieal assay (4,4'.dithiodipyridine present).

i

MAION'VL('oA C.ON('N t~.M) Fig. 2. Effect of malonyl-CoA concentralinn on the activation of CPT. Membranes were obtained and treated as for Fig. I to obtain 7(}00× g pcllcls at~er treatment ~ith PEG 6I~M).CPT was assayed at a fixed [palmitoyI-CoA] of 40 #M in the absence or the presence of the indicated concentrations of malonyl-('oA, e, spcctrophotometric assay; i , radinchemical assay f4,4"-dithiodipyridine absent).

380 gree o f activation by m a l o n y l - C o A was cor~sis~.ently in the r a n g e of 3 0 - 4 0 % . T h e p r e s e n c e or ab~ence of 4,4'-dithiodipyridine in the assay b u f f e r did not a p p r e ciably alter these trends. Fig. 2 shows t h a t activation by m a l o n y I - C o A was essentially c o n s t a n t over the r a n g e 1-100`aM. Above 1 4 0 - 1 5 0 ,aM m a l o n y l - C o A , the activation phenon~enon d i s a p p e a r e d a n d very high c o n c e n trations of m a l o n y l - C o A c a u s e d inhibition ol the enzyme. T o some extent, t h e r e f o r e , m a l o n y l - C o A a n d palmitoyl-Co,~, have interactive efects with this C P T p r e p a r a t i o n . CPT~ in situ in liver m i t o c h o n d r i a l m e m b r a n e s also shows interactive kinetics b e t w e e n the long chain acyl-CoA s u b s t r a t e a n d m a l o n y I - C o A [15].

Discussion Previous studies [3,4] are now e x t e n d e d to show t h a t a s u b - p o p u l a t i o n o f the C P T z in c h o l a t e extracts o f m e m b r a n e s b e c o m e s activatable by m a l o n y I - C o A und e r the P E G 6 0 0 0 - i n d u c e d association conditions. W e previously s p e c u l a t e d t h a t 100% inhibition of CPT2 by m a l o n y l - C o A in the artificial association system w a s not achieved b e c a u s e o f failure to correctly o r i e n t a t e all available C P T catalytic units with m a l o n y l - C o A binding units in the c h o l a t e micelles [3]. W e n o w f u r t h e r suggest t h a t ' i n c o r r e c t ' o r i e n t a t i o n m a y actually p e r m i t activation of the C P T a by m a l o n y l - C o A . T h i s may p a r t i c u l a r l y p e r t a i n w h e n the o u t e r m e m b r a n e f a c t o r is o b t a i n e d f r o m f a s t e d rats. U n d e r t h o s e c o n d i tions only approx. 15% o f available C P T b e c o m e s inhibitable by m a l o n y l - C o A in the ' r e c o n s t i t u t i o n ' system [4]. W h a t e v e r m o l e c u l a r c o m p l e x e s c o n t r i b u t e to the m a l o n y l - C o A activatable a n d inactivatable f o r m s o f the enzyme, they m u s t have s o m e d i f f e r e n c e s in physical p r o p e r t i e s since they c a n to some extent be separ a t e d by c e n t r i f u g a t i o n at 7000 × g in the p r e s e n c e o f P E G 6000. It is not k n o w r if this activation p h e n o m e n o n mimics any n o r m a l physiological i n t e r a c t i o n in situ in the m e m b r a n e b e t w e e n C P T I a n d the malo n y l - C o A sensitivity c o n f e r i n g factor. If a s u b - p o p u l a -

tion of C P T I b e c a m e activatable by low c o n c e n t r a t i o n s of m a l o n y I - C o A in fasting o r diabetes, this m a y provide a n e x p l a n a t i o n why the ICso for inhibition of C P T I by m a l o n y l - C o A is i n c r e a s e d in these states [7,12,15-17].

Acknowledgement This w o r k was s u p p o r t e d by thc Medical R e s e a r c h Council. References 1 Murthy. M.S.R. and Pande, S.V. (1987) Proc. Natl. Aead. Sci. USA 84, 379-382. 2 Ghadiminejad, 1. and Saggerson, E.D. (1990) Biochem. J. 270, 787-794. 3 Ghadiminejad, I. and Saggerson, E.D. (1990) FEBS Left. 269, 406-408. 4 Ghadiminejad, 1. and Saggerson. E.D. (1991) Biochim. Biophys. Acta 1083, 166-172. 5 McGarry, J.D. and Foster, D.W. (1980) Annu. Rev. Bioehem. 49, 395-420. 6 Zammit, V.A., Corstorphine, C.G. and Kolodzlej, M.P. (1989) Biochem. J. 263, 89-95. 7 Kolodziej, M.P. and Zammit, V.A. (1990) Biochem. J. 267, 85-90. 8 Kerner, J. and Bieber, L. (1990) Biochemistry 29, 4326-4334. 9 Zammit, V.A., Corstorphine, C.G. and Kelliher, M.G. (1988) Biochem. J. 250, 415-420. 10 Woeltje, K.F., Esser, V., Weis, B.C., Cox, W.F., $chroeder, J.G., Liao, S., Foster, D.W. and MeGarry, J.D. (1990) J. Biol. Chem. 265, 10714-10719. 11 Woeltje, K.F., Kuwajima, M., Foster, D.W. and McGarry, J.D. (1987) J. Biol. Chem. 262, 9822-9827. 12 Saggerson, E.D., Carpenter, C.A. and Tselentis, B.S. (1982) Biochem. J. 208, 667-672. 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 11 Saggerson, E.D. and Carpenter, C.A. t1982) FEBS Lett. 137, 124-128. 15 Saggerson, E.D. and Carpenter, C.A. (1981) FEBS Left. 132, 166-168. 16 Saggerson, E.D. and Carpenter, C.A. (1981) FEBS Len. 129, 225-228. 17 Gamble, M.S. and Cook, G.A. (1985) J. Biol. Chem. 260, 95169519.