Some effects of thyroxin on oxidative phosphorylation in submitochondrial particles and intact mitochondria

voL. 27 (i958)

PRELIMINARY NOTES

667

w a s v e n t e d to t h e a t m o s p h e r e . A n 8 ft. glass "U" s h a p e d c o l u m n w i t h a n i n t e r n a l d i a m e t e r of 6 m m c o n t a i n i n g one p a r t of t h e a l k y d resin to four p a r t s of s u p p o r t i n g m a t e r i a l (acid-washed Celite 545, 1 2 ° - 1 4 o m e s h ) w a s m a i n t a i n e d a t 186 °. T h e flow r a t e of h e l i u m w a s 4 ° m l / m i n a t 38 lb/in. * pressure. B y utilizing v a r i o u s m e m b e r s of t h e a l k y d resins as p a r t i t i o n agents, it n o w a p p e a r s possible t o o b t a i n good r e s o l u t i o n of t h e i n d i v i d u a l c o m p o n e n t s of m i x t u r e s of f a t t y acid esters of c h a i n l e n g t h u p to a t least C,8 w i t h i n a r e a s o n a b l e period of t i m e . T h i s w o u l d involve altering t h e p a r a m e t e r s of c o l u m n l e n g t h , g a s flow, t e m p e r a t u r e a n d m e s h size. A m o r e detailed r e p o r t conc e r n i n g t h e s e e x p e r i m e n t s will be f o r t h c o m i n g shortly. S. R. LIPSKY Department of Medicine, Yale University School o[ Medicine, R . A . LANDOWNE

New Haven, Conn, (U.S.A,) 1 A. T. JAMES AND A. J. P. MARTIN, Biochem. ]., 63 (1956) I44, C. ORR AND J. CALLEN, p e r s o n a l c o m m u n i c a t i o n . Received D e c e m b e r i o t h , 1957

Some effects of thyroxin on oxidative phosphorylation in submitochondrial particles and intact mitochondria A l t h o u g h t h y r o x i n h a s b e e n f o u n d b y manyZ, 2,3 to c a u s e u n c o u p l i n g of o x i d a t i v e p h o s p h o r y l a t i o n in liver m i t o c h o n d r i a , t h e s e effects h a v e u s u a l l y required s o m e f o r m of d a m a g e to t h e m i t o c h o n d r i a l s t r u c t u r e , a n d TAPLEY, COOPER AND L E H N I N G E R 3 were u n a b l e to o b s e r v e u n c o u p l i n g u s i n g subm i t o c h o n d r i a l particles o b t a i n e d b y d i g i t o n i n t r e a t m e n t of m i t o c h o n d r i a . I t is t h e p u r p o s e of t h i s c o m m u n i c a t i o n to r e p o r t t h a t t h y r o x i n will c a u s e a n increase in t h e P : O ratios o b t a i n e d w i t h either i n t a c t m i t o c h o n d r i a or s u b m i t o c h o n d r i a l particles a n d t h a t u n d e r conditions w h e r e t h e rate of o x i d a t i o n is n o t l i m i t e d b y t h e rate of p h o s p h o r y l a t i o n t h i s increase in P : O ratio occurs t o g e t h e r w i t h a m a r k e d i n c r e a s e in t h e r a t e of O 2 u p t a k e . Secondly, v a r i o u s f o r m s of p r e t r e a t m e n t of either i n t a c t m i t o c h o n d r i a or s u b m i t o c h o n d r i a l particles lead to m a r k e d inhibition of phosp h o r y l a t i o n b y t h y r o x i n , a n d u n d e r t h e s e c i r c u m s t a n c e s t h e r e is no increase in r a t e of oxidation. M i t o c h o n d r i a were p r e p a r e d f r o m r a t liver4, s a n d s u b m i t o c h o n d r i a l particles were p r e p a r e d b y sonic t r e a t m e n t e. O~ u p t a k e w a s m e a s u r e d w i t h t h e Clark O , electrode a n d P u p t a k e w i t h t h e s~P-incorporation procedureS,L T a b l e I s h o w s t h a t , u s i n g i n t a c t m i t o c h o n d r i a w i t h s u c c i n a t e as a s u b s t r a t e , t h e P : O ratio w a s i n c r e a s e d b y t h y r o x i n in e x p e r i m e n t s s t a r t e d b y a d d i n g t h e m i t o c h o n d r i a to a c o m p l e t e r e a c t i o n m e d i u m . T h e increase in P : O ratio w a s a c c o m p a n i e d b y a decline in t h e rate of 02 u p t a k e . W h e n t h e m i t o c h o n d r i a were p r e i n c u b a t e d in t h e r e a c t i o n m e d i u m for 4 m i n prior t o a d d i t i o n of t h y r o x i n a n d s u b s t r a t e , a m a r k e d inhibition of p h o s p h o r y l a t i o n w a s evident, b u t t h i s " u n c o u p l i n g " w a s a c c o m p a n i e d b y a drop in t h e r a t e of O~ u p t a k e . T h e second p a r t of t h e T a b l e s h o w s r e s u l t s o b t a i n e d w i t h t h y r o x i n u s i n g s u b m i t o c h o n d r i a l particles. F o r b o t h s u b s t r a t e s , t h y r o x i n c a u s e d a n increase in P : O ratio, a n d t h i s w a s a c c o m p a n i e d b y a rise in t h e r a t e of oxidation. P r e i n c u b a t i o n of t h e particles in t h e reaction m e d i u m for 4 m i n followed b y t h e a d d i t i o n of t h y r o x i n a n d t h e n either s u c c i n a t e or D P N H * resulted in a m a r k e d i n h i b i t i o n of p h o s p h o r y l a t i o n c o m p a r e d w i t h t h e p r e i n c u b a t e d control. I n a d d i t i o n to t h y r o x i n , t r i i o d o t h y r o n i n e * * , d i i o d o t h y r o n i n e * * a n d t e t r a c h l o r o t h y r o n i n e * * were t e s t e d w i t h b o t h m i t o c h o n d r i a a n d s u b m i t o c h o n d r i a l particles. Of t h e t h r e e t h y r o x i n analog u e s only t r i i o d o t h y r o n i n e w a s f o u n d to h a v e appreciable a c t i v i t y a n d it w a s less effective t h a n t h y r o x i n . T h e effects o b s e r v e d t h e r e f o r e a p p e a r to be specific for t h y r o x i n . T h e e x p e r i m e n t s w i t h t h y r o x i n described a b o v e s e e m to be b e s t e x p l a i n e d in t e r m s of t h r e e effects. O n e is t h e increase in t h e efficiency of t h e p h o s p h o r y l a t i o n process t h a t w a s e v i d e n t w i t h b o t h i n t a c t m i t o c h o n d r i a a n d s u b m i t o c h o n d r i a l f r a g m e n t s . I n o t h e r e x p e r i m e n t s it w a s f o u n d t h a t t h i s increase in efficiency w a s n o t d u e to a n inhibition of A T P a s e a c t i v i t y . A second effect of t h y r o x i n w a s to increase t h e overall r a t e of oxidation. T h i s did n o t occur w i t h i n t a c t m i t o c h o n d r i a b u t w a s e v i d e n t w i t h a v a r i e t y of s u b s t r a t e s u s i n g t h e m o r e loosely coupled s u b m i t o c h o n d r i a l particles a n d h a s also been a p p a r e n t in m u c h early w o r k w i t h whole t i s s u e s a n d i n t a c t a n i m a l s s. * T h e following a b b r e v i a t i o n s are u s e d : A T P for a d e n o s i n e t r i p h o s p h a t e ; A D P for a d e n o s i n e d i p h o s p h a t e ; A M P for a d e n o s i n e m o n o p h o s p h a t e ; D P N H for r e d u c e d d i p h o s p h o p y r i d i n e n u cleotide; P for i n o r g a n i c o r t h o p h o s p h a t e . ** G e n e r o u s l y s u p p l i e d b y Dr. JAN WOLFF.

668

PRELIMINARY NOTES

VOL. 2 7 (1958)

TABLE I THE INFLUENCE

OF

THYROXIN ON OXIDATIVE PHOSPHORYLATION

I n t h e e x p e r i m e n t s w i t h m i t o c h o n d r i a , t h e r e a c t i o n m i x t u r e c o n t a i n e d 2 /,moles A D P , 15 /*moles AMP, 5 / , m o l e s succinate, 2o/*moles p h o s p h a t e , p H 7.0 (lO s c o u n t s / m i n ) , 200/*moles sucrose, 0.7 m g p r o t e i n :N, a n d w h e r e used, 2. lO -5 M t h y r o x i n ( s o d i u m salt). W i t h s u b m i t o c h o n d r i a l particles, t h e m i x t u r e c o n t a i n e d i o / * m o l e s A D P , i o / * m o l e s AMP, l O / * m o l e s MgCl~, 2 0 / * m o l e s p h o s p h a t e , p H 7.o (IO6 c o u n t s / m i n ) , o.19 m g p r o t e i n N, a n d w h e r e used, 5 /*moles succinate, 2.5 /*moles D P N H a n d 5" lO-5 M t h y r o x i n ( s o d i u m salt). I n all cases, t h e final v o l u m e w a s 1.9 m l a n d t h e t e m p e r a t u r e 28 °. T h y r o x i n , w h e n used in p r e i n c u b a t e d s a m p l e s , was a d d e d 15 sec prior to t h e s u b s t r a t e . I n c u b a t i o n time, 3o-18o sec g i v i n g 0.2-o. 4 / * a t o m of Oe u p t a k e .

Components Mitochondria Mitochondria + thyroxin Mitochondria Mitochondria + thyroxin S u b m i t o c h o n d r i a l particles S u b m i t o c h o n d r i a l particles S u b m i t o c h o n d r i a l particles S u b m i t o c h o n d r i a l particles S u b m i t o c h o n d r i a l particles S u b m i t o c h o n d r i a l particles S u b m i t o c h o n d r i a l particles S u b m i t o c h o n d r i a l particles

Substrate PreincubationOxygenuptake P uptake (rain) (#atora/min) (l~mole]min)

+ thyroxin + thyroxin + thyroxin + thyroxin

Succinate Succinate Succinate Succinate Succinate Succinate Succinate Succinate DPNH DPNH DPNH DPNH

o o 4 4 o o 4 4 o o 4 4

o.47o o.428 0.308 o.218 o.2ol 0.257 0.086 0.073 o.415 o.633 0.479 0.492

o.623 o.643 0.403 o.o81 o.iii o.192 0.044 0.029 0.243 0.443 0.255 o.18o

P: 0

i .33 i .5o i .31 0.37 0.55 0.75 o.51 0.40 0.59 0.70 o.53 0.37

T h e failure to o b s e r v e t h i s effect w i t h m i t o c h o n d r i a c a n be e x p l a i n e d b y t h e fact t h a t for fresh m i t o c h o n d r i a , w h i c h are t e s t e d in t h e p r e s e n c e of excess p h o s p h a t e , a c c e p t o r a n d s u b s t r a t e , t h e r a t e of o x i d a t i o n s e e m s to be d e t e r m i n e d b y t h e r a t e of p h o s p h o r y l a t i o n as long as t h e efficiency r e m a i n s c o n s t a n t . T h u s in t h e a b s e n c e of a d d e d t h y r o x i n t h e r a t e of o x i d a t i o n is a l r e a d y m a x i m a l a n d w h e n t h y r o x i n is a d d e d a n increase in o x i d a t i v e rate could n o t occur. S u p p o r t for t h i s interp r e t a t i o n c a n be o b t a i n e d f r o m e x p e r i m e n t s with i n t a c t m i t o c h o n d r i a w a s h e d in a h y p o t o n i c p h o s p h a t e buffer. U n d e r t h e s e c i r c u m s t a n c e s t h e P: O ratios were lower t h a n for u n t r e a t e d c o n t r o l s a n d a d d i t i o n of t h y r o x i n g a v e increases b o t h in t h e P : O ratio a n d in t h e r a t e of oxidation. T h e t h i r d effect of t h y r o x i n e v i d e n t in t h e a b o v e e x p e r i m e n t s as in p r e v i o u s w o r k w i t h m i t o c h o n d r i a is a n inhibition of p h o s p h o r y l a t i o n . T h i s a p p e a r s to be a less direct effect, since s o m e f o r m of p r e t r e a t m e n t is n e c e s s a r y to o b s e r v e t h i s response. A l t h o u g h a t t h i s t i m e no e x p l a n a t i o n c a n be offered for t h e c h a n g e f r o m s t i m u l a t i o n to i n h i b i t i o n of p h o s p h o r y l a t i o n w h i c h t a k e s place as a result of p r e i n c u b a t i o n , t h e following evidence m a y be p e r t i n e n t . If t h y r o x i n is allowed to p r e i n c u b a t e in t h e r e a c t i o n m e d i u m w i t h a s m a l l a m o u n t of soluble p r o t e i n prior to t h e a d d i t i o n of m i t o c h o n d r i a or s u b m i t o c h o n d r i a l particles, i n h i b i t i o n r a t h e r t h a n s t i m u l a t i o n of p h o s p h o r y l a t i o n is f o u n d to occur. T h e i n h i b i t o r y effect of t h y r o x i n a f t e r p r e i n c u b a t i o n m a y t h u s be d u e to t h e c o m b i n a t i o n of t h e t h y r o x i n w i t h soluble p r o t e i n released f r o m t h e m i t o c h o n d r i a or s u b m i t o c h o n d r i a l particles d u r i n g p r e i n c u b a t i o n . A m o r e detailed r e p o r t of t h i s w o r k is being prepared.

Laboratory o~ Cellular Physiology and Metabolism, National Heart Institute, National Institutes o/Health, Bethesda, Md. (U.S.A .)

j. RAMSEY BRONK

t C. MARTIUS AND B. HESS, Arch. Biochem. Biophys., 33 (I95I) 486. G. F. ]!¢~ALEYAND H. A. LARDY, jr. Biol. Chem., 204 (1953) 435. 3 D. F. TAPLEY, C. COOPER AND A. L. LEHNINGER, Biockim. Biophys. Acta, i8 (1955) 597. 4 W. C. SCHNEIDXR, J. Biol. Chem., 176 (1948) 259. 5 W . W. KIELLEY AND R. K. KIELLEY, J. Biol. Chem., 191 (1951) 485 • 6 j . R. BRONK AND W. W. KIELLEY, Biochim. Biophys. Acta, 24 (1957) 44 o. 7 W . W. KIELLEY AND J. R. BRONX, J. Biol. Chem., in t h e press. 8 S. B. BARKER, Physiol. Revs., 31 (I951) 205. R e c e i v e d J a n u a r y 2nd, 1958