- Email: [email protected]
The isolation of a sixth Kober chromogen from the urine of pregnant women and its provisional identification as 18-hydroxyoestrone
230
PRELIMINARY NOTES
VOL. 2 6 ( 1 9 5 7)
I. T h e dissociation in t i m e (at least 3 ° min) b e t w e e n t h e s y n t h e s i s of p r o t e i n s a n d D N A synthesis, a n d t h e radically different g r o w t h c u r v e s for p r o t e i n a n d D N A , confirm t h e i n d e p e n d e n c e of t h e m e c h a n i s m of s y n t h e s i s of t h e t w o t y p e s of p h a g e c o n s t i t u e n t s , a n d s u g g e s t t h e e x i s t e n c e of c o m p l e x i n t e r m e d i a r y processes a s s u r i n g t h e t r a n s m i s s i o n of t h e genetic i n f o r m a t i o n of t h e p h a g e D N A to t h e proteins. 2. T h e s y n t h e s i s of t h e D N A of t h e p h a g e of Bacillus megatherium s e e m s to begin d u r i n g t h e period in w h i c h o t h e r i n d u c e d lysogenic s y s t e m s are p h o t o r e s t o r a b l e a n d i m m u n e for h o m o l o g o u s p h a g e s (JACOBS). T h e idea t h a t i m m u n i t y a n d p h o t o r e s t o r a b i l i t y are tied to t h e m a i n t e n a n c e of p r o p h a g e on its specific site should, therefore, be r e - e x a m i n e d in t h e light of t h i s fact.
Laboratoire de Physiologie animal*, Universitd de Bruxelles (Belgique)
R. JEBNSR
i p. LEMOINE AND C. LAVAND'HOMME, p e r s o n a l c o m m u n i c a t i o n . 2 D. KANAZIR AND M. ERRERA, Biochim. Biophys. Acta, 14 (1954) 62. 3 F. JACOB, Les bact~ries lysog~nes et la notions de provirus, M a s s o n et Cie, Paris, 1954. Received J u l y i 3 t h , I957
The isolation of a sixth Kober chromogen from the urine of pregnant women and its provisional identification as 18-hydroxyoestrone B y p a r t i t i o n c h r o m a t o g r a p h y on Celite c o l u m n s w i t h t h e s o l v e n t s y s t e m 7 ° % (v/v) m e t h a n o l in w a t e r - 2 o % (v/v) n - h e x a n e in benzene, a K o b e r c h r o m o g e n (KC-6) m o r e " p o l a r " t h a n x6ah y d r o x y o e s t r o n e w a s d e t e c t e d in k e t o n i c - p h e n o l i c f r a c t i o n s o b t a i n e d f r o m t h e u r i n e of p r e g n a n t w o m e n as described b y MARRXAN, WATSON AND PANATTOmI a n d MARRIAN, LOKE, WATSON AND PANATTON#. T r e a t m e n t of c o n c e n t r a t e s of KC-6, p r e p a r e d from s u c h fractions b y p a r t i t i o n c h r o m a t o g r a p h y , w i t h s m a l l v o l u m e s of chilled c h l o r o f o r m yielded a n e a r l y white, s p a r i n g l y soluble, solid (KC-6A) in yields of a b o u t 2 - 3 m g / I o o 1 urine, a n d a soluble fraction, believed to c o n t a i n a n o t h e r K o b e r c h r o m o g e n (KC-6B), which, h o w e v e r , h a s n o t y e t been isolated. Purification of K C - 6 A b y f u r t h e r leaching w i t h chilled chloroform, c r y s t a l l i z a t i o n f r o m m e t h a n o l - b e n z e n e a n d f r o m e t h a n o l yielded w h i t e crystals, m.p. 255-257 ° (uncorr. ; evac. sealed capillary), [u]~ + I46° (ethanol). ( F o u n d : C, 75.4; H , 7.9. Calc. for C l s H 2 t O s : C, 75.5; H, 7.7). I n t h e K o b e r reaction of BROWN8 as modified b y BAULD4, u s i n g t h e l a t t e r ' s "oestriol r e a g e n t " , K C - 6 A g a v e a p i n k colour, t h e optical d e n s i t y of w h i c h a t 512.5 m/z w a s o n l y a b o u t 2o % of t h a t g i v e n b y a n e q u a l a m o u n t of oestriol u n d e r t h e s a m e conditions. I n t h e " b l u e t e t r a z o l i u m " t e s t of MADER AND BUCK~, K C - 6 A s h o w e d negligible r e d u c i n g power, i n d i c a t i n g t h a t it is n o t a n u-ketol. O n r e d u c t i o n w i t h s o d i u m b o r o h y d r i d e in m e t h a n o l i c solution K C - 6 A yielded a n o n - k e t o n i c p r o d u c t (Girard reaction), w h i c h h a d t h e s a m e R F as K C - 6 A w h e n c h r o m a t o g r a p h e d on p a p e r in t h e s o l v e n t s y s t e m : b e n z e n e , 7o; chloroform, 4o; m e t h a n o l , 7o; w a t e r , 35- T h e optical d e n s i t y a t 512.5 m F of t h e p i n k colour given in t h e K o b e r r e a c t i o n b y t h i s r e d u c t i o n p r o d u c t was a b o u t 13o % g r e a t e r t h a n t h a t g i v e n b y a n e q u a l a m o u n t of K C - 6 A u n d e r t h e s a m e conditions. T h e infrared s p e c t r u m of K C - 6 A (KC1 disc) s h o w e d no b a n d a t 1377 c m -1. Since in o t h e r steroids a b a n d a t t h i s w a v e n u m b e r h a s been ascribed to t h e m e t h y l g r o u p a t t a c h e d to C-13 (JONES AND COLE6, JONES, COLE AND NOLINT), t h i s finding s u g g e s t e d t h e possibility t h a t in K C - 6 A t h e g r o u p a t t a c h e d to C-13 m i g h t h a v e a n o x y g e n s u b s t i t u e n t . A f t e r t r e a t m e n t of K C - 6 A w i t h N - N a O H at r o o m t e m p e r a t u r e for 4 h, o.90 m o l a r e q u i v a l e n t s of f o r m a l d e h y d e ( d e t e r m i n e d b y t h e c h r o m o t r o p i c acid reaction) were evolved on acidification a n d distillation; a n d a non-volatile, k e t o n i c - p h e n o l i c p r o d u c t was o b t a i n e d w h i c h g a v e no colour in t h e K o b e r reaction, b u t w h i c h h a d t h e s a m e R F as oestrone w h e n c h r o m a t o g r a p h e d on p a p e r in t h e s o l v e n t s y s t e m : benzene, 5 ° ; n - h e x a n e , 5 ° ; m e t h a n o l , 7 ° ; water, 3 o. B y a n a l o g y w i t h t h e a c t i o n of alkali on m e t h y l h e d e r a g o n a t e a n d on icterogenin (BARTON AND DB MAyOS), it s e e m s probable t h a t K C - 6 A possesses a p r i m a r y /~-ketol grouping. H e n c e it is provisionally c o n c l u d e d t h a t K C - 6 A is I 8 - h y d r o x y o e s t r o n e , a n d t h a t t h e non-volatile k e t o n i c - p h e n o l i c p r o d u c t f o r m e d f r o m it on alkali t r e a t m e n t is I8-noroestrone. Since KAHNT, NEHER AND WETTSTEIN 9 h a v e s h o w n t h a t l i - d e o x y c o r t i c o s t e r o n e b e c o m e s h y d r o x y l a t e d a t C-I8 a n d C-i 9 on i n c u b a t i o n w i t h o x - a d r e n a l h o m o g e n a t e s , a n a t t e m p t was m a d e to d e m o n s t r a t e t h e f o r m a t i o n of K C - 6 A f r o m oestrone u n d e r t h e s a m e conditions. I n several e x p e r i m e n t s t h e f o r m a t i o n of a b o u t 0.2 % of a K o b e r c h r o m o g e n i n d i s t i n g u i s h a b l e from K C - 6 A in its p a r t i t i o n c h r o m a t o g r a p h i c b e h a v i o u r w a s d e m o n s t r a t e d . F u r t h e r m o r e , on t r e a t m e n t w i t h alkali t h e c h r o m a t o g r a p h i c a l l y purified K o b e r c h r o m o g e n yielded f o r m a l d e h y d e in a b o u t
VOL.
26
(1957)
PRELIMINARY NOTES
231
t h e e x p e c t e d a m o u n t a n d a non-volatile p r o d u c t w a s f o r m e d w h i c h w a s i n d i s t i n g u i s h a b l e b y p a p e r c h r o m a t o g r a p h y f r o m t h e alkali d e g r a d a t i o n p r o d u c t of KC-6A. T h e s e findings a r e c o m p a t ible w i t h t h e view t h a t K C - 6 A is i S - h y d r o x y o e s t r o n e , a n d t h a t it is a m e t a b o l i c p r o d u c t of oestrone t h a t is f o r m e d in t h e a d r e n a l g l a n d s . Full e x p e r i m e n t a l details of this w o r k will be p u b l i s h e d elsewhere. T h e a u t h o r s are grateful to t h e Medical R e s e a r c h Council for a g r a n t f r o m w h i c h t h e e x p e n s e s of t h i s w o r k were defrayed, to Dr. R. K. CALLOW of t h e National I n s t i t u t e for Medical R e s e a r c h , w h o d e t e r m i n e d t h e infrared s p e c t r u m of KC-6A, a n d to Mr. J. GORDON for skilled t e c h n i c a l assistance. K. H. LOKE Department o] Biochemistry, University o/Edinburgh (Scotland) ELIZABETH J. D. WATSON G. F. MARRIAN 1 G. F. MARRIAN, E. J. D. WATSON AND M. PANATTONI, Biochem. J., 65 (1957) 12. 2 G. F. MARRIAN, K. H. LOKE, E. J. D. WATSON AND M. PANATTONI, Biochem. J., 66 (1957) 60. a j . B. BROWN, J. Endocrinol., 8 (1952) 196. 4 W . S. BAULD, Biochem. J., 56 (I954) 426. 5 W . j . MADER AND R. R. BUCK, Anal. Chem., 24 (I952) 666. 6 R. N. JONES AND A. R. H. COLE, J. Am. Chem. Soc., 74 (1952) 5648. 7 R. N. JONES, A. R. H. COLE AND B. NOLIN, J. Am. Chem. Sot., 74 (1952) 5662. s D. H. R. BARTON AND P. DE MAYO, J. Chem. Soc., (1954) 887. 9 F. W . KAHNT, R. NEHER AND A. WETTSTEIN, Helv. Chim. Aaa, 38 (1955) 1237. Received J u l y z8th, 1957
The relation of the site of action of amytal to oxidative phosphorylation* I t h a s b e e n s h o w n p r e v i o u s l y I t h a t a m y t a l (5-ethyl-5-isoamylbarbituric acid) i n h i b i t s t h e D P N H e y t o c h r o m e c r e d u c t a s e of isolated r a t liver m i t o c h o n d r i a , w i t h o u t affecting t h e o x i d a t i o n of succinate. I t w a s p o s t u l a t e d t h a t t h e a m y t a l block is located b e t w e e n D P N H a n d t h e a n t i m y c i n A - s e n s i t i v e site of t h e r e s p i r a t o r y chain. T h i s p o s t u l a t e w a s s u b s e q u e n t l y c o n f i r m e d b y CHANCE I. TABLE I EFFECTS OF AMYTAL AND ANTIMYCIN A ON RESPIRATION, OXIDATIVE PHOSPHORYLATION AND ATPAsE IN RAT LIVER MITOCHONDRIA Test systems: R e s p i r a t i o n a n d p h o s p h o r y l a t i o n : E a c h s a m p l e c o n t a i n e d m i t o c h o n d r i a f r o m 4oo m g w e t wt. r a t liver, 3o/~moles p o t a s s i u m g l u t a m a t e , 5o/~moles p o t a s s i u m o r t h o p h o s p h a t e (pH 7.5), 1/~mole A T P , 6o /*moles glucose, io /~moles MgCI 2, 2oo /amoles KC1, IOO /amoles sucrose, a n excess of y e a s t h e x o k i n a s e , and, w h e r e indicated, 3.6 /~moles a m y t a l , I p g a n t i m y c i n A, o.2 /~mole 2,4dinitrophenol. F i n a l v o l u m e , 2 ml. I n c u b a t i o n a t 3 o ° C in W a r b u r g vessels. Gas phase, air; c e n t e r well, o.2 m l 2 M K O H . I n c u b a t i o n t i m e , 25 rain, including 5 rain Eaermo-equilibration. ATPase: E a c h s a m p l e c o n t a i n e d m i t o c h o n d r i a f r o m 267 m g liver, 32 /~moles A T P , 2oo /*moles tris buffer ( p H 7.5), 4 ° / ~ m o l e s KC1 a n d 75 ° / t m o l e s sucrose. A m y t ~ l , a n t i m y c i n A, a n d dinitrophenol, a d d e d in c o n c e n t r a t i o n s as i n d i c a t e d above. F i n a l v o l u m e , 4 ml. I n c u b a t i o n a t 3 ° °C in open vessels w i t h s h a k i n g . I n c u b a t i o n t i m e , io rain.
Additions None Dinitrophenol Amy~al Dinitrophenol, a m y t a l Antimycin A Dinitrophenol, a n t i m y c i n A
RespiroMon, twgoms oxygenlmin/g liver
P/O ratio
A TPase pmolcs Pi/min/g liver
I.I6
2.2 7
0.0
i .02 o.o3 0.o 3 o.o3 0.08
o. 17 ----
6.9 o.9i o.98
* A b b r e v i a t i o n s : A T P , a d e n o s i n e t r i p h o s p h a t e ; D P N , d i p h o s p h o p y r i d i n e nucleotide; D P N H , 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 c l e o t i d e ; Pi, inorganic o r t h o p h o s p h a t e .
PRELIMINARY NOTES
VOL. 2 6 ( 1 9 5 7)
I. T h e dissociation in t i m e (at least 3 ° min) b e t w e e n t h e s y n t h e s i s of p r o t e i n s a n d D N A synthesis, a n d t h e radically different g r o w t h c u r v e s for p r o t e i n a n d D N A , confirm t h e i n d e p e n d e n c e of t h e m e c h a n i s m of s y n t h e s i s of t h e t w o t y p e s of p h a g e c o n s t i t u e n t s , a n d s u g g e s t t h e e x i s t e n c e of c o m p l e x i n t e r m e d i a r y processes a s s u r i n g t h e t r a n s m i s s i o n of t h e genetic i n f o r m a t i o n of t h e p h a g e D N A to t h e proteins. 2. T h e s y n t h e s i s of t h e D N A of t h e p h a g e of Bacillus megatherium s e e m s to begin d u r i n g t h e period in w h i c h o t h e r i n d u c e d lysogenic s y s t e m s are p h o t o r e s t o r a b l e a n d i m m u n e for h o m o l o g o u s p h a g e s (JACOBS). T h e idea t h a t i m m u n i t y a n d p h o t o r e s t o r a b i l i t y are tied to t h e m a i n t e n a n c e of p r o p h a g e on its specific site should, therefore, be r e - e x a m i n e d in t h e light of t h i s fact.
Laboratoire de Physiologie animal*, Universitd de Bruxelles (Belgique)
R. JEBNSR
i p. LEMOINE AND C. LAVAND'HOMME, p e r s o n a l c o m m u n i c a t i o n . 2 D. KANAZIR AND M. ERRERA, Biochim. Biophys. Acta, 14 (1954) 62. 3 F. JACOB, Les bact~ries lysog~nes et la notions de provirus, M a s s o n et Cie, Paris, 1954. Received J u l y i 3 t h , I957
The isolation of a sixth Kober chromogen from the urine of pregnant women and its provisional identification as 18-hydroxyoestrone B y p a r t i t i o n c h r o m a t o g r a p h y on Celite c o l u m n s w i t h t h e s o l v e n t s y s t e m 7 ° % (v/v) m e t h a n o l in w a t e r - 2 o % (v/v) n - h e x a n e in benzene, a K o b e r c h r o m o g e n (KC-6) m o r e " p o l a r " t h a n x6ah y d r o x y o e s t r o n e w a s d e t e c t e d in k e t o n i c - p h e n o l i c f r a c t i o n s o b t a i n e d f r o m t h e u r i n e of p r e g n a n t w o m e n as described b y MARRXAN, WATSON AND PANATTOmI a n d MARRIAN, LOKE, WATSON AND PANATTON#. T r e a t m e n t of c o n c e n t r a t e s of KC-6, p r e p a r e d from s u c h fractions b y p a r t i t i o n c h r o m a t o g r a p h y , w i t h s m a l l v o l u m e s of chilled c h l o r o f o r m yielded a n e a r l y white, s p a r i n g l y soluble, solid (KC-6A) in yields of a b o u t 2 - 3 m g / I o o 1 urine, a n d a soluble fraction, believed to c o n t a i n a n o t h e r K o b e r c h r o m o g e n (KC-6B), which, h o w e v e r , h a s n o t y e t been isolated. Purification of K C - 6 A b y f u r t h e r leaching w i t h chilled chloroform, c r y s t a l l i z a t i o n f r o m m e t h a n o l - b e n z e n e a n d f r o m e t h a n o l yielded w h i t e crystals, m.p. 255-257 ° (uncorr. ; evac. sealed capillary), [u]~ + I46° (ethanol). ( F o u n d : C, 75.4; H , 7.9. Calc. for C l s H 2 t O s : C, 75.5; H, 7.7). I n t h e K o b e r reaction of BROWN8 as modified b y BAULD4, u s i n g t h e l a t t e r ' s "oestriol r e a g e n t " , K C - 6 A g a v e a p i n k colour, t h e optical d e n s i t y of w h i c h a t 512.5 m/z w a s o n l y a b o u t 2o % of t h a t g i v e n b y a n e q u a l a m o u n t of oestriol u n d e r t h e s a m e conditions. I n t h e " b l u e t e t r a z o l i u m " t e s t of MADER AND BUCK~, K C - 6 A s h o w e d negligible r e d u c i n g power, i n d i c a t i n g t h a t it is n o t a n u-ketol. O n r e d u c t i o n w i t h s o d i u m b o r o h y d r i d e in m e t h a n o l i c solution K C - 6 A yielded a n o n - k e t o n i c p r o d u c t (Girard reaction), w h i c h h a d t h e s a m e R F as K C - 6 A w h e n c h r o m a t o g r a p h e d on p a p e r in t h e s o l v e n t s y s t e m : b e n z e n e , 7o; chloroform, 4o; m e t h a n o l , 7o; w a t e r , 35- T h e optical d e n s i t y a t 512.5 m F of t h e p i n k colour given in t h e K o b e r r e a c t i o n b y t h i s r e d u c t i o n p r o d u c t was a b o u t 13o % g r e a t e r t h a n t h a t g i v e n b y a n e q u a l a m o u n t of K C - 6 A u n d e r t h e s a m e conditions. T h e infrared s p e c t r u m of K C - 6 A (KC1 disc) s h o w e d no b a n d a t 1377 c m -1. Since in o t h e r steroids a b a n d a t t h i s w a v e n u m b e r h a s been ascribed to t h e m e t h y l g r o u p a t t a c h e d to C-13 (JONES AND COLE6, JONES, COLE AND NOLINT), t h i s finding s u g g e s t e d t h e possibility t h a t in K C - 6 A t h e g r o u p a t t a c h e d to C-13 m i g h t h a v e a n o x y g e n s u b s t i t u e n t . A f t e r t r e a t m e n t of K C - 6 A w i t h N - N a O H at r o o m t e m p e r a t u r e for 4 h, o.90 m o l a r e q u i v a l e n t s of f o r m a l d e h y d e ( d e t e r m i n e d b y t h e c h r o m o t r o p i c acid reaction) were evolved on acidification a n d distillation; a n d a non-volatile, k e t o n i c - p h e n o l i c p r o d u c t was o b t a i n e d w h i c h g a v e no colour in t h e K o b e r reaction, b u t w h i c h h a d t h e s a m e R F as oestrone w h e n c h r o m a t o g r a p h e d on p a p e r in t h e s o l v e n t s y s t e m : benzene, 5 ° ; n - h e x a n e , 5 ° ; m e t h a n o l , 7 ° ; water, 3 o. B y a n a l o g y w i t h t h e a c t i o n of alkali on m e t h y l h e d e r a g o n a t e a n d on icterogenin (BARTON AND DB MAyOS), it s e e m s probable t h a t K C - 6 A possesses a p r i m a r y /~-ketol grouping. H e n c e it is provisionally c o n c l u d e d t h a t K C - 6 A is I 8 - h y d r o x y o e s t r o n e , a n d t h a t t h e non-volatile k e t o n i c - p h e n o l i c p r o d u c t f o r m e d f r o m it on alkali t r e a t m e n t is I8-noroestrone. Since KAHNT, NEHER AND WETTSTEIN 9 h a v e s h o w n t h a t l i - d e o x y c o r t i c o s t e r o n e b e c o m e s h y d r o x y l a t e d a t C-I8 a n d C-i 9 on i n c u b a t i o n w i t h o x - a d r e n a l h o m o g e n a t e s , a n a t t e m p t was m a d e to d e m o n s t r a t e t h e f o r m a t i o n of K C - 6 A f r o m oestrone u n d e r t h e s a m e conditions. I n several e x p e r i m e n t s t h e f o r m a t i o n of a b o u t 0.2 % of a K o b e r c h r o m o g e n i n d i s t i n g u i s h a b l e from K C - 6 A in its p a r t i t i o n c h r o m a t o g r a p h i c b e h a v i o u r w a s d e m o n s t r a t e d . F u r t h e r m o r e , on t r e a t m e n t w i t h alkali t h e c h r o m a t o g r a p h i c a l l y purified K o b e r c h r o m o g e n yielded f o r m a l d e h y d e in a b o u t
VOL.
26
(1957)
PRELIMINARY NOTES
231
t h e e x p e c t e d a m o u n t a n d a non-volatile p r o d u c t w a s f o r m e d w h i c h w a s i n d i s t i n g u i s h a b l e b y p a p e r c h r o m a t o g r a p h y f r o m t h e alkali d e g r a d a t i o n p r o d u c t of KC-6A. T h e s e findings a r e c o m p a t ible w i t h t h e view t h a t K C - 6 A is i S - h y d r o x y o e s t r o n e , a n d t h a t it is a m e t a b o l i c p r o d u c t of oestrone t h a t is f o r m e d in t h e a d r e n a l g l a n d s . Full e x p e r i m e n t a l details of this w o r k will be p u b l i s h e d elsewhere. T h e a u t h o r s are grateful to t h e Medical R e s e a r c h Council for a g r a n t f r o m w h i c h t h e e x p e n s e s of t h i s w o r k were defrayed, to Dr. R. K. CALLOW of t h e National I n s t i t u t e for Medical R e s e a r c h , w h o d e t e r m i n e d t h e infrared s p e c t r u m of KC-6A, a n d to Mr. J. GORDON for skilled t e c h n i c a l assistance. K. H. LOKE Department o] Biochemistry, University o/Edinburgh (Scotland) ELIZABETH J. D. WATSON G. F. MARRIAN 1 G. F. MARRIAN, E. J. D. WATSON AND M. PANATTONI, Biochem. J., 65 (1957) 12. 2 G. F. MARRIAN, K. H. LOKE, E. J. D. WATSON AND M. PANATTONI, Biochem. J., 66 (1957) 60. a j . B. BROWN, J. Endocrinol., 8 (1952) 196. 4 W . S. BAULD, Biochem. J., 56 (I954) 426. 5 W . j . MADER AND R. R. BUCK, Anal. Chem., 24 (I952) 666. 6 R. N. JONES AND A. R. H. COLE, J. Am. Chem. Soc., 74 (1952) 5648. 7 R. N. JONES, A. R. H. COLE AND B. NOLIN, J. Am. Chem. Sot., 74 (1952) 5662. s D. H. R. BARTON AND P. DE MAYO, J. Chem. Soc., (1954) 887. 9 F. W . KAHNT, R. NEHER AND A. WETTSTEIN, Helv. Chim. Aaa, 38 (1955) 1237. Received J u l y z8th, 1957
The relation of the site of action of amytal to oxidative phosphorylation* I t h a s b e e n s h o w n p r e v i o u s l y I t h a t a m y t a l (5-ethyl-5-isoamylbarbituric acid) i n h i b i t s t h e D P N H e y t o c h r o m e c r e d u c t a s e of isolated r a t liver m i t o c h o n d r i a , w i t h o u t affecting t h e o x i d a t i o n of succinate. I t w a s p o s t u l a t e d t h a t t h e a m y t a l block is located b e t w e e n D P N H a n d t h e a n t i m y c i n A - s e n s i t i v e site of t h e r e s p i r a t o r y chain. T h i s p o s t u l a t e w a s s u b s e q u e n t l y c o n f i r m e d b y CHANCE I. TABLE I EFFECTS OF AMYTAL AND ANTIMYCIN A ON RESPIRATION, OXIDATIVE PHOSPHORYLATION AND ATPAsE IN RAT LIVER MITOCHONDRIA Test systems: R e s p i r a t i o n a n d p h o s p h o r y l a t i o n : E a c h s a m p l e c o n t a i n e d m i t o c h o n d r i a f r o m 4oo m g w e t wt. r a t liver, 3o/~moles p o t a s s i u m g l u t a m a t e , 5o/~moles p o t a s s i u m o r t h o p h o s p h a t e (pH 7.5), 1/~mole A T P , 6o /*moles glucose, io /~moles MgCI 2, 2oo /amoles KC1, IOO /amoles sucrose, a n excess of y e a s t h e x o k i n a s e , and, w h e r e indicated, 3.6 /~moles a m y t a l , I p g a n t i m y c i n A, o.2 /~mole 2,4dinitrophenol. F i n a l v o l u m e , 2 ml. I n c u b a t i o n a t 3 o ° C in W a r b u r g vessels. Gas phase, air; c e n t e r well, o.2 m l 2 M K O H . I n c u b a t i o n t i m e , 25 rain, including 5 rain Eaermo-equilibration. ATPase: E a c h s a m p l e c o n t a i n e d m i t o c h o n d r i a f r o m 267 m g liver, 32 /~moles A T P , 2oo /*moles tris buffer ( p H 7.5), 4 ° / ~ m o l e s KC1 a n d 75 ° / t m o l e s sucrose. A m y t ~ l , a n t i m y c i n A, a n d dinitrophenol, a d d e d in c o n c e n t r a t i o n s as i n d i c a t e d above. F i n a l v o l u m e , 4 ml. I n c u b a t i o n a t 3 ° °C in open vessels w i t h s h a k i n g . I n c u b a t i o n t i m e , io rain.
Additions None Dinitrophenol Amy~al Dinitrophenol, a m y t a l Antimycin A Dinitrophenol, a n t i m y c i n A
RespiroMon, twgoms oxygenlmin/g liver
P/O ratio
A TPase pmolcs Pi/min/g liver
I.I6
2.2 7
0.0
i .02 o.o3 0.o 3 o.o3 0.08
o. 17 ----
6.9 o.9i o.98
* A b b r e v i a t i o n s : A T P , a d e n o s i n e t r i p h o s p h a t e ; D P N , d i p h o s p h o p y r i d i n e nucleotide; D P N H , 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 c l e o t i d e ; Pi, inorganic o r t h o p h o s p h a t e .