Isolation of succinic dehydrogenase from beef heart mitochondria

VOL. 1 4

(I954)

A SIMPLE

SHORT coMMUNICATIONS, PRELIMINARY NOTES INEXPENSIVE

BACTERIAL

COLONY

295

COUNTING

DEVICE

by C. R U S S E L L

Department o[ Biochemistry, Christie Hospital and Holt Radium Institute, 3lanchester (England) SIMS~AND JORDAN 1 h a v e described a m e t h o d of c o u n t i n g b a c t e r i a l colonies w h i c h utilise~s a n e l e c t r o m a g n e t i c device for a u t o m a t i c c o u n t i n g . Fig. 1 illustrates a n a p p a r a t u s simplified b y u s i n g a m e c h a n i c a l c o u n t e r in place of t h e e l e c t r o m a g n e t i c t y p e . T h e design of SIMS AND JORDAN is f u r t h e r i m p r o v e d u p o n b y t h e built-in i l l u m i n a t i o n provided. T h e counter, C, is a Veeder m o d e l o p e r a t e d b y a lever carZ r y i n g two s p r i n g coils, S 1 a n d S~, a U n o p e n (Size 2) U, being a t t a c h e d to t h e coil b y a piece of r u b b e r t u b i n g . T h e c o u n t i n g p r o c e d u r e is as follows :T h e pen, filled w i t h suitable m a r k i n g fluid, s u c h as I n d i a n ink, is pulled d o w n t o w a r d s t h e Petri dish to m a r k w i t h a d o t t h e position of a colony. J u s t before t h e p e n reaches t h e dish t h e c o u n t e r lever registers a c o u n t a n d s i m u l t a n e o u s l y p r o d u c e s an audible click. T h e spring, S~, e n a b l e s t h e p e n to r e a c h a n y p o i n t on t h e dish, whicb r e m a i n s in a fixed position. As t h e p e n t o u c h e s t h e dish, a second click is heard. F i n a l l y t h e 'spring S 1 r e t u r n s t h e lever to its r e s t i n g position (producing a t h i r d click) ,[ p ]o w h e n t h e d o w n w a r d pull is released, r e a d y for t h e n e x t c o u n t . 0 0 0 0 0 Tile r h y t h m i c s e q u e n c e of t h e t h r e e clicks on c o u n t i n g p r o v i d e s a n audible proof of t h e faultless w o r k i n g of t h e procedure. T h e Petri Dish, /9, need n o t be m a r k e d o u t in squares, as Iq t h e r e is no possibility of c o u n t i n g a colony twice or f o r g e t t i n g

~$2

0 0 0

t h e n u m b e r of colonies c o u n t e d . T h e l a m p house, H, is m a d e from a t i n box, t h e b o t t o m being r e m o v e d a n d v e n t i l a t i o n holes drilled n e a r t h e top. A 25 W a t t L a m p , L, gives t h e r i g h t lighting a n d v e r y little heat. T h e Petri dish r e s t s u p o n a n opalescent plate, O, secured o v e r a hole in t h e t o p of t h e box, a n d is t h u s viewed in diffuse light.

[ Fig. i.

REFERENCE A. L. S I ~ s AND R. C. JORDAN, J. Sci. Instrum., 18 (1941) 243. R e c e i v e d April 7th, 1954

ISOLATION FROM

OF

SUCCINIC

BEEF

HEART

DEHYDROGENASE MITOCHONDRIA

by D. E. G R E E N * ,

P. M. K O H O U T

AND S. M I I ' *

Institute ]or Enzyme Research, University o] Wisconsin, 3fadison, Wisconsin (U.S.A.) T h e m a i n electron t r a n s p o r t s y s t e m of aerobic cells is associated w i t h particles readily sedim e n t a b l e ill low g r a v i t a t i o n a l fields a n d insoluble in a q u e o u s m e d i a a t n e u t r a l or acid p H 1. O u r p r e s e n t k n o w l e d g e of t h i s s y s t e m is b a s e d a l m o s t e x c l u s i v e l y on s t u d i e s of t h e b e h a v i o u r of m i x t u r e s * T h i s i n v e s t i g a t i o n w a s s u p p o r t e d b y a g r a n t f r o m t h e H e a r t I n s t i t u t e of t h e N a t i o n a l I n s t i t u t e s of H e a l t h . ** P o s t - d o c t o r a l trainee of t h e H e a r t I n s t i t u t e of t h e N a t i o n a l I n s t i t u t e s of H e a l t h .

296

SHORT COMMUNICATIONS, PRELIMINARY NOTES

VOL. 1 4

(1954)

of p a r t i c u l a t e e n z y m e s c o n t a i n i n g all or m o s t of t h e c o m p o n e n t s n e c e s s a r y for t r a n s p o r t of electrons f r o m v a r i o u s s u b s t r a t e s to m o l e c u l a r o x y g e n 1-3. I n recent y e a r s several i n v e s t i g a t o r s 4-s h a v e directed t h e i r efforts t o w a r d s s e p a r a t i n g t h e different c o m p o n e n t s of t h e electron t r a n s p o r t s y s t e m b y w a y of t h e differential solubilization of particles w i t h desoxycholic acid, a n d t h e n f r a c t i o n a t i o n of t h e n l i x t u r e s of solubilized e n z y m e s . T h e r e s u l t s o b t a i n e d b y t h e s e m e t h o d s h a v ~ clearly d e m o n s t r a t e d t h e feasibility of s e p a r a t i n g t h e i n d i v i d u a l p a r t i c u l a t e c o m p o n e n t s of t h e electron t r a n s f e r s y s t e m . T h e p r e s e n t c o n l m u n i c a t i o n is t h e first in a series dealing w i t h t h e p r o b l e m of isolating p a r t i c u late e n z y m e s . T h e w o r k i n g h y p o t h e s i s is t h a t s u c h e n z y m e s after release f r o m m i t o c h o n d r i a are discrete entities in t h e s a m e sense t h a t a soluble e n z y m ~ is a defined chemical s u b s t a n c e . Tile p a r ticulate e n z y m e s differ f r o m t h e i r soluble c o u n t e r p a r t s only in r e s p e c t to t h e relatively h i g h concent r a t i o n of l y o p h o b i c g r o u p s a t t a c h e d to t h e p r o t e i n molecule w h i c h c o n d u c e to c o m p l e t e :0solubility in water. T h e tlleory of t h e isolation p r o c e d u r e s w h i c h we h a v e developed is essentially t h e following. If two particles differ in size, s h a p e or specific g r a v i t y t h e y c a n be s e p a r a t e d b y differential cent r i f u g a t i o n p r o v i d i n g t h e y do n o t associate. G i v e n a chemical difference b e t w e e n two particles, a basis is p r o v i d e d for effecting differential c h a n g e s in specific g r a v i t y since t h e e x t e n t to which particles will bind or associate w i t h alcohols, s a l t s a n d ions a t different p H v a l u e s will be d e t e r m i n e d b y t h e i r r e s p e c t i v e c h e m i c a l properties. T h e s e d e n s i t y differences t h e n p e r m i t s e p a r a t i o n of tile particles in the ultracentrifuge. B y a p p l i c a t i o n of a f r a c t i o n a t i o n t e c h n i q u e b a s e d on t h e principles discussed a b o v e succinic d e h y d r o g e n a s e h a s been isolated f r o m beef h e a r t m i t o c h o n d r i a 15-2o t i m e s m o r e a c t i v e t h a n t h e s t a r t i n g m i t o c h o n d r i a l s u s p e n s i o n . T h e following e n z y m e s are a b s e n t f r o m tile p r e p a r a t i o n : c y t o c h r o m e oxidase, D P N H d e h y d r o g e n a s e , c y t o c h r o l n e c a n d all t h e k n o w n e n z y m e s of tile citric acid cycle. T h e d e h y d r o g e n a s e c a t a l y z e s tile o x i d a t i o n of s u c c i n a t e b y ferricyanide, q u i n o n e s , o x i d a t i o n r e d u c t i o n d y e s a n d c y t o c h r o m e c b u t n o t b y m o l e c u l a r o x y g e n (even in presence of a d d e d c y t o c h r o m e c). One m g of t h e lipin-free e n z y m e c a t a l y z e s t h e o x i d a t i o n b y ferricyanide of 7/2inoles of s u c c i n a t e per m i n u t e a t 38° . A s s u m i n g t h a t a n t i m y c i n A c o m b i n e s w i t h one mole of succinic d e h y d r o g e n a s e 9 t h e v a l u e f¢,r t h e m i n i m a l m o l e c u l a r w e i g h t of t h e lipin-free e n z y m e is 8. 3. io 5. Since t h e e n z y m e c o n t a i n s 6 1 % b y w e i g h t of lipin t h e u n c o r r e c t e d v a l u e for t h e m o l e c u l a r w e i g h t is ca. 2. IO6. E a c h molecule of suecinic d e h y d r o g e n a s e as defined a b o v e c o n t a i n s 4 molecules of heroin a n d 12-16 a t o m s of n o n - h e r o i n iron. N e i t h e r copper n o r flavin is p r e s e n t in d e t e c t a b l e a m o u n t s . T h e r e is no evidence of t h e presence of c y t o c h r o m e c in tile e n z y m e as isolated. H o w e v e r each molecule of e n z y m e c a n b i n d up to 16 molecules of c y t o c h r o m e e. Once b o u n d , c y t o c h r o m e c is n o t r e m o v a b l e b y r e p e a t e d w a s h i n g b y c e n t r i f u g a t i o n of t h e p a r t i c u l a t e e n z y m e . I n 1.5 % d e s o x y c h o l a t e solution t h e r e d u c e d e n z y m e s h o w s 3 b a n d s w i t h m a x i m a a t 428, 523 a n d 558 in/, while tile oxidized e n z y m e s h o w s o n l y one b a n d w i t h a p e a k at 415 m/~. T h e b a n d s of tile p y r i d i n e hemochromogei1 of t h e e n z y m e are i n d i s t i n g u i s h a b l e f r o m t h o s e of t h e p y r i d i n e h e m o c h r o m o g e n of iron p r o t o p o r p h y r i n - - t h e m a x i m a being at 420, 522 a n d 555 m/~. On a d d i t i o n of s u c c i n a t e to t h e oxidized e n z y m e t h e p r o s t h e t i c h e m i n is r e d u c e d a t once. T h e r e d u c e d heroin is p a r t i a l l y reoxidized b y f u m a r a t e , c o m p l e t e l y reoxidized b y q u i n o n e b u t n o t reoxidized b y nlolecular o x y g e n . I n presence of a n t i m y c i n o n l y half of t h e reducible p r o s t h e t i c heroin is r e d u c e d a t once b y s u c c i n a t e - - t h e o t h e r h a l f being r e d u c e d only after s o m e m i n u t e s . W e h a v e c o n c l u d e d from t h i s o b s e r v a t i o n t h a t t h e r e are at least two t y p e s of h e m i n in succinic d e h y d r o g e n a s e - - t h e r e d u c t i o n of one being a n t i m y c i n i n s e n s i t i v e a n d t h e r e d u c t i o n of t h e o t h e r a n t i m y c i n sensitive. It s h o u l d be s t a t e d explicitly t h a t no a d d i t i o n s o t h e r t h a n t h e p a r t i c u l a t e e n z y m e are n e c e s s a r y for tile o x i d a t i o n of s u c c i n a t e b y c y t o c h r o m e c a n d b y a n y of t h e o t h e r electron acceptors. A c c o r d i n g to o u r evidence t h u s far t h e p a r t i c u l a t e succinic d e h y d r o g e n a s e w h i c h we h a v e isolated is a c o m p l e t e u n i t w i t h i n w h i c h t h e entire e n z y m i c s e q u e n c e proceeds. REFERENCES 1 D. KEILIN AND E. F. HARTREE, Proc. Roy. Soc. B., 122 (19371 298; 125 (1938) 171; 127 (I939) 167; 129 (194 ° ) 277 • 2 J~. CHANCE, Nature, 169 (1952) 215. 3 E . C. SLATER, Nature, 165 (195 o) 674. 4 F. ]3. STRAUB, Z. physiol. Chem., 268 (1941) 227. 5 G. H~)BSCHER AND M. I~IESE, Naturwissenscha[ten, 22 (19521 524, 6 C. F. STRITTMATTER AND E. G. BALL, Proc. Nat. Aead. Sci., 38 (1952) I9. 7 L. SMITH AND E. STOTZ, Federation Proc., 9 (195 o) 230. 8 ~V. W. \VAINIO, in a S y m p o s i u m , Some Conjugated Proteins, p. 19, R u t g e r s U n i v e r s i t y P r e s s (19531. 9 V. R. POTTER AND A. E . REIF, J . Biol. Chem., 205 (1953) 279. Received M a y 3rd, 1954