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The role of vitamin K1 in coupled oxidative phosphorylation
448
PRELIMINARY NOTES
VOL. 2 S
(1957)
R e a c t i o n (1) is p r e s u m a b l y c a t a l y z e d b y a flavoprotein, since d i a p h o r a s e activity, a n d t h e t r a n s f e r reaction 4 (which involves a t r a n s f e r of electrons from D P N H to t h e 3-acetyl pyridine a n a l o g of D P N ) can be d e m o n s t r a t e d in t h e a m m o n i u m sulfate f r a c t i o n a t e d a n d dialyzed e x t r a c t s . At p r e s e n t it is n o t k n o w n w h e t h e r reaction (2) is e n z y m i c or n o n - e n z y m i c in n a t u r e . A l t h o u g h , in soluble p r e p a r a t i o n s , it is p r o b a b l y n o n - e n z y m i c , t h e recent d e m o n s t r a t i o n b y COLPA-BOONSTRA. AND SLATERs of a reduced m e n a d i o n e oxidase offers a n a l t e r n a t i v e possibility. If reaction (2) proves to be n o n - e n z y m i c , t h e n t h e m e n a d i o n e r e d u c t a s e (reaction (I)) like S t r a u b ' s diaphorase, c a t a l y z e s t h e reduction of m e n a d i o n e , which in t u r n reduces c y t o c h r o m e ce. T h e possible role of t h e s e soluble e n z y m e s in coupled 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 this o r g a n i s m will be discussed in a m o r e detailed p a p e r to follow. MORTON .~,1.WEBER Department o/Bacteriology and Immunology ARNOLD F. BRODIE
and Leonard Wood Memorial Laboratory, Harvard Medical School, Boston, Mass. (U.S.A.) 1 A. F. BRODIE AND C. T. GRAY, Biochim. Biophys. Acta, 19 ([956) 384 • 2 W. D. WOSILAIT AND A. NASON, J. Biol. Chem., 208 (1954) 785 . 3 C. MARTItlS AND D. NITz-LITZOW, Biochim. Biophys. Acta, 12 (1953) 134. 4 M. M. WEBER AND N. O. KAPLAN, Science, 123 (I956) 844. J. P. COLPA-BoONSTRA AND E. C. SLATER, Biochim. Biophys. Acta, 23 (1957) 222. 6 H. R. MAHLER, A. S. FAIRHURST AND B. MACKLER, J. ,qm. Chem. Soc., 77 (195.5) 1514. Received M a y 1 l t h , 1957
The role of vitamin K t in coupled oxidative phosphorylation* Roles for v i t a m i n K a n a l o g u e s in electron t r a n s p o r t t , 2 , s a n d in o x i d a t i v e phosphorylation4,5, e h a v e been s u g g e s t e d . E v i d e n c e will be p r e s e n t e d in t h i s c o m m u n i c a t i o n t h a t v i t a m i n K 1 is a n essential c o e n z y m e n e c e s s a r y for coupled 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 e x t r a c t s of Mycobacterium phlei. T h e bacterial s y s t e m requires active subcellular particlesLS a n d t h e soluble e n z y m e s of t h e s u p e r n a t a n t w h i c h include m e n a d i o n e r e d u c t a s e s. E v i d e n c e t h a t v i t a m i n K 1 p l a y s a n essential role as a physiological electron carrier, a n d is a k e y c o m p o u n d in o x i d a t i v e p h o s p h o r y l a t i o n , n o w h a s been o b t a i n e d in several w a y s : (1) restoration 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 l i g h t - i n a c t i v a t e d p r e p a r a t i o n s b y t h e a d d i t i o n of v i t a m i n Kx; (2) t h e r e c o n s t i t u t i o n of coupled p h o s p h o r y l a t i o n b y a d d i n g K 1 a f t e r e x t e n s i v e f r a c t i o n a t i o n of t h e s u p e r n a t a n t ; a n d (3) t h e specific r e q u i r e m e n t for certain configurations which occur in v i t a m i n K1, b u t n o t in related c o m p o u n d s . E x p o s u r e of isolated particles a n d active s u p e r n a t a n t to light e m i t t e d a t 36o0 A (Gates R a y m a s t e r L a m p , t u b e B) results in a c o m p l e t e loss of o x i d a t i o n a n d p h o s p h o r y l a t i o n . Addition of v i t a m i n K 1 s u s p e n d e d in t h e inactive lipids o b t a i n e d from e x t r a c t s a f t e r c e n t r i f u g a t i o n s c o m p l e t e l y restores b o t h o x i d a t i o n a n d coupled p h o s p h o r y l a t i o n (Table I). E x p o s u r e of t h e lipid-K 1 s u s p e n s i o n to light at 36oo A results in c o m p l e t e i n a c t i v a t i o n . L i p i d - e x t r a c t a b l e m a t e r i a l o b t a i n e d f r o m u n t r e a t e d (active) s u p e r n a t a n t s h a s a n a b s o r p t i o n s p e c t r u m similar to t h a t of t h e K-like c o m p o u n d s . F u r t h e r m o r e , t h i s e x p o s u r e to light alters in a similar m a n n e r t h e characteristic a b s o r p t i o n s p e c t r u m of t h e lipid-extractable material from s u p e r n a t a n t a n d of v i t a m i n K 1. T h i s r e q u i r e m e n t for v i t a m i n K 1 can also be d e m o n s t r a t e d in t h e presence of e n z y m e s o b t a i n e d b y a m m o n i u m sulfate fractionation a n d in w h i c h r e s t o r a t i o n of a c t i v i t y previously required t h e a d d i t i o n of h e a t e d c r u d e s u p e r n a t a n t . B o t h o x i d a t i o n a n d p h o s p h a t e esterification are restored b y t h e a d d i t i o n of v i t a m i n K t. After e x p o s u r e of b o t h particle a n d s u p e r n a t a n t to light, t h e bacterial s y s t e m e x h i b i t s a specific d e p e n d e n c e on v i t a m i n K 1 for coupled o x i d a t i v e p h o s p h o r y l a t i o n . L a p a c h o l m e t h y l e s t e r a n d v i t a m i n K x diacetate, c o m p o u n d s which closely resemble v i t a m i n K x in t h e R t a n d R 3 position of t h e n a p h t h o q u i n o n e , s h o w s o m e a c t i v i t y . V i t a m i n K2, m e n a d i o n e , m e n a d i o n e dip h o s p h a t e , d i m e t h y l n a p h t h o q u i n o n e , phthicol, lapachol, q u i n o n e a n d a-tocopherol are inactive. T h e s y s t e m r e a c t i v a t e d w i t h v i t a m i n K 1 is u n c o u p l e d b y d i n i t r o p h e n o l a n d d i c u m a r o l ; it is n o t affected by a n t i m y c i n A. Addition of riboflavin m o n o p h o s p h a t e (FMN) to t h e l i g h t - t r e a t e d particles a n d s u p e r n a t a n t restores o x i d a t i o n b u t n o t t h e ability to esterify inorganic p h o s p h a t e . * 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 R e s e a r c h G r a n t E-827 from t h e N a t i o n a l Microbiological I n s t i t u t 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 , U.S. Public H e a l t h Service, a n d also by a g r a n t f r o m t h e Milton F u n d of H a r v a r d U n i v e r s i t y .
rOE. 25 (I957)
PREI.IMIN,XRY Norb:s TABI.E
440
1
"rBl.; EFI:I'~CT OF VITAMIN K t oN, I.IGHT-EXPOSEI) PARTICLES AND SI;PERNArAN 1" (.~J0OI).\)
.System
Additmn~
P
1' .- S --' S
none vit. K t
"I'1' : 3",% T P - - TS TI" + TS "l'P -~- "I'S
none vit. K l crude h e a t e d s u p e r n a t a n t t r e a t e d vit. K t
Oxygen ilaloms
/-~ Pi IlmOl¢$
t'.0
2.32
1.?~
o.77
3.4 6
2. 7
o.St
o.02
0. 3 3.11 l .t~ o.2
i.o2 o. 7 t
2.92 2.2o o.7S
The s y s t e m c o n t a i n e d 0. 3 ml (7.0 mg protein) of the p a r t i c u l a t e fraction (TP) t r e a t e d for 1.5 h w i t h a G a t e s R a y m a s t e r l a m p (30o0 A), o. 4 ml (i 7 mg protein) of s u p e r n a t a n t ('I'S) t r e a t e d in a s i m i l a r m a n n e r , 4.o t m m l e s e n z y m i c a l l y r e d u c e d D P N , 3.9 t l mol e s v i t a m i n K 1 (oil) s u s p e n d e d in i n a c t i v e lipid, i 5 t~moles MgCl v 25 p m o l e s K F a n d 7 . 8 / , m o l e s i n o r g a n i c p h o s p h a t e . The a c c e p t o r s y s t e m consisted of 2. 5 /(moles ADP, 2 0 / , m o l e s glucose, a n d i m g y e a s t h e x o k i n a s e . 1' a n d S are u n t r e a t e d p a r t i c l e s a n d s u p e r n a t a n t , r e s p e c t i v e l y . The o x y g e n u p t a k e was m e a s u r e d a t 3 C' for xo min a f t e r the a d d i t i o n of I ) P N H . The reac t i on was s t o p p e d w i t h [ 0 % TCA a n d the m i x t u r e a n a l y z e d for p h o s p h a t e d i s a p p e a r a n c e . The FMN t y p e system, a p p e a r s to r e p r e s e n t the n o n - p h o s p h o r y l a t i v e p a t h w a y in e x t r a c t s from this o r g a n i s m 7,9. V i t a m i n K 1 is inwflved as a c o e n z y m e in both ele c t ron t r a n s p o r t a n d c oupl e d o x i d a t i v e p h o s p h o r y l a t i o n . P r e l i m i n a r y e v i d e n c e i n d i c a t e s t h a t ~ P is i n c o r p o r a t e d in v i t a m i n K 1. Thi s m o n o p h o s p h a t c ester has also been p o s t u l a t e d by XVEssELs TM to be t he a c t i v a t e d i n t e r m e d i a t e . XVe should like to e x p r e s s our a p p r e c i a t i o n to Â)r. I,. FIESER tor s u p p l y i n g lapachol, l a p a c h o l m e t h y l ester, a n d d i m e t h y l n a p h t h o q u i n o n e , a n d to Dr. E. A. DolsY for his gift of v i t a m i n K 2. \Ve sh ould also like to t h a n k Dr. J. H. HANKS for his advice, a nd Mrs. SARAH B. GILSON a n d Mr. ROBERT THOMAS for t h e i r t e c h n i c a l assistance. ARNOLD 1¢. BRODIE Leonard Wood Memorial Laboratory, MORTON 31. WEBER Department o/ Bacteriology and Immunology, Harvard Medical School, CLARKF T. GRAY Boston, .~las.~. (I.r.S.A.) 1 W. D. ~VosILAIT AND A. NASON, J. Biol. Chem., 208 (1954) 785 . a ('. MARTIUS, Angew. Chem., 67 (1955) 161. 3 j . p. COLPA-BooNSTRA AND E. C. SLATER, Biochim. Biophys. Acta, 23 (1957) z22. C. MARTIOS AND D. NITz-LITzOW, Biochim. Biophys. Acta, iz (1953) 134. 5 A. F. BRODIE AND C. T. GRAY, Biochim. Biophys. Acta, x9 (1956) 146. 6 A. F. BRODIE AND (~. T. GRAY, Bacteviol. Proc., (1956) 62. 7 A. F. ]:~RODIE AND C. T. GRAY, J . Biol. Chem., 219 (1956) 853. A. [:. BRODIE AND C. T. GRAY, Science, 125 (1957) 534. 9 ~l. M. VV'EBER AND A. F. BRODIE, Biochim. Biophys..-|eta, 25 (t957) 447. I0 j . S. (;. WESSELS, Rec. tray. chim., 73 (I954) 5"9. R e c e i v e d May i t t h , 1957
Purification of a serum protein required by a m a m m a l i a n cell in tissue culture The s t u d i e s of EAGLE a n d his co-workers on the n u t r i t i o n of t i s s ue c u l t u r e cells h a v e defined t h e i r a m i n o acid a n d v i t a m i n r e q u i r e m e n t s t. The n a t u r e of t he s e r u m c o m p o n e n t s n e c e s s a r y for t h e i r g r o w t h , howev er, has not been e l u c i d a t e d . A p r o t e i n w hi c h is r e q u i r e d for t h e g r o w t h of a t i s s u e c u l t u r e cell, h u m a n a p p e n d i x - A i *, has now been purified a b o u t 15-fold from calf s e r u m by c o n v e n t i o n a l p r o t e i n f r a c t i o n a t i o n procedures. The a s s a y p r o c e d u r e used to follow t h e p u r i f i c a t i o n of t he p r o t e i n d e p e n d s upon t w o m a r k e d effects p r o d u c e d b y it on w a s h e d tissue c u l t u r e cells i n o c u l a t e d i nt o a s y n t h e t i c m e d i u m 4. U n d e r t h e c o n d i t i o n s of t h e a s s a y , in t h e a b s e n c e of the p r o t e i n factor, few cells a t t a c h to a glass s urfa c e " A p p e n d i x - A t is a single clone isolate o b t a i n e d in Dr. PUCK'S l a b o r a t o r y 2 from a s t r a i n i s o l a t e d by Dr. CI-IANG3.
PRELIMINARY NOTES
VOL. 2 S
(1957)
R e a c t i o n (1) is p r e s u m a b l y c a t a l y z e d b y a flavoprotein, since d i a p h o r a s e activity, a n d t h e t r a n s f e r reaction 4 (which involves a t r a n s f e r of electrons from D P N H to t h e 3-acetyl pyridine a n a l o g of D P N ) can be d e m o n s t r a t e d in t h e a m m o n i u m sulfate f r a c t i o n a t e d a n d dialyzed e x t r a c t s . At p r e s e n t it is n o t k n o w n w h e t h e r reaction (2) is e n z y m i c or n o n - e n z y m i c in n a t u r e . A l t h o u g h , in soluble p r e p a r a t i o n s , it is p r o b a b l y n o n - e n z y m i c , t h e recent d e m o n s t r a t i o n b y COLPA-BOONSTRA. AND SLATERs of a reduced m e n a d i o n e oxidase offers a n a l t e r n a t i v e possibility. If reaction (2) proves to be n o n - e n z y m i c , t h e n t h e m e n a d i o n e r e d u c t a s e (reaction (I)) like S t r a u b ' s diaphorase, c a t a l y z e s t h e reduction of m e n a d i o n e , which in t u r n reduces c y t o c h r o m e ce. T h e possible role of t h e s e soluble e n z y m e s in coupled 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 this o r g a n i s m will be discussed in a m o r e detailed p a p e r to follow. MORTON .~,1.WEBER Department o/Bacteriology and Immunology ARNOLD F. BRODIE
and Leonard Wood Memorial Laboratory, Harvard Medical School, Boston, Mass. (U.S.A.) 1 A. F. BRODIE AND C. T. GRAY, Biochim. Biophys. Acta, 19 ([956) 384 • 2 W. D. WOSILAIT AND A. NASON, J. Biol. Chem., 208 (1954) 785 . 3 C. MARTItlS AND D. NITz-LITZOW, Biochim. Biophys. Acta, 12 (1953) 134. 4 M. M. WEBER AND N. O. KAPLAN, Science, 123 (I956) 844. J. P. COLPA-BoONSTRA AND E. C. SLATER, Biochim. Biophys. Acta, 23 (1957) 222. 6 H. R. MAHLER, A. S. FAIRHURST AND B. MACKLER, J. ,qm. Chem. Soc., 77 (195.5) 1514. Received M a y 1 l t h , 1957
The role of vitamin K t in coupled oxidative phosphorylation* Roles for v i t a m i n K a n a l o g u e s in electron t r a n s p o r t t , 2 , s a n d in o x i d a t i v e phosphorylation4,5, e h a v e been s u g g e s t e d . E v i d e n c e will be p r e s e n t e d in t h i s c o m m u n i c a t i o n t h a t v i t a m i n K 1 is a n essential c o e n z y m e n e c e s s a r y for coupled 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 e x t r a c t s of Mycobacterium phlei. T h e bacterial s y s t e m requires active subcellular particlesLS a n d t h e soluble e n z y m e s of t h e s u p e r n a t a n t w h i c h include m e n a d i o n e r e d u c t a s e s. E v i d e n c e t h a t v i t a m i n K 1 p l a y s a n essential role as a physiological electron carrier, a n d is a k e y c o m p o u n d in o x i d a t i v e p h o s p h o r y l a t i o n , n o w h a s been o b t a i n e d in several w a y s : (1) restoration 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 l i g h t - i n a c t i v a t e d p r e p a r a t i o n s b y t h e a d d i t i o n of v i t a m i n Kx; (2) t h e r e c o n s t i t u t i o n of coupled p h o s p h o r y l a t i o n b y a d d i n g K 1 a f t e r e x t e n s i v e f r a c t i o n a t i o n of t h e s u p e r n a t a n t ; a n d (3) t h e specific r e q u i r e m e n t for certain configurations which occur in v i t a m i n K1, b u t n o t in related c o m p o u n d s . E x p o s u r e of isolated particles a n d active s u p e r n a t a n t to light e m i t t e d a t 36o0 A (Gates R a y m a s t e r L a m p , t u b e B) results in a c o m p l e t e loss of o x i d a t i o n a n d p h o s p h o r y l a t i o n . Addition of v i t a m i n K 1 s u s p e n d e d in t h e inactive lipids o b t a i n e d from e x t r a c t s a f t e r c e n t r i f u g a t i o n s c o m p l e t e l y restores b o t h o x i d a t i o n a n d coupled p h o s p h o r y l a t i o n (Table I). E x p o s u r e of t h e lipid-K 1 s u s p e n s i o n to light at 36oo A results in c o m p l e t e i n a c t i v a t i o n . L i p i d - e x t r a c t a b l e m a t e r i a l o b t a i n e d f r o m u n t r e a t e d (active) s u p e r n a t a n t s h a s a n a b s o r p t i o n s p e c t r u m similar to t h a t of t h e K-like c o m p o u n d s . F u r t h e r m o r e , t h i s e x p o s u r e to light alters in a similar m a n n e r t h e characteristic a b s o r p t i o n s p e c t r u m of t h e lipid-extractable material from s u p e r n a t a n t a n d of v i t a m i n K 1. T h i s r e q u i r e m e n t for v i t a m i n K 1 can also be d e m o n s t r a t e d in t h e presence of e n z y m e s o b t a i n e d b y a m m o n i u m sulfate fractionation a n d in w h i c h r e s t o r a t i o n of a c t i v i t y previously required t h e a d d i t i o n of h e a t e d c r u d e s u p e r n a t a n t . B o t h o x i d a t i o n a n d p h o s p h a t e esterification are restored b y t h e a d d i t i o n of v i t a m i n K t. After e x p o s u r e of b o t h particle a n d s u p e r n a t a n t to light, t h e bacterial s y s t e m e x h i b i t s a specific d e p e n d e n c e on v i t a m i n K 1 for coupled o x i d a t i v e p h o s p h o r y l a t i o n . L a p a c h o l m e t h y l e s t e r a n d v i t a m i n K x diacetate, c o m p o u n d s which closely resemble v i t a m i n K x in t h e R t a n d R 3 position of t h e n a p h t h o q u i n o n e , s h o w s o m e a c t i v i t y . V i t a m i n K2, m e n a d i o n e , m e n a d i o n e dip h o s p h a t e , d i m e t h y l n a p h t h o q u i n o n e , phthicol, lapachol, q u i n o n e a n d a-tocopherol are inactive. T h e s y s t e m r e a c t i v a t e d w i t h v i t a m i n K 1 is u n c o u p l e d b y d i n i t r o p h e n o l a n d d i c u m a r o l ; it is n o t affected by a n t i m y c i n A. Addition of riboflavin m o n o p h o s p h a t e (FMN) to t h e l i g h t - t r e a t e d particles a n d s u p e r n a t a n t restores o x i d a t i o n b u t n o t t h e ability to esterify inorganic p h o s p h a t e . * 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 R e s e a r c h G r a n t E-827 from t h e N a t i o n a l Microbiological I n s t i t u t 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 , U.S. Public H e a l t h Service, a n d also by a g r a n t f r o m t h e Milton F u n d of H a r v a r d U n i v e r s i t y .
rOE. 25 (I957)
PREI.IMIN,XRY Norb:s TABI.E
440
1
"rBl.; EFI:I'~CT OF VITAMIN K t oN, I.IGHT-EXPOSEI) PARTICLES AND SI;PERNArAN 1" (.~J0OI).\)
.System
Additmn~
P
1' .- S --' S
none vit. K t
"I'1' : 3",% T P - - TS TI" + TS "l'P -~- "I'S
none vit. K l crude h e a t e d s u p e r n a t a n t t r e a t e d vit. K t
Oxygen ilaloms
/-~ Pi IlmOl¢$
t'.0
2.32
1.?~
o.77
3.4 6
2. 7
o.St
o.02
0. 3 3.11 l .t~ o.2
i.o2 o. 7 t
2.92 2.2o o.7S
The s y s t e m c o n t a i n e d 0. 3 ml (7.0 mg protein) of the p a r t i c u l a t e fraction (TP) t r e a t e d for 1.5 h w i t h a G a t e s R a y m a s t e r l a m p (30o0 A), o. 4 ml (i 7 mg protein) of s u p e r n a t a n t ('I'S) t r e a t e d in a s i m i l a r m a n n e r , 4.o t m m l e s e n z y m i c a l l y r e d u c e d D P N , 3.9 t l mol e s v i t a m i n K 1 (oil) s u s p e n d e d in i n a c t i v e lipid, i 5 t~moles MgCl v 25 p m o l e s K F a n d 7 . 8 / , m o l e s i n o r g a n i c p h o s p h a t e . The a c c e p t o r s y s t e m consisted of 2. 5 /(moles ADP, 2 0 / , m o l e s glucose, a n d i m g y e a s t h e x o k i n a s e . 1' a n d S are u n t r e a t e d p a r t i c l e s a n d s u p e r n a t a n t , r e s p e c t i v e l y . The o x y g e n u p t a k e was m e a s u r e d a t 3 C' for xo min a f t e r the a d d i t i o n of I ) P N H . The reac t i on was s t o p p e d w i t h [ 0 % TCA a n d the m i x t u r e a n a l y z e d for p h o s p h a t e d i s a p p e a r a n c e . The FMN t y p e system, a p p e a r s to r e p r e s e n t the n o n - p h o s p h o r y l a t i v e p a t h w a y in e x t r a c t s from this o r g a n i s m 7,9. V i t a m i n K 1 is inwflved as a c o e n z y m e in both ele c t ron t r a n s p o r t a n d c oupl e d o x i d a t i v e p h o s p h o r y l a t i o n . P r e l i m i n a r y e v i d e n c e i n d i c a t e s t h a t ~ P is i n c o r p o r a t e d in v i t a m i n K 1. Thi s m o n o p h o s p h a t c ester has also been p o s t u l a t e d by XVEssELs TM to be t he a c t i v a t e d i n t e r m e d i a t e . XVe should like to e x p r e s s our a p p r e c i a t i o n to Â)r. I,. FIESER tor s u p p l y i n g lapachol, l a p a c h o l m e t h y l ester, a n d d i m e t h y l n a p h t h o q u i n o n e , a n d to Dr. E. A. DolsY for his gift of v i t a m i n K 2. \Ve sh ould also like to t h a n k Dr. J. H. HANKS for his advice, a nd Mrs. SARAH B. GILSON a n d Mr. ROBERT THOMAS for t h e i r t e c h n i c a l assistance. ARNOLD 1¢. BRODIE Leonard Wood Memorial Laboratory, MORTON 31. WEBER Department o/ Bacteriology and Immunology, Harvard Medical School, CLARKF T. GRAY Boston, .~las.~. (I.r.S.A.) 1 W. D. ~VosILAIT AND A. NASON, J. Biol. Chem., 208 (1954) 785 . a ('. MARTIUS, Angew. Chem., 67 (1955) 161. 3 j . p. COLPA-BooNSTRA AND E. C. SLATER, Biochim. Biophys. Acta, 23 (1957) z22. C. MARTIOS AND D. NITz-LITzOW, Biochim. Biophys. Acta, iz (1953) 134. 5 A. F. BRODIE AND C. T. GRAY, Biochim. Biophys. Acta, x9 (1956) 146. 6 A. F. BRODIE AND (~. T. GRAY, Bacteviol. Proc., (1956) 62. 7 A. F. ]:~RODIE AND C. T. GRAY, J . Biol. Chem., 219 (1956) 853. A. [:. BRODIE AND C. T. GRAY, Science, 125 (1957) 534. 9 ~l. M. VV'EBER AND A. F. BRODIE, Biochim. Biophys..-|eta, 25 (t957) 447. I0 j . S. (;. WESSELS, Rec. tray. chim., 73 (I954) 5"9. R e c e i v e d May i t t h , 1957
Purification of a serum protein required by a m a m m a l i a n cell in tissue culture The s t u d i e s of EAGLE a n d his co-workers on the n u t r i t i o n of t i s s ue c u l t u r e cells h a v e defined t h e i r a m i n o acid a n d v i t a m i n r e q u i r e m e n t s t. The n a t u r e of t he s e r u m c o m p o n e n t s n e c e s s a r y for t h e i r g r o w t h , howev er, has not been e l u c i d a t e d . A p r o t e i n w hi c h is r e q u i r e d for t h e g r o w t h of a t i s s u e c u l t u r e cell, h u m a n a p p e n d i x - A i *, has now been purified a b o u t 15-fold from calf s e r u m by c o n v e n t i o n a l p r o t e i n f r a c t i o n a t i o n procedures. The a s s a y p r o c e d u r e used to follow t h e p u r i f i c a t i o n of t he p r o t e i n d e p e n d s upon t w o m a r k e d effects p r o d u c e d b y it on w a s h e d tissue c u l t u r e cells i n o c u l a t e d i nt o a s y n t h e t i c m e d i u m 4. U n d e r t h e c o n d i t i o n s of t h e a s s a y , in t h e a b s e n c e of the p r o t e i n factor, few cells a t t a c h to a glass s urfa c e " A p p e n d i x - A t is a single clone isolate o b t a i n e d in Dr. PUCK'S l a b o r a t o r y 2 from a s t r a i n i s o l a t e d by Dr. CI-IANG3.