- Email: [email protected]
The distribution of protease activities on liver cell fractions
VOL. 24 (1957)
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tively) are high, a n d n e a r l y e q u a l l y so, d u r i n g t h e initial intervals. T h e R N A p h o s p h o r u s w o u l d a p p e a r to m o v e as follows: Sp - - + DS - - + R N P . T h e evidence on t h e m a n n e r in w h i c h ribonucleic acid p a r t i c i p a t e s in p r o t e i n s y n t h e s i s is still f r a g m e n t a r y . If s u c h a p a r t i c i p a t i o n requires t h e r a p i d renewal of t h e nucleic acid, o u r observ a t i o n s w o u l d s u g g e s t t h a t t h e ribonucleic acid of t h e R N P particles of t h e m i c r o s o m e s is n o t directly i n v o l v e d in t h e f o r m a t i o n of m i c r o s o m a l protein, t h o u g h t h e p r o t e i n precursors, a c t i v a t e d in t h e Sp fraction, m a y be a s s e m b l e d in t h e m i c r o s o m e s to form t h e final p o l y m e r . I t is p e r h a p s significant t h a t t h e c y t o p l a s m i c s u p e r n a t a n t fraction, in w h i c h t h e e n z y m i c a c t i v a t i o n of a m i n o acids s h a s b e e n s h o w n to occur ~,7, also c o n t a i n e d t h e ribonucleic acid e x h i b i t i n g t h e h i g h e s t initial u p t a k e of asp. W e shall s u b m i t m o r e detailed i n f o r m a t i o n on t h e s e s t u d i e s in d u e course. T h i s work, w h i c h benefited f r o m t h e t e c h n i c a l a s s i s t a n c e of Miss EDITH A. LAWRENCE, h a s b e e n s u p p o r t e d b y r e s e a r c h g r a n t s f r o m t h e U.S. P u b l i c H e a l t h Service, t h e N a t i o n a l Science F o u n d a t i o n a n d t h e Rockefeller F o u n d a t i o n .
Cell Chemistry Laboratory, Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, N . Y . (U.S.A.)
HAROLD T. SHIGEURA ERWIN CHARGAFF
1 E. CHARGAFF, D. ELSON AND H. T. SHIGEURA, Nature, 178 (1956) 682. H. T. SHIGEURA AND E. CHARGAFF,Federation Proc., 16 (1957) 246J. w . LITTLEFIELD, E. B. KELLER, J. G R o s s AND P. C. ZAMECNIK, ]. Biol. Chem., 217 (1955) I i i . 4 j . L. S1MKIN AND T. S. WORK, Biochem. J., 65 (1957) 307. s H. BORSOOK, 3~me Congr. intern, biochim., Bruxelles, i955, Conf. et Rapp., p. 92. 6 M. B. HOAGLAND, E. B. KELLER AND P. C. ZAMECNIK, J. Biol. Chem., 218 (1956) 345. E. W . DAVlE, V. V. KONINGSBERGER AND F. LIPMANN, Arch. Biochem. Biophys., 65 (1956) 21. R e c e i v e d M a r c h I 8 t h , 1957
The distribution of protease activities on liver cell fractions U s i n g h e m o g l o b i n as a s u b s t r a t e , DE DUVE a n d co-workersZ, 2 f o u n d t h e h i g h e s t c a t h e p s i n a c t i v i t y in t h e light m i t o c h o n d r i a fraction of liver h o m o g e n a t e s . Earlier MAVER AND GRECO 3 h a d f o u n d t h a t t h e t o t a l m i t o c h o n d r i a fraction c o n t a i n s t h e h i g h e s t p e r c e n t a g e of t h e t o t a l p r o t e a s e a c t i v i t y of liver h o m o g e n a t e s n o t only w h e n h e m o g l o b i n , b u t also w h e n b e n z o y l - l - a r g i n i n a m i d e w a s u s e d as a s u b s t r a t e . As c a t h e p s i n is t h e collective n a m e for t h e tissue p r o t e i n a s e s a n d o t h e r proteases, e.g. dip e p t i d a s e s a n d a c a r b o x y p e p t i d a s e , also occur in t h e t i s s u e s we t h o u g h t it w o u l d be i n t e r e s t i n g to s t u d y t h e p r o t e a s e activities of t h e liver cell f r a c t i o n s w i t h s y n t h e t i c s u b s t r a t e s , specific for v a r i o u s proteases. 1:5 r a t liver h o m o g e n a t e s were p r e p a r e d in o.25 M sucrose. T h e h o m o g e n a t e w a s layered on 0.35 M sucrose a n d c e n t r i f u g e d for 5-5 rain a t 9oo × g in order to s e d i m e n t t h e n u c l e a r fraction. T h e s u p e r n a t a n t c o n t a i n i n g t h e o t h e r subcellular c o m p o n e n t s w a s c e n t r i f u g e d for 12 m i n a t 18,ooo x g. T h e t o t a l m i t o c h o n d r i a s e d i m e n t e d in t h i s w a y could be s e p a r a t e d into t h e light a n d t h e h e a v y fraction b y r e s u s p e n d i n g t h e m in o.25 M sucrose, c e n t r i f u g i n g for 8 rain a t i i , o o o × g, a n d r e s u s p e n d i n g t h e slightly p i n k light m i t o c h o n d r i a layer (easily d i s t i n g u i s h a b l e f r o m t h e d a r k yellow h e a v y m i t o c h o n d r i a layer) b y c a u s i n g a slow r o t a t o r y m o v e m e n t in t h e s u p e r n a t a n t b y m e a n s of a s m a l l pestle fitted into t h e c e n t r i f u g e t u b e . T h e s u s p e n s i o n o b t a i n e d in t h i s w a y w a s t h e n t r a n s f e r r e d to a n o t h e r c e n t r i f u g e t u b e a n d t h e light m i t o c h o n d r i a s p u n d o w n b y c e n t r i f u g i n g for 12 m i n at i8,ooo × g. T h e whole p r o c e d u r e w a s carried o u t at a b o u t 2 ° C. T h e final s u p e r n a t a n t o b t a i n e d b y s e d i m e n t i n g t h e t o t a l m i t o c h o n d r i a w a s n o t f r a c t i o n a t e d f u r t h e r ; t h u s it still c o n t a i n e d t h e m i c r o s o m e s . I n order to a v o i d d a m a g e as m u c h as possible t h e s e d i m e n t e d f r a c t i o n s were n o t w a s h e d . W i t h m o s t s u b s t r a t e s t h e p r o t e a s e activities of t h e fractions were d e t e r m i n e d b y m e a n s of t h e r e c e n t l y described m i c r o - m o d i f i c a t i o n of S ~ r e n s e n ' s f o r m a l d e h y d e m e t h o d 4. I n t h e case of b e n z o y l - l - a r g i n i n a m i d e t h e a c t i v i t y w a s m e a s u r e d b y d e t e r m i n a t i o n of t h e liberated a m m o n i a s. T h e rate of h y d r o l y s i s of h e m o g l o b i n b y t h e fractions, s t u d i e d in order to check o u r f r a c t i o n a t i o n t e c h n i q u e as c o m p a r e d w i t h t h a t e m p l o y e d b y DE DUVE, w a s d e t e r m i n e d b y m e a n s of t h e ANSON methode, 7.
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VOL. 2 4 (1957)
All h o m o g e n a t e s a n d s u s p e n s i o n s of f r a c t i o n s were s t u d i e d in the presence of "Uriton X-J oo ~, a d d e d a few m i n u t e s before t h e y were a d d e d to t h e o t h e r c o m p o n e n t s of t he r e a c t i o n m i x t u r e * . As m i c r o - m e t h o d s were used for the d e t e r m i n a t i o n of the r a t e of h y d r o l y s i s of t h e p e p t i d e bonds, the t o t a l v o l u m e s of t h e r e a c t i o n m i x t u r c s could be k e p t as low as a few ml, c o n t a i n i n g a m o u n t s of f r a c t i o n s c o r r e s p o n d i n g to a few h u n d r e d m g liver, while t he a m o u n t s of t he v a r i o u s s u b s t r a t e s r a n g e d from 3 to 15 mg. The p H ' s c o r r e s p o n d e d to t h e p H - o p t i m a of t he h y d r o l y s i s of tile substrates. The r e s u l t s can be s u m m a r i z e d as follows: The g l y c y l g l y e i n e d i p e p t i d a s e ( d e t e r m i n e d in t h e pr esence of Co-ions) a n d t h e t r i g l y c i n e p e p t i d a s e a c t i v i t y ( d e t e r m i n e d in t h e a b s e n c e of Co-ions) a p p e a r e d to be c o n c e n t r a t e d for ioO°,o in t h e s u p e r n a t a n t . On t he o t h e r ha nd, c a t h e p s i n :\ a c t i v i t y , m e a s u r e d w i t h cbz-l-glutamyl-l-tyrosine, was c o m p l e t e l y a b s e n t from t h e s u p e r n a t a n t . Most was f o u n d in t h e m i t o c h o n d r i a , t h e r a t i o b e t w e e n t h e a m o u n t s found in t he l i g h t a n d t he h e a v v f r a c t i o n b e i n g a b o u t 6: 4. The l a t t e r r e s u l t s o m e w h a t r e s e m b l e s t h e re s ul t s o b t a i n e d w i t h h e m o globin as s n b s t r a t e . I n our e x p e r i m e n t s t h e r a t i o for l i g h t a n d h e a v y m i t o c h o n d r i a w a s a b o u t 5 :]. A c c o r d i n g to DE D U V E 2 t h e r a t i o is a b o u t 2:1 in purified p a r e n c h y m a t o u s cells. W e found a h i g h e r a c t i v i t y , e x p r e s s e d as p e r c e n t a g e of t h e a c t i v i t y of the h o m o g e n a t e , in t h e n u c l e a r f r a c t i o n t h a n DE DUVE a n d a lower in t h e s u p e r n a t a n t . H o w e v e r , in our e x p e r i m e n t s t h e n u c l e a r f r a c t i o n s were n o t w a s h e d , wh ile in DE DOVE'S e x p e r i m e n t s t h e y were w a s he d. I t is r e m a r k a b l e t h a t w i t h h e m o g l o b i n as a s u b s t r a t e , we as well as DE DUVE found a c o n s i d e r a b l e a c t i v i t y in t h e s u p e r n a t a n t while w i t h c b z - l - g l u t a m y l - l - t y r o s i n e as a s u b s t r a t e we were u n a b l e t o d e t e c t a n y a c t i v i t y e ve n in a v e r y c o n c e n t r a t e d s u p e r n a t a n t , viz. t h e s u p e r n a t a n t of a i :1.5 h o m o g e n a t e in o.25 M sucrose. C a t h e p s i n B a c t i v i t y m e a s u r e d w i t h b e n z o y l - l - a r g i n i n a m i d e as a s u b s t r a t e w a s d i s t r i b u t e d in all fractions. T h e a m o u n t in t h e s u p e r n a t a n t w a s o n l y a l i t t l e l ow e r t h a n in t h e t o t a l m i t o c h o n d r i a f r action, while t h e r a t i o b e t w e e n t h e l i g h t a n d h e a v y m i t o c h o n d r i a w a s on t h e a v e r a g e a b o u t 5:4. Again, different r e s u l t s c o n c e r n i n g t h e c a r b o x y p e p t i d a s e a c t i v i t y were o b t a i n e d w i t h c b z - g l y c y l l - p h e n y l a l a n i n e as a s u b s t r a t e . No a c t i v i t y could be d e t e c t e d in t h e s u p e r n a t a n t (even from a i :1. 5 h o m o g e n a t e ) , while 80 to 9 0 % of t h e t o t a l a c t i v i t y of t h e h o m o g e n a t e w a s p r e s e n t in t h e m i t o c h o n d r i a . I n t h i s case, t h e h e a v y m i t o e h o n d r i a f r a c t i o n w a s m u c h more a c t i v e t h a n t h e l i g h t f r a c t i o n : t h e r a t i o w a s on t h e a v e r a g e a b o u t IO :I. W i t h t h e e x c e p t i o n of t h e g l y e y l g l y e i n e d i p e p t i d a s e a n d t r i g l y e i n e p e p t i d a s e a c t i v i t i e s , some a c t i v i t y w a s a l w a y s fo und in t h e n u c l e a r fraction. T h i s m a y be c a u s e d b y t h e c o n t a m i n a t i o n of these u n w a s h e d f r a c t i o n s w i t h m i t o c h o n d r i a , t h o u g h it is n o t e x c l u d e d t h a t t h e n u c l e u s also contains the enzymes studied. T h e s e o b s e r v a t i o n s raised m a n y q u e s t i o n s t h a t c a n n o t be dis c us s e d in t h i s note. a.
Laboratory/or Physiological Chemistry, The University, Utrecht (The Netherlands)
RADEMAKER
J. ]3. J. SOONS
1 C. DE DUVE, B. C. PRESSMAN, R. GIANETTO, R. WATTIAU AND V. APt'ELMANS, Biochem. J., 60 (1955) 604. 2 R. V~rATTIAU, P. ]~AUDHUIN, A. M. BERLEUR ANn C. DE DUVE, Biochem. J., 63 (1956) 608. 3 M. 1V~AVERAND A. GRECO, J. Natl. Cancer Inst., 12 (i951) 37. 4 W . RADEMAKER AND J. t3. J. SOONS, Biochim. Biophys. Acta, 24 (1957) 209. 5 j . p. GREENSTEIN AND F. M. LEUTHARDT, J. Natl. Cancer Inst., 6 (1946) 3o3 . 6 M. L. ANSON, J. Gen. Physiol., 2o (1937) 565 • 7 R. GIANETTO AND C. DE DUVE, Biochem. J., 59 (1955) 433. 8 R. V~TATTIAU AND C. DE DOVE, Biochem. J., 63 (1956) 606. R e c e i v e d F e b r u a r y 22nd, 1957 " W e are v e r y m u c h i n d e b t e d to Prof. C. DE DOVE for a s a m p l e of T r i t o n - X - I o o .
PRELIMINARY NOTES
451
tively) are high, a n d n e a r l y e q u a l l y so, d u r i n g t h e initial intervals. T h e R N A p h o s p h o r u s w o u l d a p p e a r to m o v e as follows: Sp - - + DS - - + R N P . T h e evidence on t h e m a n n e r in w h i c h ribonucleic acid p a r t i c i p a t e s in p r o t e i n s y n t h e s i s is still f r a g m e n t a r y . If s u c h a p a r t i c i p a t i o n requires t h e r a p i d renewal of t h e nucleic acid, o u r observ a t i o n s w o u l d s u g g e s t t h a t t h e ribonucleic acid of t h e R N P particles of t h e m i c r o s o m e s is n o t directly i n v o l v e d in t h e f o r m a t i o n of m i c r o s o m a l protein, t h o u g h t h e p r o t e i n precursors, a c t i v a t e d in t h e Sp fraction, m a y be a s s e m b l e d in t h e m i c r o s o m e s to form t h e final p o l y m e r . I t is p e r h a p s significant t h a t t h e c y t o p l a s m i c s u p e r n a t a n t fraction, in w h i c h t h e e n z y m i c a c t i v a t i o n of a m i n o acids s h a s b e e n s h o w n to occur ~,7, also c o n t a i n e d t h e ribonucleic acid e x h i b i t i n g t h e h i g h e s t initial u p t a k e of asp. W e shall s u b m i t m o r e detailed i n f o r m a t i o n on t h e s e s t u d i e s in d u e course. T h i s work, w h i c h benefited f r o m t h e t e c h n i c a l a s s i s t a n c e of Miss EDITH A. LAWRENCE, h a s b e e n s u p p o r t e d b y r e s e a r c h g r a n t s f r o m t h e U.S. P u b l i c H e a l t h Service, t h e N a t i o n a l Science F o u n d a t i o n a n d t h e Rockefeller F o u n d a t i o n .
Cell Chemistry Laboratory, Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, N . Y . (U.S.A.)
HAROLD T. SHIGEURA ERWIN CHARGAFF
1 E. CHARGAFF, D. ELSON AND H. T. SHIGEURA, Nature, 178 (1956) 682. H. T. SHIGEURA AND E. CHARGAFF,Federation Proc., 16 (1957) 246J. w . LITTLEFIELD, E. B. KELLER, J. G R o s s AND P. C. ZAMECNIK, ]. Biol. Chem., 217 (1955) I i i . 4 j . L. S1MKIN AND T. S. WORK, Biochem. J., 65 (1957) 307. s H. BORSOOK, 3~me Congr. intern, biochim., Bruxelles, i955, Conf. et Rapp., p. 92. 6 M. B. HOAGLAND, E. B. KELLER AND P. C. ZAMECNIK, J. Biol. Chem., 218 (1956) 345. E. W . DAVlE, V. V. KONINGSBERGER AND F. LIPMANN, Arch. Biochem. Biophys., 65 (1956) 21. R e c e i v e d M a r c h I 8 t h , 1957
The distribution of protease activities on liver cell fractions U s i n g h e m o g l o b i n as a s u b s t r a t e , DE DUVE a n d co-workersZ, 2 f o u n d t h e h i g h e s t c a t h e p s i n a c t i v i t y in t h e light m i t o c h o n d r i a fraction of liver h o m o g e n a t e s . Earlier MAVER AND GRECO 3 h a d f o u n d t h a t t h e t o t a l m i t o c h o n d r i a fraction c o n t a i n s t h e h i g h e s t p e r c e n t a g e of t h e t o t a l p r o t e a s e a c t i v i t y of liver h o m o g e n a t e s n o t only w h e n h e m o g l o b i n , b u t also w h e n b e n z o y l - l - a r g i n i n a m i d e w a s u s e d as a s u b s t r a t e . As c a t h e p s i n is t h e collective n a m e for t h e tissue p r o t e i n a s e s a n d o t h e r proteases, e.g. dip e p t i d a s e s a n d a c a r b o x y p e p t i d a s e , also occur in t h e t i s s u e s we t h o u g h t it w o u l d be i n t e r e s t i n g to s t u d y t h e p r o t e a s e activities of t h e liver cell f r a c t i o n s w i t h s y n t h e t i c s u b s t r a t e s , specific for v a r i o u s proteases. 1:5 r a t liver h o m o g e n a t e s were p r e p a r e d in o.25 M sucrose. T h e h o m o g e n a t e w a s layered on 0.35 M sucrose a n d c e n t r i f u g e d for 5-5 rain a t 9oo × g in order to s e d i m e n t t h e n u c l e a r fraction. T h e s u p e r n a t a n t c o n t a i n i n g t h e o t h e r subcellular c o m p o n e n t s w a s c e n t r i f u g e d for 12 m i n a t 18,ooo x g. T h e t o t a l m i t o c h o n d r i a s e d i m e n t e d in t h i s w a y could be s e p a r a t e d into t h e light a n d t h e h e a v y fraction b y r e s u s p e n d i n g t h e m in o.25 M sucrose, c e n t r i f u g i n g for 8 rain a t i i , o o o × g, a n d r e s u s p e n d i n g t h e slightly p i n k light m i t o c h o n d r i a layer (easily d i s t i n g u i s h a b l e f r o m t h e d a r k yellow h e a v y m i t o c h o n d r i a layer) b y c a u s i n g a slow r o t a t o r y m o v e m e n t in t h e s u p e r n a t a n t b y m e a n s of a s m a l l pestle fitted into t h e c e n t r i f u g e t u b e . T h e s u s p e n s i o n o b t a i n e d in t h i s w a y w a s t h e n t r a n s f e r r e d to a n o t h e r c e n t r i f u g e t u b e a n d t h e light m i t o c h o n d r i a s p u n d o w n b y c e n t r i f u g i n g for 12 m i n at i8,ooo × g. T h e whole p r o c e d u r e w a s carried o u t at a b o u t 2 ° C. T h e final s u p e r n a t a n t o b t a i n e d b y s e d i m e n t i n g t h e t o t a l m i t o c h o n d r i a w a s n o t f r a c t i o n a t e d f u r t h e r ; t h u s it still c o n t a i n e d t h e m i c r o s o m e s . I n order to a v o i d d a m a g e as m u c h as possible t h e s e d i m e n t e d f r a c t i o n s were n o t w a s h e d . W i t h m o s t s u b s t r a t e s t h e p r o t e a s e activities of t h e fractions were d e t e r m i n e d b y m e a n s of t h e r e c e n t l y described m i c r o - m o d i f i c a t i o n of S ~ r e n s e n ' s f o r m a l d e h y d e m e t h o d 4. I n t h e case of b e n z o y l - l - a r g i n i n a m i d e t h e a c t i v i t y w a s m e a s u r e d b y d e t e r m i n a t i o n of t h e liberated a m m o n i a s. T h e rate of h y d r o l y s i s of h e m o g l o b i n b y t h e fractions, s t u d i e d in order to check o u r f r a c t i o n a t i o n t e c h n i q u e as c o m p a r e d w i t h t h a t e m p l o y e d b y DE DUVE, w a s d e t e r m i n e d b y m e a n s of t h e ANSON methode, 7.
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PRt'~LIMIN~R'~ ~ NOTP~S
VOL. 2 4 (1957)
All h o m o g e n a t e s a n d s u s p e n s i o n s of f r a c t i o n s were s t u d i e d in the presence of "Uriton X-J oo ~, a d d e d a few m i n u t e s before t h e y were a d d e d to t h e o t h e r c o m p o n e n t s of t he r e a c t i o n m i x t u r e * . As m i c r o - m e t h o d s were used for the d e t e r m i n a t i o n of the r a t e of h y d r o l y s i s of t h e p e p t i d e bonds, the t o t a l v o l u m e s of t h e r e a c t i o n m i x t u r c s could be k e p t as low as a few ml, c o n t a i n i n g a m o u n t s of f r a c t i o n s c o r r e s p o n d i n g to a few h u n d r e d m g liver, while t he a m o u n t s of t he v a r i o u s s u b s t r a t e s r a n g e d from 3 to 15 mg. The p H ' s c o r r e s p o n d e d to t h e p H - o p t i m a of t he h y d r o l y s i s of tile substrates. The r e s u l t s can be s u m m a r i z e d as follows: The g l y c y l g l y e i n e d i p e p t i d a s e ( d e t e r m i n e d in t h e pr esence of Co-ions) a n d t h e t r i g l y c i n e p e p t i d a s e a c t i v i t y ( d e t e r m i n e d in t h e a b s e n c e of Co-ions) a p p e a r e d to be c o n c e n t r a t e d for ioO°,o in t h e s u p e r n a t a n t . On t he o t h e r ha nd, c a t h e p s i n :\ a c t i v i t y , m e a s u r e d w i t h cbz-l-glutamyl-l-tyrosine, was c o m p l e t e l y a b s e n t from t h e s u p e r n a t a n t . Most was f o u n d in t h e m i t o c h o n d r i a , t h e r a t i o b e t w e e n t h e a m o u n t s found in t he l i g h t a n d t he h e a v v f r a c t i o n b e i n g a b o u t 6: 4. The l a t t e r r e s u l t s o m e w h a t r e s e m b l e s t h e re s ul t s o b t a i n e d w i t h h e m o globin as s n b s t r a t e . I n our e x p e r i m e n t s t h e r a t i o for l i g h t a n d h e a v y m i t o c h o n d r i a w a s a b o u t 5 :]. A c c o r d i n g to DE D U V E 2 t h e r a t i o is a b o u t 2:1 in purified p a r e n c h y m a t o u s cells. W e found a h i g h e r a c t i v i t y , e x p r e s s e d as p e r c e n t a g e of t h e a c t i v i t y of the h o m o g e n a t e , in t h e n u c l e a r f r a c t i o n t h a n DE DUVE a n d a lower in t h e s u p e r n a t a n t . H o w e v e r , in our e x p e r i m e n t s t h e n u c l e a r f r a c t i o n s were n o t w a s h e d , wh ile in DE DOVE'S e x p e r i m e n t s t h e y were w a s he d. I t is r e m a r k a b l e t h a t w i t h h e m o g l o b i n as a s u b s t r a t e , we as well as DE DUVE found a c o n s i d e r a b l e a c t i v i t y in t h e s u p e r n a t a n t while w i t h c b z - l - g l u t a m y l - l - t y r o s i n e as a s u b s t r a t e we were u n a b l e t o d e t e c t a n y a c t i v i t y e ve n in a v e r y c o n c e n t r a t e d s u p e r n a t a n t , viz. t h e s u p e r n a t a n t of a i :1.5 h o m o g e n a t e in o.25 M sucrose. C a t h e p s i n B a c t i v i t y m e a s u r e d w i t h b e n z o y l - l - a r g i n i n a m i d e as a s u b s t r a t e w a s d i s t r i b u t e d in all fractions. T h e a m o u n t in t h e s u p e r n a t a n t w a s o n l y a l i t t l e l ow e r t h a n in t h e t o t a l m i t o c h o n d r i a f r action, while t h e r a t i o b e t w e e n t h e l i g h t a n d h e a v y m i t o c h o n d r i a w a s on t h e a v e r a g e a b o u t 5:4. Again, different r e s u l t s c o n c e r n i n g t h e c a r b o x y p e p t i d a s e a c t i v i t y were o b t a i n e d w i t h c b z - g l y c y l l - p h e n y l a l a n i n e as a s u b s t r a t e . No a c t i v i t y could be d e t e c t e d in t h e s u p e r n a t a n t (even from a i :1. 5 h o m o g e n a t e ) , while 80 to 9 0 % of t h e t o t a l a c t i v i t y of t h e h o m o g e n a t e w a s p r e s e n t in t h e m i t o c h o n d r i a . I n t h i s case, t h e h e a v y m i t o e h o n d r i a f r a c t i o n w a s m u c h more a c t i v e t h a n t h e l i g h t f r a c t i o n : t h e r a t i o w a s on t h e a v e r a g e a b o u t IO :I. W i t h t h e e x c e p t i o n of t h e g l y e y l g l y e i n e d i p e p t i d a s e a n d t r i g l y e i n e p e p t i d a s e a c t i v i t i e s , some a c t i v i t y w a s a l w a y s fo und in t h e n u c l e a r fraction. T h i s m a y be c a u s e d b y t h e c o n t a m i n a t i o n of these u n w a s h e d f r a c t i o n s w i t h m i t o c h o n d r i a , t h o u g h it is n o t e x c l u d e d t h a t t h e n u c l e u s also contains the enzymes studied. T h e s e o b s e r v a t i o n s raised m a n y q u e s t i o n s t h a t c a n n o t be dis c us s e d in t h i s note. a.
Laboratory/or Physiological Chemistry, The University, Utrecht (The Netherlands)
RADEMAKER
J. ]3. J. SOONS
1 C. DE DUVE, B. C. PRESSMAN, R. GIANETTO, R. WATTIAU AND V. APt'ELMANS, Biochem. J., 60 (1955) 604. 2 R. V~rATTIAU, P. ]~AUDHUIN, A. M. BERLEUR ANn C. DE DUVE, Biochem. J., 63 (1956) 608. 3 M. 1V~AVERAND A. GRECO, J. Natl. Cancer Inst., 12 (i951) 37. 4 W . RADEMAKER AND J. t3. J. SOONS, Biochim. Biophys. Acta, 24 (1957) 209. 5 j . p. GREENSTEIN AND F. M. LEUTHARDT, J. Natl. Cancer Inst., 6 (1946) 3o3 . 6 M. L. ANSON, J. Gen. Physiol., 2o (1937) 565 • 7 R. GIANETTO AND C. DE DUVE, Biochem. J., 59 (1955) 433. 8 R. V~TATTIAU AND C. DE DOVE, Biochem. J., 63 (1956) 606. R e c e i v e d F e b r u a r y 22nd, 1957 " W e are v e r y m u c h i n d e b t e d to Prof. C. DE DOVE for a s a m p l e of T r i t o n - X - I o o .