15.10. 1975
Specialia
w h e n t h e liver a n d muscle glycogen supplies h a v e b e e n exhausted.
Summary. T h e t i m e f u n c t i o n of free f a t t y acids a n d of c o r t i c o s t e r o n e in p l a s m a in fed a n d f a s t e d r a t s was inv e s t i g a t e d . Also a s t u d y of t h e influence of h y p o x i a o n t h e c o n c e n t r a t i o n of p l a s m a free f a t t y acids was c a r r i e d out. W h e r e a s f a s t i n g does n o t seem to s t i m u l a t e lipolysis m a r k e d l y before 12 h, 2 h of h y p o x i a e l e v a t e t h e free
1137
f a t t y acid level to its m a x i m u m . I n t h e f a s t e d c o n d i t i o n , all values are s i g n i f i c a n t l y h i g h e r t h a n c o r t i c o s t e r o n e in t h e fed a n i m a l . W. MLEKUSCH, ~V. TRUPPE, ~V. BEYER u n d B. PALETTA
lnstitut /i~r Medizinische Chemie und -Pregl-Labor der Universitiit Graz, Universitdtsplatz 2, A-8010 Graz (Austria), 76 April 7975.
E v i d e n c e for P u r i n e B i o s y n t h e s i s in H u m a n L e u k o c y t e s P r e v i o u s studies h a v e s h o w n t h a t i s o l a t e d h u m a n l e u k o c y t e s are able to i n c o r p o r a t e 14C-labelled f o r m a t e a n d a d e n i n e into t h e i r nucleic acid b a s e s 1. DIAMOND e t al. 2 o b s e r v e d also a n increased 14C-glycine i n c o r p o r a t i o n i n t o a d e n i n e a n d g u a n i n e of R N A a n d D N A of l e u k o c y t e s f r o m g o u t y p a t i e n t s . H o w e v e r , a d e n i n e a n d g u a n i n e are n o t m e t a b o l i c e n d p r o d u c t s of p u r i n e m e t a b o l i s m a n d t h e i r c o n c e n t r a t i o n c a n n o t give a d e q u a t e i n f o r m a t i o n on t h e b e h a v i o u r of p u r i n e m e t a b o l i s m ; m o r e o v e r , t h e glycine i n c o r p o r a t i o n r a t e d e t e r m i n e d in l e u k o c y t e s in v i t r o does n o t necessarily reflect t h e a c t u a l i m p o r t a n c e of de n o v o p u r i n e b i o s y n t h e s i s . R e c e n t l y , REaM a f o u n d t h a t B u r k i t t l y m p h o m a a n d h u m a n spleen cells could s y n t h e t i z e t h e first a n d second i n t e r m e d i a t e c o m p o u n d s of t h e p u r i n e b i o s y n t h e t i c p a t h w a y , a n d VV'OOD a n d S~2EOMILLER4 a s s a y e d a n d d e t e r m i n e d some p r o p e r t i e s of 5 - p h o s p h o r i bosyl-l-pyrophosphate amidotransferase, the enzyme t h a t c a t a l y z e s t h e first step i n v o l v e d in de n o v o p u r i n e b i o s y n t h e s i s , in h u m a n l y m p h o b l a s t s m a i n t a i n e d in tissue culture. T h e p u r p o s e of t h e p r e s e n t s t u d y was to investigate whether human leukocytes and lymphocytes are c a p a b l e of de n o v o p u r i n e b i o s y n t h e s i s . ~Ve a s s a y e d t h e a c t i v i t y of 5 - p h o s p h o r i b o s y l - l - p y r o p h o s p h a t e a m i d o t r a n s f e r a s e in l e u k o c y t e s a n d l y m p h o c y t e s isolated b y t h e u s u a l p r o c e d u r e s from 11 h e a l t h y v o l u n t e e r s u b j e c t s a c c o r d i n g to t h e m e t h o d of WYNOAARDEN a n d ASHTON a. T h e assays were p e r f o r m e d on l e u k o c y t e s a n d l y m p h o c y t e s e x t r a c t s dialyzed 24 h a g a i n s t distilled w a t e r in order to r e m o v e h e p a r i n a n d o t h e r molecules i n t e r f e r i n g or i n h i b i t i n g t h e e n z y m e . E n z y m e a c t i v i t y was expressed as units/10" cells, w h e r e one u n i t of e n z y m e a c t i v i t y is t h e a m o u n t t h a t p r o d u c e s a n increase of 0.001 a b s o r b a n c e u n i t / m i n a t 363 n m a t p H 8 a n d r o o m t e m p e r a t u r e (25 ~ E n z y m e a c t i v i t y h a s b e e n d e t e c t e d in b o t h k i n d s of cells e x a m i n e d . T h e m e a n v a l u e s r e s u l t e d 1.34 ~ 0.58 units/106 cells in l e u k o c y t e s a n d 0.86 ~ 0.66 units/106 cells in l y m p h o c y t e s (Table). P r e f o r m e d p u r i n e s c o n v e r t e d b y cellular m e t a b o l i s m to r i b o n u c l e o t i d e s h a v e b e e n c o n s i d e r e d t h e o n l y source of p u r i n e n u c l e o t i d e s for m a n y h u m a n cellsa,% Most of t h e conclusions on t h i s d e p e n d e n c e are, h o w e v e r , b a s e d o n i n d i r e c t or n e g a t i v e evidence. S t u d y of t h e e a r l y steps of de n o v o p u r i n e b i o s y n t h e s i s in h u m a n cells, such 5-Phosphoribosyl-l-pyrophosphate amidotransferase activity in leukocytes and lymphoeytes of healthy human subjects
as fibroblasts, l e u k o c y t e s a n d platelets, h a s b e e n l i m i t e d to e v a l u a t i n g t h e a c c u m u l a t i o n of f o r m y l g l y c i n a m i d e r i b o n u c l e o t i d e 3,6 or o b s e r v i n g t h e failure to i n c o r p o r a t e glycine or f o r m a t e i n t o p u r i n e n u c l e o t i d e s in v i t r o 6, while t h e p r e s e n c e a n d t h e p r o p e r t i e s of 5 - p h o s p h o r i b o s y l - 1 pyrophosphate amidotransferase have not been extensively e x a m i n e d 4. H o w e v e r , t h e i n t e r p r e t a t i o n of isotopei n c o r p o r a t i o n studies i n v o l v i n g p u r i n e s y n t h e s i s r e a c t i o n s requires some care c o n c e r n i n g t h e c o n c e n t r a t i o n of precursors a n d i n t e r m e d i a t e c o m p o u n d s 1, 6, a n d t h e p o s s i b i l i t y t h a t some s u b s t r a t e n e c e s s a r y for de n o v o s y n t h e s i s m a y b e e n lacking% O u r results p r o v i d e e v i d e n d e t h a t h u m a n l e u k o c y t e s a n d l y m p h o c y t e s poss2ss t h e e n z y m e a c t i v i t y t h a t c a t a l y z e s t h e f o r m a t i o n of t h e first i n t e r m e d i a t e c o m p o u n d of t h e p u r i n e b i o s y n t h e t i c p a t h w a y . T h e f i n d i n g of t h e p r e s e n c e of 5 - p h o s p h o r i b o s y l - l - p y r o p h o s p h a t e a m i d o t r a n s f e r a s e in h u m a n l e u k o c y t e s a n d l y m p h o c y t e s suggests, therefore, t h a t t h e s e cells h a v e t h e c a p a c i t y for de n o v o p u r i n e b i o s y n t h e s i s a n d are n o t solely d e p e n d e n t on t h e liver for t h e i r s u p p l y of purines. De n o v o p u r i n e s y n t h e s i s m a y t h e r e f o r e be more w i d e s p r e a d than previously reported, and human leukocytes and l y m p h o c y t c s m a y be a n i m p o r t a n t e x t r a h e p a t i c site for de n o v e p u r i n e b i o s y n t h e s i s in m a n . T h e s t u d y of t h e p u r i n e b i o s y n t h e t i c p a t h w a y in h u m a n l e u k o c y t e s a n d l y m p h o c y t e s seems of p a r t i c u l a r i n t e r e s t since i t could p r o v i d e a v a l u a b l e e x p e r i m e n t a l m o d e l to i n v e s t i g a t e t h e factors i n v o l v e d in t h e r e g u l a t i o n of de n o v o p u r i n e bios y n t h e s i s a n d clarify p u r i n e m e t a b o l i s m a l t e r a t i o n s in p a t i e n t s w i t h h y p e r u r i c e m i a , gout, or l e u k e m i a a n d o t h e r m y e l o p r o l i f e r a t i v e diseases.
Summary. H u m a n l e u k o c y t e s a n d l y m p h o c y t e s h a v e s h o w n to b e e q u i p p 3 d w i t h 5 - p h o s p h o r i b o s y l - l - p y r o p h o s phate amidotransrease, the enzyme which catalyzes the s y n t h e s i s of t h e first i n t e r m e d i a t e of t h e p u r i n e p a t h w a y , t h u s p r o v i d i n g e v i d e n c e t h a t t h e s e cells h a v e t h e c a p a c i t y for de n o v o p u r i n e b i o s y n t h e s i s . R. MARCOLONGO, G. POMPUCCI a n d V. MICHELI
Cattedra di Reumatologia and Institute o/ Biochemistry, University o/ Siena, Via Ponizetti 7, Siena (italy), 26 May 7975. 1 j. L. SCOTT, J. clin. hlvest. 41, 67 (1962). 2 }{. S. DIAMOND, M. FRIEDLANI), D. HOLBERSTAM a n d 1). KAPLAN,
Cell type
Enzyme activity (units/l 0~cells)
Leukocytes Lymphocytes
1.34 -c 0.58 0.86 -c 0.66
Ann. rheum. Dis. 28, 27.5 (1969). 3 G. H. REEM, J. clin. Invest. 57, 1058 (1972). 4 A. W. WOOD and J. E. SEEGMILLER, J. biol. Chem. 248, 138 (1973). J. B. WYNOAAm)EN and D. M. ASHTDN, J. biol. Chem. 234, 1492 (1959). A. \V. MURRAY, Ann. Rev. lgioehem. 40, 811 (1971).
Specialia
w h e n t h e liver a n d muscle glycogen supplies h a v e b e e n exhausted.
Summary. T h e t i m e f u n c t i o n of free f a t t y acids a n d of c o r t i c o s t e r o n e in p l a s m a in fed a n d f a s t e d r a t s was inv e s t i g a t e d . Also a s t u d y of t h e influence of h y p o x i a o n t h e c o n c e n t r a t i o n of p l a s m a free f a t t y acids was c a r r i e d out. W h e r e a s f a s t i n g does n o t seem to s t i m u l a t e lipolysis m a r k e d l y before 12 h, 2 h of h y p o x i a e l e v a t e t h e free
1137
f a t t y acid level to its m a x i m u m . I n t h e f a s t e d c o n d i t i o n , all values are s i g n i f i c a n t l y h i g h e r t h a n c o r t i c o s t e r o n e in t h e fed a n i m a l . W. MLEKUSCH, ~V. TRUPPE, ~V. BEYER u n d B. PALETTA
lnstitut /i~r Medizinische Chemie und -Pregl-Labor der Universitiit Graz, Universitdtsplatz 2, A-8010 Graz (Austria), 76 April 7975.
E v i d e n c e for P u r i n e B i o s y n t h e s i s in H u m a n L e u k o c y t e s P r e v i o u s studies h a v e s h o w n t h a t i s o l a t e d h u m a n l e u k o c y t e s are able to i n c o r p o r a t e 14C-labelled f o r m a t e a n d a d e n i n e into t h e i r nucleic acid b a s e s 1. DIAMOND e t al. 2 o b s e r v e d also a n increased 14C-glycine i n c o r p o r a t i o n i n t o a d e n i n e a n d g u a n i n e of R N A a n d D N A of l e u k o c y t e s f r o m g o u t y p a t i e n t s . H o w e v e r , a d e n i n e a n d g u a n i n e are n o t m e t a b o l i c e n d p r o d u c t s of p u r i n e m e t a b o l i s m a n d t h e i r c o n c e n t r a t i o n c a n n o t give a d e q u a t e i n f o r m a t i o n on t h e b e h a v i o u r of p u r i n e m e t a b o l i s m ; m o r e o v e r , t h e glycine i n c o r p o r a t i o n r a t e d e t e r m i n e d in l e u k o c y t e s in v i t r o does n o t necessarily reflect t h e a c t u a l i m p o r t a n c e of de n o v o p u r i n e b i o s y n t h e s i s . R e c e n t l y , REaM a f o u n d t h a t B u r k i t t l y m p h o m a a n d h u m a n spleen cells could s y n t h e t i z e t h e first a n d second i n t e r m e d i a t e c o m p o u n d s of t h e p u r i n e b i o s y n t h e t i c p a t h w a y , a n d VV'OOD a n d S~2EOMILLER4 a s s a y e d a n d d e t e r m i n e d some p r o p e r t i e s of 5 - p h o s p h o r i bosyl-l-pyrophosphate amidotransferase, the enzyme t h a t c a t a l y z e s t h e first step i n v o l v e d in de n o v o p u r i n e b i o s y n t h e s i s , in h u m a n l y m p h o b l a s t s m a i n t a i n e d in tissue culture. T h e p u r p o s e of t h e p r e s e n t s t u d y was to investigate whether human leukocytes and lymphocytes are c a p a b l e of de n o v o p u r i n e b i o s y n t h e s i s . ~Ve a s s a y e d t h e a c t i v i t y of 5 - p h o s p h o r i b o s y l - l - p y r o p h o s p h a t e a m i d o t r a n s f e r a s e in l e u k o c y t e s a n d l y m p h o c y t e s isolated b y t h e u s u a l p r o c e d u r e s from 11 h e a l t h y v o l u n t e e r s u b j e c t s a c c o r d i n g to t h e m e t h o d of WYNOAARDEN a n d ASHTON a. T h e assays were p e r f o r m e d on l e u k o c y t e s a n d l y m p h o c y t e s e x t r a c t s dialyzed 24 h a g a i n s t distilled w a t e r in order to r e m o v e h e p a r i n a n d o t h e r molecules i n t e r f e r i n g or i n h i b i t i n g t h e e n z y m e . E n z y m e a c t i v i t y was expressed as units/10" cells, w h e r e one u n i t of e n z y m e a c t i v i t y is t h e a m o u n t t h a t p r o d u c e s a n increase of 0.001 a b s o r b a n c e u n i t / m i n a t 363 n m a t p H 8 a n d r o o m t e m p e r a t u r e (25 ~ E n z y m e a c t i v i t y h a s b e e n d e t e c t e d in b o t h k i n d s of cells e x a m i n e d . T h e m e a n v a l u e s r e s u l t e d 1.34 ~ 0.58 units/106 cells in l e u k o c y t e s a n d 0.86 ~ 0.66 units/106 cells in l y m p h o c y t e s (Table). P r e f o r m e d p u r i n e s c o n v e r t e d b y cellular m e t a b o l i s m to r i b o n u c l e o t i d e s h a v e b e e n c o n s i d e r e d t h e o n l y source of p u r i n e n u c l e o t i d e s for m a n y h u m a n cellsa,% Most of t h e conclusions on t h i s d e p e n d e n c e are, h o w e v e r , b a s e d o n i n d i r e c t or n e g a t i v e evidence. S t u d y of t h e e a r l y steps of de n o v o p u r i n e b i o s y n t h e s i s in h u m a n cells, such 5-Phosphoribosyl-l-pyrophosphate amidotransferase activity in leukocytes and lymphoeytes of healthy human subjects
as fibroblasts, l e u k o c y t e s a n d platelets, h a s b e e n l i m i t e d to e v a l u a t i n g t h e a c c u m u l a t i o n of f o r m y l g l y c i n a m i d e r i b o n u c l e o t i d e 3,6 or o b s e r v i n g t h e failure to i n c o r p o r a t e glycine or f o r m a t e i n t o p u r i n e n u c l e o t i d e s in v i t r o 6, while t h e p r e s e n c e a n d t h e p r o p e r t i e s of 5 - p h o s p h o r i b o s y l - 1 pyrophosphate amidotransferase have not been extensively e x a m i n e d 4. H o w e v e r , t h e i n t e r p r e t a t i o n of isotopei n c o r p o r a t i o n studies i n v o l v i n g p u r i n e s y n t h e s i s r e a c t i o n s requires some care c o n c e r n i n g t h e c o n c e n t r a t i o n of precursors a n d i n t e r m e d i a t e c o m p o u n d s 1, 6, a n d t h e p o s s i b i l i t y t h a t some s u b s t r a t e n e c e s s a r y for de n o v o s y n t h e s i s m a y b e e n lacking% O u r results p r o v i d e e v i d e n d e t h a t h u m a n l e u k o c y t e s a n d l y m p h o c y t e s poss2ss t h e e n z y m e a c t i v i t y t h a t c a t a l y z e s t h e f o r m a t i o n of t h e first i n t e r m e d i a t e c o m p o u n d of t h e p u r i n e b i o s y n t h e t i c p a t h w a y . T h e f i n d i n g of t h e p r e s e n c e of 5 - p h o s p h o r i b o s y l - l - p y r o p h o s p h a t e a m i d o t r a n s f e r a s e in h u m a n l e u k o c y t e s a n d l y m p h o c y t e s suggests, therefore, t h a t t h e s e cells h a v e t h e c a p a c i t y for de n o v o p u r i n e b i o s y n t h e s i s a n d are n o t solely d e p e n d e n t on t h e liver for t h e i r s u p p l y of purines. De n o v o p u r i n e s y n t h e s i s m a y t h e r e f o r e be more w i d e s p r e a d than previously reported, and human leukocytes and l y m p h o c y t c s m a y be a n i m p o r t a n t e x t r a h e p a t i c site for de n o v e p u r i n e b i o s y n t h e s i s in m a n . T h e s t u d y of t h e p u r i n e b i o s y n t h e t i c p a t h w a y in h u m a n l e u k o c y t e s a n d l y m p h o c y t e s seems of p a r t i c u l a r i n t e r e s t since i t could p r o v i d e a v a l u a b l e e x p e r i m e n t a l m o d e l to i n v e s t i g a t e t h e factors i n v o l v e d in t h e r e g u l a t i o n of de n o v o p u r i n e bios y n t h e s i s a n d clarify p u r i n e m e t a b o l i s m a l t e r a t i o n s in p a t i e n t s w i t h h y p e r u r i c e m i a , gout, or l e u k e m i a a n d o t h e r m y e l o p r o l i f e r a t i v e diseases.
Summary. H u m a n l e u k o c y t e s a n d l y m p h o c y t e s h a v e s h o w n to b e e q u i p p 3 d w i t h 5 - p h o s p h o r i b o s y l - l - p y r o p h o s phate amidotransrease, the enzyme which catalyzes the s y n t h e s i s of t h e first i n t e r m e d i a t e of t h e p u r i n e p a t h w a y , t h u s p r o v i d i n g e v i d e n c e t h a t t h e s e cells h a v e t h e c a p a c i t y for de n o v o p u r i n e b i o s y n t h e s i s . R. MARCOLONGO, G. POMPUCCI a n d V. MICHELI
Cattedra di Reumatologia and Institute o/ Biochemistry, University o/ Siena, Via Ponizetti 7, Siena (italy), 26 May 7975. 1 j. L. SCOTT, J. clin. hlvest. 41, 67 (1962). 2 }{. S. DIAMOND, M. FRIEDLANI), D. HOLBERSTAM a n d 1). KAPLAN,
Cell type
Enzyme activity (units/l 0~cells)
Leukocytes Lymphocytes
1.34 -c 0.58 0.86 -c 0.66
Ann. rheum. Dis. 28, 27.5 (1969). 3 G. H. REEM, J. clin. Invest. 57, 1058 (1972). 4 A. W. WOOD and J. E. SEEGMILLER, J. biol. Chem. 248, 138 (1973). J. B. WYNOAAm)EN and D. M. ASHTDN, J. biol. Chem. 234, 1492 (1959). A. \V. MURRAY, Ann. Rev. lgioehem. 40, 811 (1971).
