15. 8. 1977
Specialia
997
Effects of N 2, OV-dibutyril cyclic G M P on the n u c l e o s i d e p h o s p h o t r a n s f e r a s e activity of the retina of the chick e m b r y o s G. Tesoriere, G. Calvaruso a n d IR. V e n t o
Institute o~ Biological Chemistry, University o/ Palermo, 1-90127 Palermo (Italy), 27 October 1976 Summary. I n t h e r e t i n a of t h e chick e m b r y o , 2 d i f f e r e n t f o r m s of nucleoside p h o s p h o t r a n s f e r a s e t a k e p a r t in t h e p h o s p h o r y l a t i o n of t h y m i d i n e . One is a n u n s t a b l e f o r m w i t h h i g h e r molecular weight. The o t h e r w i t h lower m. w t is a stable form. This p a p e r s h o w s t h a t N ~, O V - d i b u t y r i l cyclic G M P causes a m a r k e d d e c r e m e n t of t h e a c t i v i t y of t h e u n s t a b l e nueleoside phosphotransferase. T h e p h o s p h o r y l a t i o n of t h y m i d i n e is c a t a l y z e d in m a m m a l i a n tissues a n d E. coli b y t h i m i d i n e kinase. I n p l a n t s a n d in c e r t a i n m i c r o o r g a n i s m s ~-5, t h i s r e a c t i o n d e p e n d s u p o n a n o t h e r a c t i v i t y , a nucleoside p h o s p h o t r a n s f e r a s e w h i c h c a t a l y z e s t h e reversible t r a n s f e r of e s t e r p h o s p h a t e f r o m nucleoside m o n o p h o s p h a t e or d i p h o s p h a t e t o nucleoside. P r e v i o u s l y 6 we f o u n d in t h e r e t i n a of t h e chick e m b r y o s 2 d i f f e r e n t f o r m s of nucleoside p h o s p h o t r a n s f e r a s e w h i c h are able to p h o s p h o r y l a t e t h y m i d i n e . B o t h t h e s e f o r m s h a v e a non-specific a c t i v i t y a n d t h e y p r e f e r t h e nucleosides m o n o p h o s p h a t e as p h o s p h a t e donors. The
Effects of N 2, O2'-dibutyril cyclic GMP (DBcGMP) on the thymidine phosphoryIating activity of the chick embryo retina Substrate
Thymidine phosphorylated nmoles/mg of protein DBcGMP 1 mM 2 mM
No addition ADP AMP UDP UMP GMP CMP TMP
0.18 ~- 0.02 1.18 =t_ 0.12 1.65 :~ 0.18 2.85 -L 0.30 3.60 ~c 0.26 3.30 ~z 0.24 3.10 j= 0.28 3.75 =t_ 0.32
Data are the mean i
0.15 zJ= 0.02 1.41 zt= 0.14 2.24 ~_ 0.25 1.99 j= 0.22 1.90 zL 0.18 2.21 zt= 0.20 1.86 ~_ 0.20 2.25 ~= 0.24
0.10 J= 0.02 1.65 =~ 0.18 3.08 ~ 0.30 1.56 ~ 0.17 1.58 J- 0.19 2.14 ~_ 0.18 1.52 ~ 0.17 1.76 ~_ 0.20
SE of 6 separate experiments.
0.6
0.4
o
E => '--
0.~
f o r m w i t h a h i g h e r m . w t is v e r y u n s t a b l e to dilution, s t o r a g e a n d gel filtration, a n d it is u n a b l e to utilize t h e a d e n i n e n u c l e o t i d e s as p h o s p h a t e donors. U T P seems to protect this form by the inactivation. The other form w i t h lower m . w t is s t a b l e a n d utilizes also t h e a d e n i n e n u e l e o t i d e s as p h o s p h a t e donors. A M P s e e m s to be t h e s u b s t r a t e more active. I n t h i s p a p e r , we e x a m i n e t h e effects of N2, O2'-dibutyril G M P (DBcGMP) on t h e nucleoside p h o s p h o t r a n s f e r a s e a c t i v i t y of t h e chick e m b r y o retina. The r e t i n a s were r a p i d l y r e m o v e d f r o m 12-day-old chick e m b r y o s , w a s h e d g e n t l y in cold 0.1 M tris-HC1 buffer (pH 8.0) a n d h o m o g e n i z e d in P o t t e r - E l v e h j e m in t h e s a m e m e d i u m (0.25 m l / r e t i n a ) . The h o l n o g e n a t e was c e n t r i f u g e d a t 105,000 • g for 30 mill a n d t h e s u p e r n a t a n t was utilized for t h e i n c u b a t i o n samples. The s t a n d a r d r e a c t i o n m i x t u r e c o n t a i n e d , in a final v o l u m e of 500 ~xl, 40 m M tris-HC1 (pH 8), 5 m M MgC12, 10 m M s u b s t r a t e , 20 FM (0.5 vCi) (Me-3H) t h y m i d i n e a n d 200 F1 of t h e e n z y m e e x t r a c t . A f t e r i n c u b a t i o n a t 37~ for 1 h, t h e r e a c t i o n was s t o p p e d b y boiling t h e s a m p l e s for 3 Inin. F o r t h e e v a l u a t i o n of t h e t h y m i d i n e n u c l e o f i d e s formed, we h a v e e m p l o y e d c o l u m n s (3 x 1 cm) of Dowex-1 form a t e , p r e p a r e d a c c o r d i n g to H u r l b e r t e t al.L A t first t h y m i d i n e w a s e l u t e d f r o m t h e c o l u m n b y 38 ml of 2 N formic acid. Successively t h e t h y m i d i n e n u c l e o t i d e s were e l u t e d b y 10 ml of 1 N a m m o n i u m f o r m a t e - 4 N formic acid. This last f r a c t i o n was e v a p o r a t e d 'in v a c u o ' a t 10 ~ a n d t h e residue was dissolved in 0.5 m l of wafer. A l i q u o t s were e v a l u a t e d for r a d i o a c t i v i t y in a NuclearChicago s c i n t i l l a t i o n c o u n t e r . Corrections for q u e n c h i n g were m a d e b y using e x t e r n a l s t a n d a r d i z a t i o n . As determ i n e d b y c h r o m a t o g r a p h i c p r o c e d u r e , t h y m i d i l i c acid a c c o u n t e d for a t least 95% of t h e p h o s p h o r y l a t e d products. D a t a r e p o r t e d in t h e t a b l e s h o w t h e effects of D B c G M P on t h e t h y m i d i n e p h o s p h o r y l a t i n g a c t i v i t y w h e n t h e r e a c t i o n was m e a s u r e d in t h e 105,000 g s u p e r n a t a n t . D B c G M P causes a n i n c r e m e n t of t h i s a c t i v i t y w h e n A D P a n d p a r t i c u l a r l y A1V~P are e m p l o y e d as p h o s p h a t e donors, while it has a n i n h i b i t i v e effect u s i n g t h e o t h e r s u b s t r a t e s r e p o r t e d in t h e table. This i n h i b i t i o n is g r e a t e r w h e n U M P was e m p l o y e d . The e l u t i o n p a t t e r n of nucleoside p h o s p h o t r a n s f e r a s e on S e p h a d e x G-200 c o l u m n is
I
cI
10
20 "fube number
30
z[O
Fig. 1. Gel filtration pattern of the nucleoside phosphotransferase activity on a Sephadex G-200 column. For this experiment, a homogenate was obtained from 40 retinas and the resulting 105,000 g supernatant was applied to a column (32 • 2 era) of Sephadex G-200. The column was equilibrated and Muted with 5 mM tris-HC1 buffer (pH 8.0) containing 0.2 mM UTP and 2 mM MgCI~. Fractions of 5 ml were collected. 200 [xl of each fraction were utilized for the xneasurement of the nucleoside phosphotransferase activity by using as substrate UMP ( 9169 or AMP (O--O).
1 E. F. Brunngraber and E. Chargaff, J. biol. Chem. 242, 4834 (1967). 2 J.C. Georgatsos, Arehs Biochem. Biophys. 121, 619 (1967). 3 M. Tunis and E. Chargaff, 13ioehim. biophys, Aeta 37, 257 (1960). 4 M. Tunis and E, Chargaff, Biochim. biophys. Acta 37, 267 (1960). 5 T. Shiosaka, H. Okuda and S. Fujii, Bioehim. biophys. Aeta 246, 171 (1971). 6 G. Tesoriere, R. Vento and G. Calvaruso, submitted to Bioehim. biophys. Aeta. 7 R. t3. Hurlbert, H. Schmitz, A. F. Brumm and V. R. Potter, j'. biol. Chem. 209, 23 (1954).
998
Specialia
r e p o r t e d in figure 1. F o r t h i s e x p e r i m e n t , t h e S e p h a d e x was swelled a n d e l u t e d w i t h a s o l u t i o n of 5 m M tris-HC1 b u f f e r (pH 8.0) c o n t a i n i n g 0.2 m M U T P a n d 2 m M 1V[gCl~, b e c a u s e Mg ++ a n d p a r t i c u l a r l y U T P stabilize t h e 0.6
0.4
~0.2
E v--
D8 GMPc Fig. 2. Effects of DBcGMP on the 2 forms of nucleoside phosphotransferase of chick embryo retina. Peak I (tube numbers 11-15)
and peak II (tube numbers 19-23) were collected separately and 200 ill of each peak were utilized for the incubation sample. The activity was measured by using as phosphate donor UMP ( 9169 for peak I and AMP (D--O) for peak II. Data are the means ~ SE of 6 separate experiments.
EXPERIENTIA 33/8
u n s t a b l e f o r m of nucleoside p h o s p h o t r a n s f e r a s e 6. T h e figure shows 2 p e a k s of a c t i v i t y : t h e first corresponds, as p r e v i o u s l y d e m o n s t r a t e d 6, t o t h e u n s t a b l e n u c l e o s i d e p h o s p h o t r a n s f e r a s e a n d i t prefers U M P as p h o s p h a t e d o n o r , while t h e s e c o n d i s r e p r e s e n t e d b y a s t a b l e f o r m w h i c h e m p l o y e s p r e f e r e n t i a l l y A M P as s u b s t r a t e . As s h o w n in figure 2, D B c G M P m a r k e d l y i n h i b i t s t h e n u c l e o s i d e p h o s p h o t r a n s f e r a s e of p e a k I, w h i l e a n y s i g n i f i c a t i v e effect was n o t o b s e r v e d for t h e a c t i v i t y of p e a k I I . P r e v i o u s l y ~ we h a v e h y p o t h e s i z e d t h a t t h e n u c l e o s i d e p h o s p h o t r a n s f e r a s e is p r e s e n t in t h e c h i c k e m b r y o r e t i n a a t l e a s t i n 2 d i f f e r e n t forms, w h i c h could b e a n expression of t h e s a m e e n z y m e a t d i f f e r e n t aggreg a t i o n states. I t is possible t h a t D B c G M P f a c i l i t a t e s t h e c o n v e r s i o n of t h e f o r m w i t h h i g h e r m . w t i n t o a d i s a g g r e g a t e d state. T h i s s t a t e c o u l d b e r e p r e s e n t e d b y t h e s t a b l e nucleoside p h o s p h o t r a n s f e r a s e , a n e n z y m a t i c a c t i v i t y w h i c h is able to utilize as p h o s p h a t e d o n o r s also t h e a d e n i n e nucleotides. T h e s e c o n s i d e r a t i o n s c o u l d e x p l a i n w h y t h e D B c G M P causes a n i n c r e m e n t of t h e t h y m i d i n e p h o s p h o r y l a t i n g r a t e w h e n t h e r e a c t i o n is m e a s u r e d , b y u s i n g A M P as p h o s p h a t e d o n o r , in t h e 105,000 g s u p e r n a t a n t . F u r t h e r m o r e , b e c a u s e i t seems t h a t t h e nucleoside p h o s p h o t r a n s f e r a s e t a k e s p a r t in t h e c o n t r o l of t h e e n d o g e n o u s pools of nucleosides a n d n u c l e o t i d e s , t h e effects of D B c G M P o n t h i s a c t i v i t y could i n d i c a t e t h e p a r t i c i p a t i o n of t h i s c o m p o u n d i n t h e r e g u l a t i o n of nucleotide metabolism.
Absorption andlbiotransformation of L( + ) vtt _ a r t a r i c , ~ a c i d in rats L. F. C h a s s e a u d , W . H. D o w n a n d D. K i r k p a t r i c k
Huntingdon Research Centre, Department o/ Metabolism and Pharmacokinetics, Huntingdon PE18 6ES (England), 18 January 1977 Summary. Oral or p a r e n t e r a l doses of m o n o s o d i u m l * C - L ( + ) - t a r t r a t e (400 mg/kg) are r a p i d l y e x c r e t e d b y r a t s a n d a p r o p o r t i o n c o m p l e t e l y m e t a b o l i z e d t o CO v T h e oral dose was w e l l - a b s o r b e d . T a r t a r i c acid a n d its s a l t s are used in m e d i c i n e a n d in t h e food i n d u s t r y . I n h u m a n s , t h e acid is t h o u g h t t o b e p o o r l y a b s o r b e d 1 a n d w h e n g i v e n orally, t o b e m e t a b o lized b y t h e g u t flora 2, 3, since i t is r e a d i l y m e t a b o l i z e d b y m i c r o o r g a n i s m s s u c h as P s e u d o m o n a s p u t i d a ~ a n d P e n i 90 J
o 70
.~ 5o
g o
o :
E
r
24 Time
48h
Cumulative excretion of radioactivity in the urine ( I , 0) and expired air (D, C) of rats dosed orally or i.v. respectively with monosodium 14C-L(+)-tartrate (400 mg/kg).
cillium charlesii 5, w h i c h c o n v e r t i t t o g l y c e r a t e a n d CO v S t u d i e s in dogs a n d r a b b i t s h a v e s h o w n t h a t oral doses of t a r t a r i c acid were e x c r e t e d in t h e u r i n e as u n c h a n g e d c o m p o u n d , t h e p r o p o r t i o n of w h i c h d e c r e a s e d w i t h i n c r e a s i n g doses 6. M u c h of t h e t a r t a r i c acid used is o b t a i n e d as a b y p r o d u c t of w i n e m a n u f a c t u r e a n d is therefore the naturally-occurring L(+) formL Thus the a b s o r p t i o n a n d b i o t r a n s f o r m a t i o n of t a r t a r i c acid h a s b e e n e v a l u a t e d u s i n g t h e 14C-L(+) form. Materials. (1,4-1*C)-DL-Tartaric acid c~f specific a c t i v i t y 2 - 1 0 m C i / m m o l e s was o b t a i n e d f r o m T h e R a d i o c h e m i c a l Centre, A m e r s h a m , E n g l a n d , a n d w a s resolved i n t o t h e L( + ) - i s o m e r s. T h e r e s u l t i n g m o n o s o d i u m 14C-L(+)1 L. S. Goodman and A. Gilman, The Pharmacological Basis of Therapeutics, 4th ed. Macmillan, London 1970. 2 F.P. Underhill, F. I. Peterman, T. C. Jaleski and C. S. Leonard, J. Pharmac. exp. Ther. 43, 381 (1931). 3 P. Finkle, J. biol. Chem. 700, 349 (1933). 4 L.D. Kohn and W. B. Jakoby, J. biol. Chem. 243, 2465 (1968). 5 K.P. Klatt, P. D. Rick and J. E. Gander, Archs Biochem. Biophys. 134, 335 (1969). 6 F.P. Underhill, C. S. Leonard, E. G. Gross and T. C. Jaleski, J, Pharmae. exp. Ther. 43, 359 (1931). 7 M. H, M. Arnold, Acidulants for Food and Beverages. Food Trade Press Ltd., London 1975. 8 J. Read and W. G. Reid, J. Soc. chem. Ind. 47, 8 (1928).
Specialia
997
Effects of N 2, OV-dibutyril cyclic G M P on the n u c l e o s i d e p h o s p h o t r a n s f e r a s e activity of the retina of the chick e m b r y o s G. Tesoriere, G. Calvaruso a n d IR. V e n t o
Institute o~ Biological Chemistry, University o/ Palermo, 1-90127 Palermo (Italy), 27 October 1976 Summary. I n t h e r e t i n a of t h e chick e m b r y o , 2 d i f f e r e n t f o r m s of nucleoside p h o s p h o t r a n s f e r a s e t a k e p a r t in t h e p h o s p h o r y l a t i o n of t h y m i d i n e . One is a n u n s t a b l e f o r m w i t h h i g h e r molecular weight. The o t h e r w i t h lower m. w t is a stable form. This p a p e r s h o w s t h a t N ~, O V - d i b u t y r i l cyclic G M P causes a m a r k e d d e c r e m e n t of t h e a c t i v i t y of t h e u n s t a b l e nueleoside phosphotransferase. T h e p h o s p h o r y l a t i o n of t h y m i d i n e is c a t a l y z e d in m a m m a l i a n tissues a n d E. coli b y t h i m i d i n e kinase. I n p l a n t s a n d in c e r t a i n m i c r o o r g a n i s m s ~-5, t h i s r e a c t i o n d e p e n d s u p o n a n o t h e r a c t i v i t y , a nucleoside p h o s p h o t r a n s f e r a s e w h i c h c a t a l y z e s t h e reversible t r a n s f e r of e s t e r p h o s p h a t e f r o m nucleoside m o n o p h o s p h a t e or d i p h o s p h a t e t o nucleoside. P r e v i o u s l y 6 we f o u n d in t h e r e t i n a of t h e chick e m b r y o s 2 d i f f e r e n t f o r m s of nucleoside p h o s p h o t r a n s f e r a s e w h i c h are able to p h o s p h o r y l a t e t h y m i d i n e . B o t h t h e s e f o r m s h a v e a non-specific a c t i v i t y a n d t h e y p r e f e r t h e nucleosides m o n o p h o s p h a t e as p h o s p h a t e donors. The
Effects of N 2, O2'-dibutyril cyclic GMP (DBcGMP) on the thymidine phosphoryIating activity of the chick embryo retina Substrate
Thymidine phosphorylated nmoles/mg of protein DBcGMP 1 mM 2 mM
No addition ADP AMP UDP UMP GMP CMP TMP
0.18 ~- 0.02 1.18 =t_ 0.12 1.65 :~ 0.18 2.85 -L 0.30 3.60 ~c 0.26 3.30 ~z 0.24 3.10 j= 0.28 3.75 =t_ 0.32
Data are the mean i
0.15 zJ= 0.02 1.41 zt= 0.14 2.24 ~_ 0.25 1.99 j= 0.22 1.90 zL 0.18 2.21 zt= 0.20 1.86 ~_ 0.20 2.25 ~= 0.24
0.10 J= 0.02 1.65 =~ 0.18 3.08 ~ 0.30 1.56 ~ 0.17 1.58 J- 0.19 2.14 ~_ 0.18 1.52 ~ 0.17 1.76 ~_ 0.20
SE of 6 separate experiments.
0.6
0.4
o
E => '--
0.~
f o r m w i t h a h i g h e r m . w t is v e r y u n s t a b l e to dilution, s t o r a g e a n d gel filtration, a n d it is u n a b l e to utilize t h e a d e n i n e n u c l e o t i d e s as p h o s p h a t e donors. U T P seems to protect this form by the inactivation. The other form w i t h lower m . w t is s t a b l e a n d utilizes also t h e a d e n i n e n u e l e o t i d e s as p h o s p h a t e donors. A M P s e e m s to be t h e s u b s t r a t e more active. I n t h i s p a p e r , we e x a m i n e t h e effects of N2, O2'-dibutyril G M P (DBcGMP) on t h e nucleoside p h o s p h o t r a n s f e r a s e a c t i v i t y of t h e chick e m b r y o retina. The r e t i n a s were r a p i d l y r e m o v e d f r o m 12-day-old chick e m b r y o s , w a s h e d g e n t l y in cold 0.1 M tris-HC1 buffer (pH 8.0) a n d h o m o g e n i z e d in P o t t e r - E l v e h j e m in t h e s a m e m e d i u m (0.25 m l / r e t i n a ) . The h o l n o g e n a t e was c e n t r i f u g e d a t 105,000 • g for 30 mill a n d t h e s u p e r n a t a n t was utilized for t h e i n c u b a t i o n samples. The s t a n d a r d r e a c t i o n m i x t u r e c o n t a i n e d , in a final v o l u m e of 500 ~xl, 40 m M tris-HC1 (pH 8), 5 m M MgC12, 10 m M s u b s t r a t e , 20 FM (0.5 vCi) (Me-3H) t h y m i d i n e a n d 200 F1 of t h e e n z y m e e x t r a c t . A f t e r i n c u b a t i o n a t 37~ for 1 h, t h e r e a c t i o n was s t o p p e d b y boiling t h e s a m p l e s for 3 Inin. F o r t h e e v a l u a t i o n of t h e t h y m i d i n e n u c l e o f i d e s formed, we h a v e e m p l o y e d c o l u m n s (3 x 1 cm) of Dowex-1 form a t e , p r e p a r e d a c c o r d i n g to H u r l b e r t e t al.L A t first t h y m i d i n e w a s e l u t e d f r o m t h e c o l u m n b y 38 ml of 2 N formic acid. Successively t h e t h y m i d i n e n u c l e o t i d e s were e l u t e d b y 10 ml of 1 N a m m o n i u m f o r m a t e - 4 N formic acid. This last f r a c t i o n was e v a p o r a t e d 'in v a c u o ' a t 10 ~ a n d t h e residue was dissolved in 0.5 m l of wafer. A l i q u o t s were e v a l u a t e d for r a d i o a c t i v i t y in a NuclearChicago s c i n t i l l a t i o n c o u n t e r . Corrections for q u e n c h i n g were m a d e b y using e x t e r n a l s t a n d a r d i z a t i o n . As determ i n e d b y c h r o m a t o g r a p h i c p r o c e d u r e , t h y m i d i l i c acid a c c o u n t e d for a t least 95% of t h e p h o s p h o r y l a t e d products. D a t a r e p o r t e d in t h e t a b l e s h o w t h e effects of D B c G M P on t h e t h y m i d i n e p h o s p h o r y l a t i n g a c t i v i t y w h e n t h e r e a c t i o n was m e a s u r e d in t h e 105,000 g s u p e r n a t a n t . D B c G M P causes a n i n c r e m e n t of t h i s a c t i v i t y w h e n A D P a n d p a r t i c u l a r l y A1V~P are e m p l o y e d as p h o s p h a t e donors, while it has a n i n h i b i t i v e effect u s i n g t h e o t h e r s u b s t r a t e s r e p o r t e d in t h e table. This i n h i b i t i o n is g r e a t e r w h e n U M P was e m p l o y e d . The e l u t i o n p a t t e r n of nucleoside p h o s p h o t r a n s f e r a s e on S e p h a d e x G-200 c o l u m n is
I
cI
10
20 "fube number
30
z[O
Fig. 1. Gel filtration pattern of the nucleoside phosphotransferase activity on a Sephadex G-200 column. For this experiment, a homogenate was obtained from 40 retinas and the resulting 105,000 g supernatant was applied to a column (32 • 2 era) of Sephadex G-200. The column was equilibrated and Muted with 5 mM tris-HC1 buffer (pH 8.0) containing 0.2 mM UTP and 2 mM MgCI~. Fractions of 5 ml were collected. 200 [xl of each fraction were utilized for the xneasurement of the nucleoside phosphotransferase activity by using as substrate UMP ( 9169 or AMP (O--O).
1 E. F. Brunngraber and E. Chargaff, J. biol. Chem. 242, 4834 (1967). 2 J.C. Georgatsos, Arehs Biochem. Biophys. 121, 619 (1967). 3 M. Tunis and E. Chargaff, 13ioehim. biophys, Aeta 37, 257 (1960). 4 M. Tunis and E, Chargaff, Biochim. biophys. Acta 37, 267 (1960). 5 T. Shiosaka, H. Okuda and S. Fujii, Bioehim. biophys. Aeta 246, 171 (1971). 6 G. Tesoriere, R. Vento and G. Calvaruso, submitted to Bioehim. biophys. Aeta. 7 R. t3. Hurlbert, H. Schmitz, A. F. Brumm and V. R. Potter, j'. biol. Chem. 209, 23 (1954).
998
Specialia
r e p o r t e d in figure 1. F o r t h i s e x p e r i m e n t , t h e S e p h a d e x was swelled a n d e l u t e d w i t h a s o l u t i o n of 5 m M tris-HC1 b u f f e r (pH 8.0) c o n t a i n i n g 0.2 m M U T P a n d 2 m M 1V[gCl~, b e c a u s e Mg ++ a n d p a r t i c u l a r l y U T P stabilize t h e 0.6
0.4
~0.2
E v--
D8 GMPc Fig. 2. Effects of DBcGMP on the 2 forms of nucleoside phosphotransferase of chick embryo retina. Peak I (tube numbers 11-15)
and peak II (tube numbers 19-23) were collected separately and 200 ill of each peak were utilized for the incubation sample. The activity was measured by using as phosphate donor UMP ( 9169 for peak I and AMP (D--O) for peak II. Data are the means ~ SE of 6 separate experiments.
EXPERIENTIA 33/8
u n s t a b l e f o r m of nucleoside p h o s p h o t r a n s f e r a s e 6. T h e figure shows 2 p e a k s of a c t i v i t y : t h e first corresponds, as p r e v i o u s l y d e m o n s t r a t e d 6, t o t h e u n s t a b l e n u c l e o s i d e p h o s p h o t r a n s f e r a s e a n d i t prefers U M P as p h o s p h a t e d o n o r , while t h e s e c o n d i s r e p r e s e n t e d b y a s t a b l e f o r m w h i c h e m p l o y e s p r e f e r e n t i a l l y A M P as s u b s t r a t e . As s h o w n in figure 2, D B c G M P m a r k e d l y i n h i b i t s t h e n u c l e o s i d e p h o s p h o t r a n s f e r a s e of p e a k I, w h i l e a n y s i g n i f i c a t i v e effect was n o t o b s e r v e d for t h e a c t i v i t y of p e a k I I . P r e v i o u s l y ~ we h a v e h y p o t h e s i z e d t h a t t h e n u c l e o s i d e p h o s p h o t r a n s f e r a s e is p r e s e n t in t h e c h i c k e m b r y o r e t i n a a t l e a s t i n 2 d i f f e r e n t forms, w h i c h could b e a n expression of t h e s a m e e n z y m e a t d i f f e r e n t aggreg a t i o n states. I t is possible t h a t D B c G M P f a c i l i t a t e s t h e c o n v e r s i o n of t h e f o r m w i t h h i g h e r m . w t i n t o a d i s a g g r e g a t e d state. T h i s s t a t e c o u l d b e r e p r e s e n t e d b y t h e s t a b l e nucleoside p h o s p h o t r a n s f e r a s e , a n e n z y m a t i c a c t i v i t y w h i c h is able to utilize as p h o s p h a t e d o n o r s also t h e a d e n i n e nucleotides. T h e s e c o n s i d e r a t i o n s c o u l d e x p l a i n w h y t h e D B c G M P causes a n i n c r e m e n t of t h e t h y m i d i n e p h o s p h o r y l a t i n g r a t e w h e n t h e r e a c t i o n is m e a s u r e d , b y u s i n g A M P as p h o s p h a t e d o n o r , in t h e 105,000 g s u p e r n a t a n t . F u r t h e r m o r e , b e c a u s e i t seems t h a t t h e nucleoside p h o s p h o t r a n s f e r a s e t a k e s p a r t in t h e c o n t r o l of t h e e n d o g e n o u s pools of nucleosides a n d n u c l e o t i d e s , t h e effects of D B c G M P o n t h i s a c t i v i t y could i n d i c a t e t h e p a r t i c i p a t i o n of t h i s c o m p o u n d i n t h e r e g u l a t i o n of nucleotide metabolism.
Absorption andlbiotransformation of L( + ) vtt _ a r t a r i c , ~ a c i d in rats L. F. C h a s s e a u d , W . H. D o w n a n d D. K i r k p a t r i c k
Huntingdon Research Centre, Department o/ Metabolism and Pharmacokinetics, Huntingdon PE18 6ES (England), 18 January 1977 Summary. Oral or p a r e n t e r a l doses of m o n o s o d i u m l * C - L ( + ) - t a r t r a t e (400 mg/kg) are r a p i d l y e x c r e t e d b y r a t s a n d a p r o p o r t i o n c o m p l e t e l y m e t a b o l i z e d t o CO v T h e oral dose was w e l l - a b s o r b e d . T a r t a r i c acid a n d its s a l t s are used in m e d i c i n e a n d in t h e food i n d u s t r y . I n h u m a n s , t h e acid is t h o u g h t t o b e p o o r l y a b s o r b e d 1 a n d w h e n g i v e n orally, t o b e m e t a b o lized b y t h e g u t flora 2, 3, since i t is r e a d i l y m e t a b o l i z e d b y m i c r o o r g a n i s m s s u c h as P s e u d o m o n a s p u t i d a ~ a n d P e n i 90 J
o 70
.~ 5o
g o
o :
E
r
24 Time
48h
Cumulative excretion of radioactivity in the urine ( I , 0) and expired air (D, C) of rats dosed orally or i.v. respectively with monosodium 14C-L(+)-tartrate (400 mg/kg).
cillium charlesii 5, w h i c h c o n v e r t i t t o g l y c e r a t e a n d CO v S t u d i e s in dogs a n d r a b b i t s h a v e s h o w n t h a t oral doses of t a r t a r i c acid were e x c r e t e d in t h e u r i n e as u n c h a n g e d c o m p o u n d , t h e p r o p o r t i o n of w h i c h d e c r e a s e d w i t h i n c r e a s i n g doses 6. M u c h of t h e t a r t a r i c acid used is o b t a i n e d as a b y p r o d u c t of w i n e m a n u f a c t u r e a n d is therefore the naturally-occurring L(+) formL Thus the a b s o r p t i o n a n d b i o t r a n s f o r m a t i o n of t a r t a r i c acid h a s b e e n e v a l u a t e d u s i n g t h e 14C-L(+) form. Materials. (1,4-1*C)-DL-Tartaric acid c~f specific a c t i v i t y 2 - 1 0 m C i / m m o l e s was o b t a i n e d f r o m T h e R a d i o c h e m i c a l Centre, A m e r s h a m , E n g l a n d , a n d w a s resolved i n t o t h e L( + ) - i s o m e r s. T h e r e s u l t i n g m o n o s o d i u m 14C-L(+)1 L. S. Goodman and A. Gilman, The Pharmacological Basis of Therapeutics, 4th ed. Macmillan, London 1970. 2 F.P. Underhill, F. I. Peterman, T. C. Jaleski and C. S. Leonard, J. Pharmac. exp. Ther. 43, 381 (1931). 3 P. Finkle, J. biol. Chem. 700, 349 (1933). 4 L.D. Kohn and W. B. Jakoby, J. biol. Chem. 243, 2465 (1968). 5 K.P. Klatt, P. D. Rick and J. E. Gander, Archs Biochem. Biophys. 134, 335 (1969). 6 F.P. Underhill, C. S. Leonard, E. G. Gross and T. C. Jaleski, J, Pharmae. exp. Ther. 43, 359 (1931). 7 M. H, M. Arnold, Acidulants for Food and Beverages. Food Trade Press Ltd., London 1975. 8 J. Read and W. G. Reid, J. Soc. chem. Ind. 47, 8 (1928).
