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5′-Deoxycytidylic acid deaminase enzymic production of 5′-deoxyuridylic acid
VOL. 9.9 (z958)
PRELIMINARY NOTES
459
5"-Deoxycytidylic acid deaminase Enzymic production of 5;-deoxyuridylic acid" D u r i n g a s t u d y of enzymic t r a n s p h o s p h o r y l a t i o n reactions between 5'-deoxynucleotides and a d e n o s i n e t r i p h o s p h a t e 1 it h a s been found t h a t dCMP *~ is deaminated to d U M P in the presence of h o m o g e n a t e s and also in the presence of e x t r a c t s of acetone p o w d e r s f r o m unfertilized eggs and f r o m e m b r y o s (2- 4 cells, blastulae, gastrulae) of the Sea Urchin P a r a c e n t r o t u s l i v i d u s 2. A spectrop h o t o m e t r i c assay for following t h e d e a m i n a t i o n has been worked out. This assay is based on the spectral differences in the ultraviolet of cytosine and uracil compounds. Table I gives the spectral variations d u r i n g the reaction. T w o isosbestic points are found at 232 m/z and at 267 m t c U n d e r the conditions of the assay of Table I, the theoretical zlA2s0 and zlA2~0 a r e - - 0 . 3 4 ° and + o . 1 7 ° respectively for complete deamination, a s s u m i n g t h a t the d U M P had the same molar extinction coefficient as 5'-uridylic acid. I n the e x p e r i m e n t described in Table I parallel determinations of inorganic p h o s p h a t e 3 and of reducing sugars4 were made on suitable aliquots of the incubation mixture. B o t h the inorganic p h o s p h a t e and the reducing sugars remained unchanged during the whole incubation period. N H s d e t e r m i n a t i o n s 5 showed exact stoichiometry to the a m o u n t of dCMP deaminated. The d e a m i n a t i o n reaction has an o p t i m u m p H a r o u n d 7.2. TABLE I Acetone-powder e x t r a c t containing 15 mg proteinS; dCMP, 6 t~moles; Tris, 18o imloles. Final vol. 3 ml; pH, 7.2. I n c u b a t i o n at 3o°. At the indicated times, o.i ml was t a k e n out and o.t ml lO% HC10~ was added. After high-speed centrifugation, o.i ml of the clear colorless s u p e r n a t a n t was diluted with 2. 4 ml o.oi N HCI. Absorbance at Time (min)
232m ~
25o m#
a67 mlt
280 mlt
o IO 20 40 60 80
0.420 0.430 o.42o o.42o o.4Io 0.420
0.440 0.500 o.55o o.590 0.620 0.620
0.900 0,900 0.89o o.90o 0.900 0.900
0.960 0.840 0.740 o.66o o.61o o.61o
A
=
O
LJ =
+O.180
a/j =
0
d
=
--'0.350
The d U M P formed was isolated in three different w a y s : p a p e r c h r o m a t o g r a p h y 2, ionophoresis on p a p e r 2 a n d ion-exchange c h r o m a t o g r a p h y . I n a s e m i m a c r o : p r e p a r a t i o n e x p e r i m e n t i oo/~moles dCMP were incubated with the enzymic p r e p a r a t i o n u n d e r the conditions of the assay of Table I. The i n c u b a t i o n was s t o p p e d w h e n 8o /*moles of the nucleotide had been deaminated. F r o m t h e acid-soluble c o m p o n e n t s of this incubation m i x t u r e 7° tlmoles d U M P were separated b y ionexchange c h r o m a t o g r a p h y on D o w e x - I x 8, lOO-2OO mesh, in the f o r m a t e form. After charcoal c o n c e n t r a t i o n of the c o l u m n eluate containing the uracil nucleotide, and b a r i u m - e t h a n o l precipitation, 5 ° Izmoles d U M P were obtained. Table I I gives some s p e c t r o p h o t o m e t r i c c o n s t a n t s of the nucleotide. The molar extinction coefficient at 26o mt, and p H 2 was 9,8oo ± 4oo. Deoxyribose analysis s and total p h o s p h a t e analysis gave a ratio deoxyribose to p h o s p h a t e of I to I. No inorganic p h o s p h a t e and no ribose 7 were present. After hydrolysis with HCIO 4 at Ioo °, uracil was split off the nucleotide. The uracil so obtained was c o m p a r e d b y p a p e r c h r o m a t o g r a p h y in three different solvents with a sample of p u r e uracil. The t w o c o m p o u n d s b e h a v e d in exactly the same way. Using a 5'-nucleotidase from viper venom, all the p h o s p h a t e was hydrolyzed from the d UMP. This fact d e m o n s t r a t e s t h a t the nucleotide is a 5'-nucleotide. The nucleoside, set free from the d U M P by the viper-venom 5'-nucleotidase, was c o m p a r e d with a sample of deoxyuridinc by paper c h r o m a t o g r a p h y in 4 solvents. This nucleoside was indistinguishable from the sample of dcoxvuridinc. * .\ided by a g r a n t from the National Cancer I n s t i t u t e No. C-34ol (CI) SS% of the National I n s t i t u t e s of Health, l'nited States Public Health Service. ** Ahbreviations: 5'-deoxycytidylic acid, d('MP; 5'-deoxyuridylic acid, d U M P ; t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e , Tris.
460
VOL. 29 (1958)
PRELIMINARY NOTES T A B L E II
pH
2max ml*
~min mlz
2 7 12
260 260 26O
230 230 240
e230/**e0 0.29 0.35 0.94
**~0/e,~,0 0.78 0.80 0.83
e~0/e2,0
*:90/e~0
O.41 0.43 0.32
O.IO 0.12 O.O5
D e o x y c y t i d i n e , 5'-cytidylic acid, 2"- a n d 3'-cytidylic acid, cytidine, cytosine, 5 ' - d e o x y a d e n y l i c acid, 5 ' - d e o x y g u a n y l i c acid a n d 5'-adenylic acid are n o t d e a m i n a t e d b y t h e e n z y m i c preparation. T h e d i s t r i b u t i o n a n d t h e purification of t h i s e n z y m e are being i n v e s t i g a t e d .
Stazione Zoologica, Naples and the Institute o / H u m a n Physiology, University o[ Naples (Italy)
EDUARDO SCARANO
1 E. SCARANO AND A. OREN60, Boll. soc. ital. al. biol. sper., 33 (1957) 17292 E. SCARANO, Boll. soc. ital. al. biol. sper., in t h e press. 3 G. GOMORI, J. Lab. Clin. Med., 27 (1942) 955. 4 j . T. PARK AND M. J. JOHNSON, J. Biol. Chem., 181 (1949) 149. 5 E. J, CONWAY,Microdiffusion Analysis and Volumetric Error, C r o s b y Lockwood, L o n d o n , 1956. 8 S. BRODY, Acta Chem. Scand., 7 (1953) 5o2. 7 W. MEIYBAUM, Z. physiol. Chem., 258 (1939) 117. 8 0 . H. LowRY, N. J. ~RosEBROUGH, A. L. FARR AND 1:{. J. RANDALL, J . Biol. Chem., 193 (1951) 265. Received April 29th, I958
Dependence of amino acid binding to soluble ribonucleic acid on cytidine triphosphate* I n p r e v i o u s e x p e r i m e n t s 1 it w a s f o u n d t h a t labeled a m i n o acids are b o u n d to t h e soluble ribonucleic acid (RNA) of t h e c y t o p l a s m of r a t liver a n d of E h r l i c h m o u s e - a s e i t e s t u m o r , as a n interm e d i a t e step in t h e over-all process of i n c o r p o r a t i o n of a m i n o acids into protein. T h e f o r m a t i o n of a m i n o a c i d - n l ~ l e o t i d e i n t e r m e d i a t e s h a s been described r e c e n t l y b y a n u m b e r of o t h e r investigators2-L Metabolic s t u d i e s on t h i s R N A h a v e s h o w n t h a t c y t o s i n e n u c l e o t i d e s a n d a t e r m i n a l a d e n i n e n u c l e o t i d e are a d d e d in s e q u e n c e to t h e free 3' (or a d j a c e n t 2 ' ) - h y d r o x y l g r o u p of t h e soluble R N A in p h o s p h o d i e s t e r linkage s-l°. T h e nucleoside t r i p h o s p h a t e s s e r v e as p r e c u r s o r s in t h i s reaction. T h e correlations d e s c r i b e d h e r e i n b e t w e e n t h e s e t w o r e a c t i o n s s u g g e s t t h a t t h e s e specific R N A - e n d u n i t s m a y s e r v e as f u n c t i o n a l g r o u p i n g s in t h e a m i n o acid b i n d i n g to t h e R N A . Ascites cells were lysed, h o m o g e n i z e d , a n d t h e centrifugal s u p e r n a t a n t fraction (lO5,OOO × g for 2 h) w a s p r e p a r e d ix. T h e s u p e r n a t a n t w a s b r o u g h t to p H 5.2 b y a d d i n g i M acetic acid. T h i s p r e c i p i t a t e s a n e n z y m e f r a c t i o n c o n t a i n i n g a l m o s t all of t h e R N A p r e s e n t in t h e s u p e r n a t a n t fluid. T h e p r e c i p i t a t e w a s dissolved in buffered m e d i u m A 11 a n d w a s r e p r e e i p i t a t e d a n d redissolved. T h i s fraction w a s t h e n i n c u b a t e d for 20 rain a t 37 °, p r e c i p i t a t e d a t p H 5.2, a n d dissolved in buffered m e d i u m A, a n d t h e s e t h r e e s t e p s were r e p e a t e d . T h e p r e c i p i t a t i o n s a t p H 5.2 free t h e fraction of t h e b u l k of a m i n o acids a n d free nucleotides, a n d t h e i n c u b a t i o n s r e s u l t in a n e n z y m i c loss of t h e a d e n i n e a n d c y t o s i n e nucleotide e n d u n i t s of t h e R N A c o n t a i n e d herein. T h i s p r e i n c u b a t e d e n z y m e - R N A fraction h a s a g r e a t l y r e d u c e d c a p a c i t y to i n c o r p o r a t e a t e r m i n a l a d e n i n e n u c l e o t i d e into t h e R N A in t h e p r e s e n c e of 14C-labeled A T P alone. The addition of c y t i d i n e t r i p h o s p h a t e (CTP) to t h i s s y s t e m leads to a m a r k e d increase in t h e i n c o r p o r a t i o n of t h e a d e n i n e nucleotide. I n parallel e x p e r i m e n t s it h a s b e e n s h o w n , w i t h t h e u s e of 14C-labeled CTP, t h a t t h e c y t o s i n e nucleotide is i n c o r p o r a t e d into R N A a n d t h e r e b y p r o v i d e s f u r t h e r sites for t h e a d d i t i o n of t h e t e r m i n a l a d e n i n e nucleotide. * T h i s w o r k was s u p p o r t e d in p a r t b y g r a n t s f r o m t h e A m e r i c a n Cancer Society, t h e U. S. A t o m i c E n e r g y C o m m i s s i o n , a n d t h e U. S. Public H e a l t h Service. T h i s is p u b l i c a t i o n No. 919 ot t h e H a r v a r d Cancer Commission.
PRELIMINARY NOTES
459
5"-Deoxycytidylic acid deaminase Enzymic production of 5;-deoxyuridylic acid" D u r i n g a s t u d y of enzymic t r a n s p h o s p h o r y l a t i o n reactions between 5'-deoxynucleotides and a d e n o s i n e t r i p h o s p h a t e 1 it h a s been found t h a t dCMP *~ is deaminated to d U M P in the presence of h o m o g e n a t e s and also in the presence of e x t r a c t s of acetone p o w d e r s f r o m unfertilized eggs and f r o m e m b r y o s (2- 4 cells, blastulae, gastrulae) of the Sea Urchin P a r a c e n t r o t u s l i v i d u s 2. A spectrop h o t o m e t r i c assay for following t h e d e a m i n a t i o n has been worked out. This assay is based on the spectral differences in the ultraviolet of cytosine and uracil compounds. Table I gives the spectral variations d u r i n g the reaction. T w o isosbestic points are found at 232 m/z and at 267 m t c U n d e r the conditions of the assay of Table I, the theoretical zlA2s0 and zlA2~0 a r e - - 0 . 3 4 ° and + o . 1 7 ° respectively for complete deamination, a s s u m i n g t h a t the d U M P had the same molar extinction coefficient as 5'-uridylic acid. I n the e x p e r i m e n t described in Table I parallel determinations of inorganic p h o s p h a t e 3 and of reducing sugars4 were made on suitable aliquots of the incubation mixture. B o t h the inorganic p h o s p h a t e and the reducing sugars remained unchanged during the whole incubation period. N H s d e t e r m i n a t i o n s 5 showed exact stoichiometry to the a m o u n t of dCMP deaminated. The d e a m i n a t i o n reaction has an o p t i m u m p H a r o u n d 7.2. TABLE I Acetone-powder e x t r a c t containing 15 mg proteinS; dCMP, 6 t~moles; Tris, 18o imloles. Final vol. 3 ml; pH, 7.2. I n c u b a t i o n at 3o°. At the indicated times, o.i ml was t a k e n out and o.t ml lO% HC10~ was added. After high-speed centrifugation, o.i ml of the clear colorless s u p e r n a t a n t was diluted with 2. 4 ml o.oi N HCI. Absorbance at Time (min)
232m ~
25o m#
a67 mlt
280 mlt
o IO 20 40 60 80
0.420 0.430 o.42o o.42o o.4Io 0.420
0.440 0.500 o.55o o.590 0.620 0.620
0.900 0,900 0.89o o.90o 0.900 0.900
0.960 0.840 0.740 o.66o o.61o o.61o
A
=
O
LJ =
+O.180
a/j =
0
d
=
--'0.350
The d U M P formed was isolated in three different w a y s : p a p e r c h r o m a t o g r a p h y 2, ionophoresis on p a p e r 2 a n d ion-exchange c h r o m a t o g r a p h y . I n a s e m i m a c r o : p r e p a r a t i o n e x p e r i m e n t i oo/~moles dCMP were incubated with the enzymic p r e p a r a t i o n u n d e r the conditions of the assay of Table I. The i n c u b a t i o n was s t o p p e d w h e n 8o /*moles of the nucleotide had been deaminated. F r o m t h e acid-soluble c o m p o n e n t s of this incubation m i x t u r e 7° tlmoles d U M P were separated b y ionexchange c h r o m a t o g r a p h y on D o w e x - I x 8, lOO-2OO mesh, in the f o r m a t e form. After charcoal c o n c e n t r a t i o n of the c o l u m n eluate containing the uracil nucleotide, and b a r i u m - e t h a n o l precipitation, 5 ° Izmoles d U M P were obtained. Table I I gives some s p e c t r o p h o t o m e t r i c c o n s t a n t s of the nucleotide. The molar extinction coefficient at 26o mt, and p H 2 was 9,8oo ± 4oo. Deoxyribose analysis s and total p h o s p h a t e analysis gave a ratio deoxyribose to p h o s p h a t e of I to I. No inorganic p h o s p h a t e and no ribose 7 were present. After hydrolysis with HCIO 4 at Ioo °, uracil was split off the nucleotide. The uracil so obtained was c o m p a r e d b y p a p e r c h r o m a t o g r a p h y in three different solvents with a sample of p u r e uracil. The t w o c o m p o u n d s b e h a v e d in exactly the same way. Using a 5'-nucleotidase from viper venom, all the p h o s p h a t e was hydrolyzed from the d UMP. This fact d e m o n s t r a t e s t h a t the nucleotide is a 5'-nucleotide. The nucleoside, set free from the d U M P by the viper-venom 5'-nucleotidase, was c o m p a r e d with a sample of deoxyuridinc by paper c h r o m a t o g r a p h y in 4 solvents. This nucleoside was indistinguishable from the sample of dcoxvuridinc. * .\ided by a g r a n t from the National Cancer I n s t i t u t e No. C-34ol (CI) SS% of the National I n s t i t u t e s of Health, l'nited States Public Health Service. ** Ahbreviations: 5'-deoxycytidylic acid, d('MP; 5'-deoxyuridylic acid, d U M P ; t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e , Tris.
460
VOL. 29 (1958)
PRELIMINARY NOTES T A B L E II
pH
2max ml*
~min mlz
2 7 12
260 260 26O
230 230 240
e230/**e0 0.29 0.35 0.94
**~0/e,~,0 0.78 0.80 0.83
e~0/e2,0
*:90/e~0
O.41 0.43 0.32
O.IO 0.12 O.O5
D e o x y c y t i d i n e , 5'-cytidylic acid, 2"- a n d 3'-cytidylic acid, cytidine, cytosine, 5 ' - d e o x y a d e n y l i c acid, 5 ' - d e o x y g u a n y l i c acid a n d 5'-adenylic acid are n o t d e a m i n a t e d b y t h e e n z y m i c preparation. T h e d i s t r i b u t i o n a n d t h e purification of t h i s e n z y m e are being i n v e s t i g a t e d .
Stazione Zoologica, Naples and the Institute o / H u m a n Physiology, University o[ Naples (Italy)
EDUARDO SCARANO
1 E. SCARANO AND A. OREN60, Boll. soc. ital. al. biol. sper., 33 (1957) 17292 E. SCARANO, Boll. soc. ital. al. biol. sper., in t h e press. 3 G. GOMORI, J. Lab. Clin. Med., 27 (1942) 955. 4 j . T. PARK AND M. J. JOHNSON, J. Biol. Chem., 181 (1949) 149. 5 E. J, CONWAY,Microdiffusion Analysis and Volumetric Error, C r o s b y Lockwood, L o n d o n , 1956. 8 S. BRODY, Acta Chem. Scand., 7 (1953) 5o2. 7 W. MEIYBAUM, Z. physiol. Chem., 258 (1939) 117. 8 0 . H. LowRY, N. J. ~RosEBROUGH, A. L. FARR AND 1:{. J. RANDALL, J . Biol. Chem., 193 (1951) 265. Received April 29th, I958
Dependence of amino acid binding to soluble ribonucleic acid on cytidine triphosphate* I n p r e v i o u s e x p e r i m e n t s 1 it w a s f o u n d t h a t labeled a m i n o acids are b o u n d to t h e soluble ribonucleic acid (RNA) of t h e c y t o p l a s m of r a t liver a n d of E h r l i c h m o u s e - a s e i t e s t u m o r , as a n interm e d i a t e step in t h e over-all process of i n c o r p o r a t i o n of a m i n o acids into protein. T h e f o r m a t i o n of a m i n o a c i d - n l ~ l e o t i d e i n t e r m e d i a t e s h a s been described r e c e n t l y b y a n u m b e r of o t h e r investigators2-L Metabolic s t u d i e s on t h i s R N A h a v e s h o w n t h a t c y t o s i n e n u c l e o t i d e s a n d a t e r m i n a l a d e n i n e n u c l e o t i d e are a d d e d in s e q u e n c e to t h e free 3' (or a d j a c e n t 2 ' ) - h y d r o x y l g r o u p of t h e soluble R N A in p h o s p h o d i e s t e r linkage s-l°. T h e nucleoside t r i p h o s p h a t e s s e r v e as p r e c u r s o r s in t h i s reaction. T h e correlations d e s c r i b e d h e r e i n b e t w e e n t h e s e t w o r e a c t i o n s s u g g e s t t h a t t h e s e specific R N A - e n d u n i t s m a y s e r v e as f u n c t i o n a l g r o u p i n g s in t h e a m i n o acid b i n d i n g to t h e R N A . Ascites cells were lysed, h o m o g e n i z e d , a n d t h e centrifugal s u p e r n a t a n t fraction (lO5,OOO × g for 2 h) w a s p r e p a r e d ix. T h e s u p e r n a t a n t w a s b r o u g h t to p H 5.2 b y a d d i n g i M acetic acid. T h i s p r e c i p i t a t e s a n e n z y m e f r a c t i o n c o n t a i n i n g a l m o s t all of t h e R N A p r e s e n t in t h e s u p e r n a t a n t fluid. T h e p r e c i p i t a t e w a s dissolved in buffered m e d i u m A 11 a n d w a s r e p r e e i p i t a t e d a n d redissolved. T h i s fraction w a s t h e n i n c u b a t e d for 20 rain a t 37 °, p r e c i p i t a t e d a t p H 5.2, a n d dissolved in buffered m e d i u m A, a n d t h e s e t h r e e s t e p s were r e p e a t e d . T h e p r e c i p i t a t i o n s a t p H 5.2 free t h e fraction of t h e b u l k of a m i n o acids a n d free nucleotides, a n d t h e i n c u b a t i o n s r e s u l t in a n e n z y m i c loss of t h e a d e n i n e a n d c y t o s i n e nucleotide e n d u n i t s of t h e R N A c o n t a i n e d herein. T h i s p r e i n c u b a t e d e n z y m e - R N A fraction h a s a g r e a t l y r e d u c e d c a p a c i t y to i n c o r p o r a t e a t e r m i n a l a d e n i n e n u c l e o t i d e into t h e R N A in t h e p r e s e n c e of 14C-labeled A T P alone. The addition of c y t i d i n e t r i p h o s p h a t e (CTP) to t h i s s y s t e m leads to a m a r k e d increase in t h e i n c o r p o r a t i o n of t h e a d e n i n e nucleotide. I n parallel e x p e r i m e n t s it h a s b e e n s h o w n , w i t h t h e u s e of 14C-labeled CTP, t h a t t h e c y t o s i n e nucleotide is i n c o r p o r a t e d into R N A a n d t h e r e b y p r o v i d e s f u r t h e r sites for t h e a d d i t i o n of t h e t e r m i n a l a d e n i n e nucleotide. * T h i s w o r k was s u p p o r t e d in p a r t b y g r a n t s f r o m t h e A m e r i c a n Cancer Society, t h e U. S. A t o m i c E n e r g y C o m m i s s i o n , a n d t h e U. S. Public H e a l t h Service. T h i s is p u b l i c a t i o n No. 919 ot t h e H a r v a r d Cancer Commission.