368 PRELIMINARY NOTES

Formation ofdeoxyguanosine 5'-phosphate from guanosine 5'-phosphate with enzymes from chick embryos

Incorporation experiments with labeled pyrimidine ribonucleosides originally dem- onstrated that deoxyribosyl compounds could be formed through a reduction of ribosyl compounds (cf. ref. I). More recently, the formation of deoxy-UMP and deoxy- CMP from the corresponding ribonucleotides was demonstrated with soluble enzymes from chick embryo and Escherichia coli, respectively 2, 3. With the cytosine derivatives, requirements for ATP, Mg++ and TPNH were found; the dependence of the reaction on TPNH was shown by treating the bacterial extract with Dowex-2-Cl-. Further- more, it was demonstrated that deoxy-CDP is the first product of the reaction, and therefore it was considered likely that the actual reduction step occurred at the diphosphate level.

For purine nucleotides, the earlier evidence from experiments in vivo was less conclusive for a direct formation of deoxyribosyl purines from ribosyl purines 4. However, results have now been obtained with extracts of 5-day-old chick embryos (25,ooo × g supernatant fraction) which demonstrate a direct transformation of guanosine ribonucleotides to deoxyribonucleotides.

Table I describes the conditions of incubation for the optimal formation of deoxy- guanosine phosphates from [8-uC]GMP and indicates that the reaction was dependent

T A B L E I

REQUIREMENTS FOR THE FORMATION OF DEOxY-GMP

The comple te s y s t e m conta ined : 27 m/~moles [8-14C~GMP (3.0" lO 6 counts/min/t*mole), 0.25 pmo le ATP, I. 5 /*moles MgC12, 3 .3 /*moles Tris , p H 8.0, and 1. 3 m g prote in . Volume , o.15 ml. Incuba -

t ion for 3 ° rain a t 37 °. For fu r the r exp l ana t i on see tex t .

Omission or addition mgmole deoxyguanosine found

None o. 189 None* o.o29 None** 0.008 Minus A T P o.ot 5 Minus MgC12 0.076 Plus 2 tm~oles E D T A 0.008

* The t r e a t m e n t wi th snake v e n o m to dephosphory l a t e deoxyguanos ine p h o s p h a t e s was omi t t ed . ** The reac t ion m i x t u r e was boiled prior to incuba t ion .

on ATP and Mg ÷+. In each experiment the reaction was stopped by boiling, o.I M KOH (0.005 ml), 0.5 M MgC12 (o.oi ml) and o.I mg of crude Crotalus adamenteus venom were then added, and the mixture was incubated at 37 ° for 60 min to de- phosphorylate all nucleotides. After heat inactivation, carrier deoxyguanosine was added, and the total supernatant fraction after centrifugation was chromatographed on paper (Whatman 3 MM) using a borate solvent 5. This procedure separated deoxy- guanosine (RF = 0.49 ) completely from guanine (RF - o.34), guanosine (RF = o.i8) and xanthine (Rv = 0.26). From the total radioactivity in the deoxyguanosine spot the amount of deoxyguanosine compounds formed could be calculated.

Abbrev ia t ions : UMP, ur id ine 5 ' - m o n o p h o s p h a t e ; CMP and CDP, cyt id ine 5 ' -mono and d i -phospha te ; GMP and GDP, guanos ine 5 ' -mono- and d i -phospha te ; ATP, adenos ine 5"-tri- p h o s p h a t e ; T P N H , reduced t r i phosphopyr id ine nucleot ide ; Tris, t r i s ( h y d r o x y m e t h y l ) a m i n o - lne thane-HC1 buffer; E D T A , e thy lened iamine te t r ace t i c acid, sod ium salt .

Biochim. Biophys. Acta, 41 (196o) 368-369

PRELIMINARY NOTES 369

For fur ther ident i f icat ion the deoxyguanos ine isola ted b y paper c h r o m a t o g r a p h y was r ech roma tog raphed on Dowex-5o-NH4+. Of the to ta l r ad ioac t iv i ty added to the column 88 % was recovered in the deoxyguanos ine peak and, fur thermore, the ra t io of u l t r av io le t absorp t ion to r ad ioac t iv i ty remained cons tan t in each of the effluent fract ions of the peak.

Evidence for ma in ta inance of the glycosyl l inkage dur ing deoxy-GMP format ion is p resented in Table I I In this exper iment 14C-randomly labeled GMP was used as the subs t r a t e for the format ion of deoxyguanos ine der ivat ives . The specific act ivi t ies of the deoxyguanos ine and of the guanine por t ion ob ta ined af ter acid degrada t ion of deoxyguanos ine were de termined. The ra t io of the specific ac t i v i t y of the guanine to t ha t of the sugar was found to be ve ry s imilar in bo th GMP and deoxyguanosine .

TABLE II

DEGRADATION OF DEOXYGUANOSINE FORMED FROM RANDOMLY LABELED GMP

Randomly labeled [laC]GMP (o.16 /,mole, 1.6. io s counts/min/#mole) was incubated at 37 ° for 45 min with 3 /,moles ATP, 12/,moles MgCI2, 27/,moles Tris, pH 8.0, and lO. 4 mg protein in a final volume of o.98 ml. The reaction was stopped by boiling. Carrier deoxyguanosine ~2/,moles) was added and the nucleoside was isolated by chromatography after treatment of the reaction

mixture with snake venom.

Specific activity (counts~rain/l, mole) Compound

Nucleoside Guanine Sugar Guanine~sugar

Randomly labeled GMP* 4,33 ° 1,75o 2,58o o.68 Deoxyguanosine formed 825 317 508 0.62

* Determined after addition of carrier.

A close s imi la r i ty is appa ren t between the react ions leading to the format ion of deoxyr ibosy l phospha tes from bo th GMP and CMP. Bo th types of react ions show sharp op t ima in their ATP and Mg++ requi rements and are inh ib i ted b y the addi t ion of an ATP-regenera t ing system, such as creat ine phospha te and kinase. I t appears p robable t ha t d e o x y - G D P is the first deoxyr ibosyl compound formed from GMP, a l though direct evidence on this poin t has not ye t been obta ined.

This inves t iga t ion was suppor t ed b y a personal g ran t from the Jane Coffin Childs Memorial F u n d and b y a g ran t (CY 3076 C2) from the Nat iona l Ins t i tu t e s of Heal th , U.S. Publ ic Hea l th Service. The au thor wishes to t hank Professor A. D. WELCH for his in teres t in the work and for helping to make his s t ay in the Uni ted S ta tes possible.

Department of Pharmacology, Yale University School of Medicine, PETER REICHARD* New Haven, Conn. (U.S.A.)

1 p. REICHARD, Advances in Enzymology, 2I (1959) 263. 2 p. RI~ICHARD, Biochim. Biophys. Acta, 27 (1958) 434. 3 p. REICHARD AND L. RUTBERG, Biochim. Biophys. Acta, 37 (196o) 554. 4 p . M. I:{OLL, H . WEINFELD, E . CARROLL AND C-. B. BROWN, J . Biol. Chem., 220 (1956) 439. s p. PLESNER, Acta Chem. Scand., 9 (1955) 197.

Received May 2nd, 1960

* Permanent address: Department of Chemistry I, Karolinska Instituter, Stockholm (Sweden).

Biochim. Biophys. Acta, 41 (196o) 368-369