1766
COMMCNICATIONS TO THE EDITOR
Yol.
so
C O M M W N I C A T T T N S'roT H E E D I T O R INCORPORATION O F THYMIDINE TRIPHOSPHATE INTO DEOXYRIBONUCLEIC ACID BY A PURIFIED MAMMALIAN ENZYME
reported in crude preparations. Requirement for DNA, Mg++, and presence of all four deoxynucleoside triphosphates, together with the fact that this enzyme is inhibited by pyrophosphate,x suggest
Sir: Previous work from this laboratory demonstrated the incorporation of thymidine into DNXI catTABLE I alyzed by the high-speed supernatant fraction from REQCIREMENTS FOR H 3 - T H Y M 1 ~ I s ET R I P H O S P € I A T E I T C O R regenerating rat liver homogenates.? The incorPORATIOS r w o D S . l poration of thymidine was stimulated by a mixture Complete system contained in 0.25 ml.: DX.1, 260 p g ; of D - U I P , DGMP, and DChlP. Enzymes cat- enzyme, 380 fig. protein; H3-TTP, 3.8 milmoles (22,3!)0 alyzing formation of higher phosphates of deoxy- c.p.m.) ; D G T P , 8 mhmoles; DATP, 10 m ~ m o l e s ;D C T P , nucleotides have been observed by several investi- 9 mpmoles; ?"lg'+, 2 pmoles; and T R I S : H C I , PH 8.0, 10 After 60 minutes incubation a t 37" 1.0 nil. colti lfl?;, gators." T o elucidate the deoxynucleotide effect pmoles. TCA was added. TCX insoluble material was washed two on thymidine incorporation, supernatant fraction times with 0.5 ml. 5% TCA, hydrolyzed 20 minutes a t 803 was examined for ability to phosphorylate D B X P , in 0.2 ml. 0.2 M NaOH, DXA and protein reprecipitated with D G M P and DCMP. n'hen fortified with 3 mA1. dilute HC1 and 5 5 T C 1 , and precipitate Trashed with 95', L8 EtOH. Insoluble material dissolved in 0.5 ml. Si) adenosine triphosphate, 5mM. ,\Ig++, and G mXI. acid and 0.1 nil. plated. (This rapid method uf n %phosphoglycerate regenerating r a t liver superna- tion gives quantitative recovery.) Total radioactivityfL tant fraction phosphorylated 3 n M . DXMP, in L ) S A . c p.m Reaction mixture D G l l P , or D C M P a t rates of approximately 1 Compiete 1,100 ,urnole hr. ,'mg. protein, forming DhTP, DGTP, or 330 Omit D G T P D C T P (respectively) as principal product. H3Omit D.I\TP 1!)0 Thymidine also was phosphorylated to H3-TTPby Omit D C T P 3Oi) regenerating liver supernatant fraction, b u t a t a Omit DGTP, D.ITP, and D C T P 17,5 much lower rate (approximately 0.03 pmole/hr. Omit h l g + 2.i mg. protein) and this rate was lower by a factor of Omit D S A b 35 a t least 10 in normal supernatant fraction.' 45 Complete, 60 minutes a t 0" Deoxynucleoside triphosphates, synthesized by Radioactivity assayed in windowless flow counters. crude supernatant fraction as outlined above, Since the same amount of material was plated from each isolated by ion-exchange,j and purified where reaction mixture no correction has been made for self-absorpnecessary by chromatography on cellulose,3c were tion. 260 g. DXA added after incubation. then incubated with a polymerizing enzyme purified about ten fold from regenerating rat liver.6 that the reaction mechanism described for the E . The experiment presented in Table I demonstrates coli enzymeg will also hold for rat liver enzyme. (8) Inhibition was 50% a t 10 m.lf and 100% a t 100 m.lf pyrrmhosa requirement for Mg++, DKA1,?and the presence of all four deoxynucleoside triphosphates for maxi- phate, tested with the crude enzyme from rereneratinp liver. Details t o be published soon. mal activity of purified enzyme in incorporating (9) M . Bessman, I . R . Lehman, E. S. Simms and A . Kornberp-, H"-TTP into DNA. Federolion Proc., 16, 153 (1957) 110) Postdoctoral Fellon of t h e National Cancer Institute, USPHS. Omission of a single deoxynucleoside triphosreduced incorporation of H3-TTP by 66- This investiRation was supported b y a grant (C-04G) from t h e Sational Cancer Institute, USPHS, t o Prof. Van R . Potter. I wish t o express -in earlier experiment with slightly less ac- sincere appreciation t o Prof. Potter f o r interest and helpful suggeqtive enzyme and a different H3-TTP preparation tions dur;ng t h e course oi this work. I a m indebted t o Miss Geraldine gave the same general result with a less definite re- DeGrazia for doing the many spectrophotometric analyses reriuired quirement for Illg++. These findings are offered in the isolation, characterization, and purification of t h e deoxynucleoside triphosphate^, and in checking oiit the validity of the rapid m r t h d as explanation for the deoxynucleotide stimulation for isolating DA-A. ( 1 ) These abbreviations are used: D S h , deoxyribonucleic acid; DALTP, D G M P , DCRIP, and T M P for the mono- and D A T P , D G T P , D C T P , and T T P for t h e triphosphates of deoxyadenosine. deoxyguanosine, deoxycytosine, and thymidine; T C A , trichloroacetic acid; and T R I S , tris- (hydroxymethy1)-aminomethane. (2) F . J . Bollum and V . R. Potter, Abstracts, 132nd meeting, American Chemical Society, 19-C (1057). (3) (a) H . Z . Sahle, P B Wilber, A . E. Cohen and M. R . Kane, Biochim. B i o p h y s A c f a , 13, 150 (1954); ( b ) L. I. Hecht, V . R . Potter and E . Herbert, ibi,L, 15, 134 (1954); (c) H. Klenow and E. Lichtler, i b i d . , 23, 0 (1957); (d) E. S. Canellakis and R . Mantsavinos, ibid , in press; (e) I . Leberman, A . Kornberg and E. S. Simms, J . Bid Chem , 215, 429 (1955); (i) A. Kornberg, "The Chemical Basis of Heredity," ed. 'A' D. McElroy and B. Glass, Johns Hopkins Press, Baltimore. I l d , 1957, p 5 7 0 ; (9)S . Ochoa and L. Heppel, i b i d . , p. 61.5 ( 4 ) F. J . Bollum, P . A hIorse and V. R Potter, unpublished. (i) R . B. Hurlbert, H. Schmitz, A . F. Brumm and V. R . Potter, J . B i d . Cheriz., 209,2 3 (19541. (0) F. J. Bollum, F e d o n t i c t i Proc., 17, in press (1958). (7) T h e requirement for Dh'4 was also demonstrated with crude supernatant fraction, see ref. 2.
h I C h R D L E LXEMORIAL LABORATORY UNIVERS~TP OF 1 1 7 ~ ~ AXADISOX 6. ~'ISCCJSSIX
~ F.~J. 130r,r,cnc10 ~ ~
REACTION OF CHLORODIFLUOROMETHANE W I T H LINDE MOLECULAR SIEVE 5A Sir :
Chlorodifluorornethane reacts a t room temperature with a synthetic zeolite (Linde Molecular Sieve 3X) Chlorofluoromethanes have been reported stable to temperatures in excess of 400°.2 When chlorodifluoromethane was adsorbed a t 25.0" on (1) Empirical formula C a O ~ A I ~ O ~ . ? S i O ? ~ isee ~ H also ~ O ; n. W .Breck e t al.. THISJ O U R N A L . 78. 5903 (1955). (2) A B.Trenivith and R. H. \Tatson, J . C h e m .SOL , 13(38 ( 1 0 5 7 ) .
~
~
April 3 , 1938
COMMUNICATIONS TO THE EDITOR
Linde Molecular Sieve 5A which had been evacuated at 10-5 mm. and 350" for 15 hours, a type I isotherm was obtained, in which the flat was attained a t a low relative pressure (PIPwas 0.00506 a t 25'). The corresponding adsorption was 0.20 g. sorbate per g. of sorbent. Each point required about 24 hours for equilibration. When desorption was commenced, a large loss of sorbate with decreasing pressure was observed, placing the desorption isotherm below the adsorption isotherm, rather than above it, the latter normally being expected. Considerable divergence of the two curves was seen down to a pressure of approximately 20 mm. Equilibration took longer on desorption and the residual solid was very gassy. The desorption isotherm was superposable on but not coincident with a carbon dioxide-Sieve 5.1 isotherm.3 Carbon dioxide was the only gaseous reaction product from this system. The sample was placed in an absorption train, and yielded only carbon dioxide gas, a t temperatures below 150°, where pyrolysis of any residual CHClF, would not be anticipated. Chlorodifluoromethane also was stored over outgassed Sieve for three weeks a t 25", and analyzed chromatographically; i t was found to contain 0.45; by volume of carbon dioxide which previously had not been present. The residual solid material showed predominantly carbon dioxide evolution when heated in a mass spectrometer to 1000". No halogen acid or carbon monoxide was seen. Similar experiments conducted with Linde Molecular Sieve 4A did not show the same change in the isotherms, but infrared spectra showed a strong carbonyl absorption a t 4 . 2 0 and ~ unassigned new bands a t 1 4 in~ the final gas. Both series of experiments resulted in a loss of surface area of the solid (e.g. from an initial value 566.6 sq.m. /g. to a new value of 290.0 sq. m./g. for carbon dioxide on 4 6 a t 25.0') but no gross structural changes in the sieves caused by the action of CHClFz were seen b y X-ray diffraction. Halogen acid products subsequently were found in the solid residuum : they presumably are present in a fully ionized form, since they were not seen in the mass spectrometer. The reaction appears similar to that observed by Park, et al.,? who obtained carbon dioxide on heating wet CHCIFa, the water in the present case being the constitutional water of the zeolite. However, the mechanism appears open since Barrer and Brook5 suggested dehydrofluorination to explain the attack of CHCIFz on natural zeolites, and Ayscough and Emel&& remarked on the formation of carbon dioxide and SiFl when CF3 radicals were studied in quartz. The evidence of Neilson and lVhite7 on the association of liquid CHClF2, and the peculiar geometry of the Molecular Sieve lattice' make i t impossible to rule out here an ex(3) "Molecular Sieve D a t a Sheets,'' Linde Air Products Co., TonaWanda, h- Y. (4) J. D. P a r k , et a i . , I n d . Eng. C h e m . , 39, 354 (1947). ( 5 ) R. hL. Barrer and D . 'A', Brook, TYans. F a r a d a y Sac., 49, 940 (1953). ( 6 ) P. B. Ayscough and H. J. Emelkus, J . Chem. Soc., 3381 (1954). (7) E. F. Neilson and D. White, THISJ O U R N A L , 79, 5020 (1957).
176T
change mechanism based on the polarization of CHClF2. GENERAL ELECTRIC RESEARCH LABORATORY SCHENECTADY, NEW YORK PETERC A N K O N 3, 1967 RECEIVED DECEMBER T H E SITES OF REDUCTION AND BASE-CATALYZED HYDROGEN-EXCHANGE I N N'-METHYLNICOTINAMIDE IODIDE'
Sir: Previous work on the dithionite-reduction of N'methylnicotinamide indicated that hydrogen is added to the 4 - p 0 s i t i o n . * , ~ , Our ~ , ~ nuclear magnetic resonance (n.ni.r,) data substantiate this, and further show that base-catalyzed hydrogen-exchange occurs at the 2- and 6-positions. Such exchange was noticed only a t the 2-position of diphosphopyridine nucleotide (DPN) The assignment of sites was accomplished by comparing the n.m.r. spectra of N1-methylriicotinamide iodide (I), 2,6-dideuterio-N1-methylriicotinamide iodide ( I I ) , n"-niethylnicotinamide chloride partially 4-deuteriated (111),3 and N*-benzylnicotinamide chloride (IV). I1 was formed b y dissolving 0.025 g. of I in 0.5 cc. of DzO, containing 0.0'75 g. of Na2C03. The exchange was completed by heating to 100". The spectrum of the ring protons of I consists of a single peak with relative area 1 unit at -114 cycles (benzene = zero cycles. Field strength 40 Lfc.), three peaks a t - 104, - 100 and -95 cycles with total relative area 2 units, four peaks a t - 77, -71, -69 and -62 cycles with total relative area 1 unit. I n the spectrum of 11, the peaks a t -114 and -100 cycles had disappeared, the peak a t -104 cycles was reduced in size, and the peaks centered about -70 cycles had collapsed into a 1:1 doublet. In 111, the peak a t -95 cycles was greatly reduced in size, and the peaks centered about - 70 cycles partially collapsed into a doublet. The spectrum of IV is similar to t h a t of I. Because of the electronegativity and the formal charge of N1, the 2- and 6-protons are shifted down field.7 The carboxamide group should similarly affect the 2- and 4-protons. Thus the peak a t - 114 cycles may be assigned to the 2-proton and the group centered about -70 cycles t o the 5-proton. The 2-proton could give a single line since there are no hydrogens on the adjacent atoms. The 5-proton line should be split by the 4- and 6protons into two overlapping doublets or approximately a 1: 2 :1 triplet. The remaining spectrum, centered about - 100 cycles, must therefore be assigned t o the 4- and 6-protons. Since N1-methylpyridinium iodide exchanges its 2- and 6-protons in aqueous NasCOa solution* and (1) This work was supported in part b y a grant from Restarch Corporation. ( 2 ) M. E. Pullman, A. San Pietro and S. P . Colowick, J . Bial. Chem., 206, 129 (1954). (3) G. W . Rafter and S.P. Colowick, ibid., 209, 773 (1954'1. (4) M . B. Yarmolinsky and S. P . Colowick, Biochim. e' B i o p h y s . A c t a , 20, 177 (1950). ( 5 ) D. Mauzerall and F. H. Westheimer, THIS J O U R K A L , 7 7 , 2201 (1955). (6) A. San Pietro, J . B i d . Chem., 217, 589 (1955). (7) This is consistent with t h e results of W. G . Schneiiler, H. J. Bernstein a n d J . A. Pople, Can. J . Chem., 36, 1487 (1957). ( 8 ) H. E. Dubb, h f . Saunders and J. H. K'ang, t o be published.
