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COMMCNICATIONS TO THE EDITOR
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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
Sir: Previous work from this laboratory demonstrated the incorporation of thymidine into DNXI catalyzed by the high-speed supernatant fraction from regenerating rat liver homogenates.? T h e incorporation of thymidine was stimulated b y a mixture of D - U I P , D G M P , and D C h l P . Enzymes catalyzing formation of higher phosphates of deoxynucleotides have been observed by several investigators." T o elucidate the deoxynucleotide effect on thymidine incorporation, supernatant fraction was examined for ability to phosphorylate D B X P , D G M P and DCMP. n'hen fortified with 3 mA1. adenosine triphosphate, 5mM. ,\Ig++, and G mXI. %phosphoglycerate regenerating r a t liver supernatant fraction phosphorylated 3 n M . DXMP, D G l l P , or D C M P a t rates of approximately 1 ,urnole hr. ,'mg. protein, forming DhTP, DGTP, or D C T P (respectively) as principal product. H3Thymidine also was phosphorylated to H3-TTPb y regenerating liver supernatant fraction, b u t a t a much lower rate (approximately 0.03 pmole/hr. mg. protein) and this rate was lower by a factor of a t least 10 in normal supernatant fraction.' Deoxynucleoside triphosphates, synthesized b y crude supernatant fraction as outlined above, isolated b y ion-exchange,j and purified where necessary b y chromatography on cellulose, 3c were then incubated with a polymerizing enzyme purified about ten fold from regenerating rat liver.6 The experiment presented in Table I demonstrates a requirement for M g + + , DKA1,?and the presence of all four deoxynucleoside triphosphates for maximal activity of purified enzyme in incorporating H"-TTP into DNA. Omission of a single deoxynucleoside triphosreduced incorporation of H 3 - T T P b y 66-in earlier experiment with slightly less active enzyme and a different H3-TTP preparation gave the same general result with a less definite requirement for Illg++. These findings are offered as explanation for the deoxynucleotide stimulation ( 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.
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 TABLE I REQCIREMENTS FOR
H 3 - T H Y M 1 ~ I s ET R I P H O S P € I A T E
PORATIOS
I TCOR-
r w o DS.l
Complete system contained in 0.25 ml.: DX.1, 260 p g ; enzyme, 380 fig. protein; H3-TTP, 3.8 milmoles (22,3!)0 c.p.m.) ; D G T P , 8 mhmoles; DATP, 10 m ~ m o l e s ;D C T P , 9 mpmoles; ?"lg'+, 2 pmoles; and T R I S : H C I , PH 8.0, 10 pmoles. After 60 minutes incubation a t 37" 1.0 nil. colti lfl?;, TCA was added. TCX insoluble material was washed two times with 0.5 ml. 5% TCA, hydrolyzed 20 minutes a t 803 in 0.2 ml. 0.2 M NaOH, DXA and protein reprecipitated with dilute HC1 and 5 5 T C 1 , and precipitate Trashed with 95', L8 EtOH. Insoluble material dissolved in 0.5 ml. Si) acid and 0.1 nil. plated. (This rapid method uf n tion gives quantitative recovery.) Reaction mixture
Total radioactivityfL in L ) S A . c p.m
Compiete 1,100 Omit D G T P 330 Omit D.I\TP 1!)0 Omit D C T P 3Oi) Omit DGTP, D.ITP, and D C T P 17,5 Omit h l g + 2.i Omit D S A b 35 45 Complete, 60 minutes a t 0" Radioactivity assayed in windowless flow counters. Since the same amount of material was plated from each reaction mixture no correction has been made for self-absorption. 260 g. DXA added after incubation.
that the reaction mechanism described for the E . coli enzymegwill also hold for rat liver enzyme. (8) Inhibition was 50% a t 10 m.lf and 100% a t 100 m.lf pyrrmhosphate, tested with the crude enzyme from rereneratinp liver. Details t o be published soon. (9) M . Bessman, I . R . Lehman, E. S. Simms and A . Kornberp-, Federolion Proc., 16, 153 (1957) 110) Postdoctoral Fellon of t h e National Cancer Institute, USPHS. 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 sincere appreciation t o Prof. Potter f o r interest and helpful suggeqtions dur;ng t h e course oi this work. I a m indebted t o Miss Geraldine DeGrazia for doing the many spectrophotometric analyses reriuired 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 for isolating DA-A. 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 ) .
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