Sept. 5 , 1957
COMMUNICATIONS TO THE EDITOR
phoretic mobilityS and by enzymatic assay with the pyruvic kinase ~ y s t e m . Both ~ ADP and ,4TP had the expected specific activity, based on that of P3*-labeled Pi used in the incubation. No ADP was formed in the absence of Pi. Inorganic pyrophosphate was not required, and in fact was rapidly hydrolyzed to P i by the liver fraction. AMP was not an intermediate in the formation of ADP from Poly A. Thus, with 50 ug. of enzyme and conditions similar to those of Table I, these quantities of P3?-labeled Pi were esterified (in pmoles) : (1) 0.035 with Poly A equivalent to 0.4 pmole of adenine, in 3 hours; ( 2 ) 0.000 with 0.5 pmole of AMP, in 3 hours; ( 3 ) 0.000 with 0.1 pmole of 9 M P and 0.02 pmole of ADP, in 11 hours.
481 1
sues suggest that nucleoside 5’-triphosph ates are the substrates for polymerization. The exact function of the phosphorolysis reaction in RNA metabolism is under investigation. NATIONAL ISSTITUTE OF ARTHRITIS R . J . HILMOE AXD METABOLIC DISEASES L. A . HEPPEL XATIOSALISSTITUTES OF HEALTH UNITEDSTATES PUBLIC HEALTHSERVICE BETHESDA, MARYLAND RECEIVEDJUXE 19, 1957
EXPONENTIAL KINETIC DEPENDENCIES I N INHIBITED AUTOXIDATIONS’
Sir:
The conventional treatment of the induction ti, in inhibited autoxidations has been based upon the steady-state assumption with reA mixture of 1.0 mg. of liver fraction, 19.3 pmoles of - ~assumpPO1--- buffer (PH 7.2, 95,000 c.p.m. of P32 per pmole), spect t o chain carrier c ~ n c e n t r a t i o n , ~an tion which accounts well for the observed direct Poly A* equivalent to 6.8 pmoles adenine and 9 pmoles of MgClz, all in 0.9 nil., was incubated at 37’ for 12 hours. proportionality between inhibitor concentration Nucleotides were adsorbed by norite in the presence of and induction p e r i ~ d . ~Furthermore, ,~ the nonperchloric acid and the washed suspension mas eluted with linear relationships between ti and inhibitmsr cona n ethanol-h7H40H-water mixture. Aliquots of the eluate centration, obtained for stabilized samples of were assayed eiizymatically for ADP, counted for P32,and chromatographed on paper for quantitative separation of cracked gasolines, have been fitted to an empirical AMP, A D P arid ATP. equation? which is isomorphous with that deduced Amount Specific activity through a steady-state treatment of a simple chainReaction product rmoles c . p . m . per omole branching reaction ~ c h e m e . ~ XMP~ 0.80 110 There is, however, a significant body of minter-1DP 0 , s5c 80,000 preted observations of accelerating increases in ti ATPb 0.45 16~,000 with increasing inhibitor concentrationsg and o€ Total esterified Pi 1.75“ decelerating decreases in t i with increasing cona Synthesized from ADP, usirig polynucleotide phoscentrations of pro-oxidant catalyst^.^^^^ These obphorylase from A . vinclandii ( l a ) , A T P and part of the A M P were formed by adenylate kinase. T h e remaining servations have not been explained in terms of From A M P was formed from Poly A by a nuclease. steady-state kinetics. absorption a t 260 nip after chromatographic separation; We wish to report the obtainment of well-defined 0.85 pmole of A D P was also found by enzymatic assay.4 From sum of A D P and 2 X .4TP. Total esterified Pi exponential dependencies of ti upon both a.ntioxidant and catalyst concentrations. The data shown determined by direct count of the washed norite suspension was 1.9 pmoles. in Table I were obtained with purified tetralin a t The liver enzyme had a pH optimum a t about 70’ using a manometric apparatus.“ These data pH 7, required Mg++ and was inhibited completely conform to the relationship t i = t i 0 exp (kX) by 0.06 M fluoride. K m was 3 X 10-3M for P i in (1) the phosphorolysis reaction. The enzyme also where X is the concentration variable The catalyzed the exchange of 0.5 ,umole of P32-labeled least-squares values for k and the standard deviaP i with ADP per hour per mg. of p r ~ t e i n . ~For tions are: set A, X = dibutylcresol concn , k = for Mg++, 2 X this reaction K m was 1.3 X 0.87 kg./millimole, s.d. & 0.01; set B, X = 10-4JI for ADP and 3 X 10-3X for Pi. An ex- cobaltous naphthenate concn., k = - 0.257 change of Pi with ATP was also noted, so far un- kg./micromole, s.d. =I= 0.003; set C, X = cobaltous explained. Experiments with other nucleoside naphthenate concn., k = - 0.100 kg./micromole, diphosphates must await removal of an interfering (1) Presented in p a r t before t h e Division of Organic Chemistry phosphatase (inactive with ADP), a t t h e 131st meeting of t h e American Chemical Society, M i a m i , Although phosphorolysis of adenylic polynu- Florida, April, 1957. T h i s work was generously supported b y a T h e Research Corporation cleotide to give ADP has been demonstrated here g r a( n2 t) from P. George, E. K. IZideal a n d A. Robertson, PYOC.R 3 y . S O L , for the first time in animal tissues, net synthesis of 8185, 288 (1940). polynucleotide could not be detected (even with (3) P. George a n d A. Kobettson, ; D i d , 309. (4) G . S. H a m m o n d , C . E . Buozer, C . E. Hamilton a u d J. K . Sen, C’*-ADP) because of contaminating nucleases. Previous results on the incorporation of CI4- T a I S J O U R N A L , 77, 31’38 (1935). ( 5 ) G. W. Kennerly a n d UT.L . Patterson, Jr., “Kinetic Sludies of labeled ATP6l7and UMPs into RNA of animal tis- Petroleum Antioxidants,” 128th meeting of t h e American C!hemical TABLE I period, PHOSPHOROLYSIS OF ADENYLIC POLPSUCLEOTIDE
+
(3) K . M a r k h a m a n d J , D. S m i t h , Biochem J . , 62, 552 (1952). (4) A. Kornberg a n d 1%’. E. Pricer, Jr., J . B i d . Chem., 193, 481 (1951). ( 5 ) T h e same activity per mg. of protein was obtained b y measurn g t h e r a t e of phosphorolysis of Poly A a n d this value was 5 % of t h a t obtained with a purified E . ccli fraction ( I b ) . ( 6 ) P. C . Zamecnik, IC.I,. Stephensun, J. I?. Scott a n d M. B. Hoagland, F e d . Pvoc., 16, 27.5 (1957). 17) I(XI
TiP
1
I
Ti'. G. LLOYD
R-P
r
AMP OH
NIT? I
1
T H E ROLE OF PURINES IN HISTIDINE BIOSYNTHESIS'
Sir: The formation of XIC.IRZ (I) in bacterial estracts incubated with ATP, an XTP-generating system, RP, and glutamine has been reported.s We have now found that I G P (JI), an essential precursor of h i ~ t i d i n eis , ~an additional product of this reaction. Furthermore, in the absence of glutamine a compound 111 accumulates which yields (1) Supported in p a r t by research griints from t h e Satiunal Science Poundation (NSF-Gl293) a n d from t h e U. S. Public Health Service (C-2864). ( 2 ) Abbreviations: XIC.lR, .i~amiu[i-l-r,-(5'~phciil,horihns4.1) 1imidazolecarboxami~e; IGP. 4 ~ ( n ~ e v j . l h ~ o - l ' . ~ ' - d i h y d r ~ ~ x y - 3 ' . p h o s phopropy1)-imidazole; X T P , adeni,sine-5'-triphosphate; AlhIP, aileiiosine-S'-monophosphate; I I I P , in~,sine-j'-monopIio.jphate; RP, ribrise-j-phosphate. i IC. r=, < ) tanig., 212, Cis7 ( I
III? ill
Sll,
