COMRIUNIC.\TIONS TO
4496
TABLE I PROI ON SL-CLEAR ~ I A C ~ E T RFSONANCE IC ADSORPI 104 CIIAKACIERISTICS 012 SOME N IT COMPOTJSDS"
Compound
N-H absorgtion a t 3Z0
Approx. tranqition t e m p . , .i of t r i p zinglet to trip- let a h let absorpsorption, tion, OC. c.p.s.h
"c-C.' \
Formamide Broad singlet 50 f loc 60 & 4 ;\cetaniide Broad singletd 175 lrie 56 f 5 X-hlethylformamide Broad singlet S o t obsd. .. t o 250 N-Methylacetamirle Broad singlet 225 f 20 A O =k 5 Succinimide >250 .. Pyrrole Very broad singlet 50 i 23 55 & 5 Methylammonium Broad tripletg H
functioned as a transformylation co-factor. Precipitation of the norite-treated extract with 50% acetone a t 0" resolved the enzymatic activity into two fractions. Both the acetone precipitate and the acetone supernate were necessary for transformylation activity. The activity of the latter was replaced by L-glutamine. Although glutamic acid and NH4+ions had a slight effect, asparagine, NH4+ or glutamic acid were ineffective. Vitamins, purines, pyrimidines, nucleotides, nucleosides, DPh'H, T P N H , D P N and T P N were incapable of replacing glutamine. The activity of the apo-enzyrne was limited by the concentration of L-glutamine in the reaction mixture. The results are shown in the table. TABLE I THEEFFECTOF GLUTAMINE ON THE FORMATION OF ARYL .IMINE BY INOSISICACIDTRAXSFORMYLASE OF PIGEON LIVER T h e reaction vessels were incubated for 30 minutes a t 37' and the reaction stopped by adding 2 ml. of 0.2 N HC1 and
V
the substitution reactions is similar to that proposed for the CH2 H2r e a ~ t i o n . ~
+
0.2 ml. of 20% trichloroacetic acid. T h e acetone precipitate of the extract represented 1 ml. of the original cell-free extract; total volume 1.0 ml. a t p H 7.5. The substrates were: S a inosinate, 0.6 p M . ; glycine, 100 pM.; leucovorin, 0.1 p M . ; CUSO.~.~HZO, 0.1 pM. ; ATP, 0.2 /AM.
( 8 ) A. Meerwein, H. Rathjen and H. Werner, Ber., 75, 1610 (1942); W. yon E . Doering, R . G. Buttery, R . G . Laughlin and N. Chandhuri, THISJ O C R N A L , 7 8 , 3 2 2 4 (1958). (9) J . Chanmugam and M . Burton, i b i d . , 78, 509 (1956). See also for references t o earlier discussions of this mechanism.
@molesaryl amine (AIC) I IIb IIIc
WHITMORE LABORATORY THEPENNSYLVANIA STATE UNIVERSITY PHILIPS. SKELL UNIVERSITY PARK,PENNSYLVANIA ROBERTC. WOODWORTH RECEIVED JULY 16, 1956
GLUTAMINE REQUIREMENT FOR T H E INOSINIC ACID TRANSFORMYLASE REACTION
Sir: Flaks and Buchanan' described the purification of pigeon liver inosinic acid transformylase which catalyzed the following reaction : I ~ h f P - 5 ' ~ glyAICR. These investigators demcine S serine onstrated that leucovorin was a required co-factor. It is the purpose of this communication to present data to show that L-glutamine is also required in this reaction. The pigeon liver extracts were prepared according to the method of Greenberg3 except that bicarbonate was omitted from the buffer and the homo' in a blender for 15 genization performed a t 4 seconds. The supernate after centrifugation a t 36,000 X g was absorbed twice with norite (20 mg./ ml.) a t 0". This extract required either A T P and C F or anhydroleucovorin per se as co-factors for the transformylase reaction. The reaction was followed by aryl amine l i b e r a t i ~ n . ~Greenbergsf has shown that C F must be activated by ATP before it
+
+
(1) J. G. Flaks and J. hl. Buchanan, THISJOURHAL, 76, 2275 (1964). (2) Abbreviations:
A T P , adenosine triphosphate; CF, leucovorin; DPN, diphosphopyridine nucleotide; T P N , triphosphopyridine nucleotide; AIC, 4-amino-5-imidazolecarboxamide; IMP-5', inosinic acid; AICR, 4-amino-5-imidazolecarboxamideribotide. (3) G. R . Greenberg, J . B i d . Chem., 190, 011 (1951). (-1) A. C. Bratton and E . K . Marshall, Jr. ibid., 128, 537 ( 1 9 x 9 ) . ( 5 ) G . R . Greenherg, THISJ O U R N A L , 76, 1458 (1931). (ti) G . K. Greenberg, F P ~P .r o r . , 13, 221 (19.54).
+ CF" + + glycine + inosin-
1 Acetone precipitate ATP ate
2
+ 0,08pM. L-glutamine + 0 . 2 p M . L-glutamine + 0 . 4 pM.L-glutamine + 0 . 8 p M . L-glutamine + 0 . 8 p M . L-asparagine + 0 . 8 p M . DL-glutamic acid 1 + 2 pM. NHaC1 1 + 0 . 8 pM.DL-glutamic acid + 2pM. NH,Cl
1 3 1 4 1 5 1 6 1 7 1
8 9
0.011 0.007 0.007 ... ,027 ... ,068 ... ... ,104 ... , . . ,077 ,104 ,054 ,011 ... ...
,011
,016 ,035 ,038 .026 ,046 ,050 ,053 ,005
... ...
...
...
. ...
...
.045 ... 5 minus C F ,028 5 minus glycine ,028 5 minus inosinate ,007 15 pM.DL-serine 14 5 ,001 15 5 minus acetone ppt. ... a Anhydroleucovorin can replace C F . Assay contained 14 pM. glycine. Acetone reprecipitated enzyme; 14 pM. glycine in reaction vessel. 10 11 12 13
5 minus A T P
+
,025 .022 ,018 ,017 .007
The following criteria were used to show that the liberation of aryl amine was due to the inosinic acid transformylase reaction : (a) serine specifically inhibited aryl amine liberation; (b) both glycine and inosinic acid were required for optimal activity (some enzyme preparations contained small amounts of these substrates) ; (c) the reaction required both A T P and CF7; (d) the liberated aryl amine could not be acetylated by acetic anhydride8; and (e) the acid-hydrolysis product of the liberated aryl amine was identified as AIC when chromatographed according to the method of Greenberg and Spilman. ( 7 ) L. Warren and J . G . Flaks, i b i d . , 16,379 (1950) ( 8 ) J . M. Ravel, R . E . Eakin and W. Shive, J . B i d C h r m . , 1 7 2 , 07 (1948).
(9) (;,
R . Oreenberg and E. I,. Spilman i h i d . , 219, 411 ( I Y 5 f i ) .
