Sept. 5 , 19.i6
4407
COMMUNICATIONS TO THE EDITOR
butenes has been used to detect other carbene reaction intermediates. For example, photolyses of ethyl diazoacetate in the presence of these olefin produces carbethoxycarbene, : CHCOOC2H6, since the ethyl 2,3-dimethylcyclopropanecarboxylates are obtained in stereospecific reactions. Copperbronze catalysis produces the same results as photolysis. Conversion of C-H bonds to C-CHs bonds has been described.* The failure to observe isomerization in the products cis- and trans-2-pentene from : CH2 and cis- and trans-2-butene, respectively, suggests that the intermediate in this substitution reaction has structure (V). This intermediate for U
CH,CH=CHC,
i>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 The 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. The acetone precipitate of the extract represented 1 ml. of the original cell-free extract; total volume 1.0 ml. a t pH 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 ... .045 5 minus ATP ,025 ,038 ... 5 minus C F .022 .026 ,028 5 minus glycine ,046 ,018 ,028 ,050 ,017 5 minus inosinate ,007 15 pM.DL-serine ,053 .007 14 5 ,001 ,005 15 5 minus acetone ppt. ... a Anhydroleucovorin can replace CF. Assay contained 14 pM. glycine. Acetone reprecipitated enzyme; 14 pM. glycine in reaction vessel. 10 11 12 13
+
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 ) .
Vol. 78
COMNUNICATIONS TO THE EDITOR
4498
The role of glutamine in the 1-carbon transfer reaction is yet to be elucidated.
TABLE I INCORPORATIOS OF ACETATE A N D OF P - H ~ D R o x I , - P - ~ ~ E T ~ I Y L MEDICINALCHEMICAL RESEARCH SECTIOVN H SLOANE6-VALEROLACTOSE (D\'.%) I N T O CHOLESTEROL I N RAT1,IVER RESEARCH DIVISIOY IY F. BARG,JR HOMOGEKATES AMERICAN CYANAMID COMPA\Y E BOGGIAVO Each flask contained 5 ml. of liver homogenate, and 1 rng. LEDERLELABORATORIES B COULOMB each of A4TPand DPN. Labeled or non-labeled substrates PEARL RIVER,NEWYORK B L HUTCHIVGS were added as indicated, Final volume was 9.5 ml. Gas RECEIVEDMAY31, 1956 phase was 95% 0,-5yo C02. In a given experiment all flasks contained aliquots of the same liver preparation. Incubation was carried out a t 37" for 4.5 hours. Cholesterol was and counted as the digitonide. THE UTILIZATION OF B-HYDROXY-B-METHYLP-HYDROXY-B-METHYL_..._ ~ ~ _ _ ~ _ isolated _ _ Recov6-VALEROLACTONE ~-VALEROLACTONEIN I N CHOLESTEROL ered BIOSYNTHESIS Activity choles~~~~
~
Sir :
Substrates
Wolf, et d . , l have shown recently t h a t the structure of the growth-promoting factor for Lactobacillus acidophilus (-1TCC 4963) discovered by Skeggs, et ~ l .and , ~ obtained by Wright, et ~ l .is, P-hydroxy~ p-methyl-6-valerolactone CH,
I
0
I
OH
1
NaOAc
0.16
DVA
mg.,
....
added total c.p.m. X 105
terol
c.pm./ mg. C
8.7
1,281)
8.7
570
1-ci4
2
0.16 mg., 80 units" concentrate 1-ci4 .... 0.10 mg.,
1.15
4,800
1-ci4
I
CH2-CHJ-C-CH2-C=O
Expt.
0.10
mg., 0 . 9 2 mg., 1.15 2,180 synthetic, non-labeled 0 . 1 0 mg. 0.92 mg., 1.15 28,100 non-labeled 2-C14 One unit of activity has since been shown to be equivnlent to 0.010 mg. of DL synthetic material.
1
i-cl4
The structural similarity of this lactone to ,Bhydroxy-/3-methylglutaric acid prompted us to investigate its possible role in cholesterol metabolism. T o this end we have studied cholesterol synthesis in TABLE I1 cell-free rat liver homogenates using the technique XCTIVITVOF HMG, DMA, A N D ~ - H Y D R O X Y - ~ - M E T H Y L - ~ described by Bucher4 and modified by Rabinowitz VALEROLACTONE (DV.4) I N CHOLESTEROL BIOSYNTHESIS BY RATLIVERHOMOGESATES and Gurin.j Material prepared from distillers' solubles, as Protocol as in Table I. -111 flasks contained aliquots from well as synthetic DL samples, suppress the incor- one pool of liver homogenate, "Corrected" values are calporation of l-C14-acetateinto cholesterol (expts. 1, culated to D V h as standard. Each compound was added a t level of 6 pM. per flask. Total cholesterol estimated by 2, Table I). Since such an effect is open to several the al." interpretations, p-hydroxy-/3-rnethyl-&valerolac- a modification of the method of ;\bell, etCholesterol recovered tone (DVA) labeled in the 2-position with C14 was Activity C" found C'4 corrected synthesizeds and used in a second experiment. The added total c.p.m./ c.p.m./mg. Total Substrates c.p.m. X 104 mg. C . C mg./flask lactone-C14 was incorporated into cholesterol to 400 139 1.61 33.0 such a degree as t o indicate preferential utilization 3'-C14-HMG 4.9 1,350 3,170 1.61 of the 2-carbon of the lactone over the 1-carbon of 4-C14-DMAI ~ - c ~ ~ - D v x1 1 . 5 33,700 33,700 1.77 acetate for sterol synthesis (expt. 2 , Table I). I n the light of these observations, and the experiThese data indicate that only 0.16% of the iso~ of 3'-C14-HMGand 3.8y0 of the radioactivity ments of Rabinowitz and Curin,j of Bloch, et ~ l . , tope and of R ~ d n e y indicating ,~~~ that @-hydroxy+ of 4-C14-DMA are incorporated into cholesterol. methylglutaric acid (HMG) and P,P-dimethyl- I n contrast, 43.47* of the isotope of 2-C14-DVA apacrylic acid (DATA) may be incorporated into pears in the sterol. If the utilization by liver tissue cholesterol, experiments were conducted t o deter- is restricted to only one of the optical isomers of mine the relative efficiency of HMG, DATA and synthetic factor (as is the case with Lactobacillus DVX in contributing isotope for cholesterol bio- acidophilus), then it appears that virtually all of the synthesis.I0 The results of one of these experi- biologically available DVA-CI4 must have been ments are shown in Table IT. transferred t o sterol. I t has been reported that (1) D . E. Wolf, C . H . Hoffman, P. E . Aldrich, H . R . Skeggs. L . D . while some H M G can be utilized in toto for the Wright and K . Folkers, THISJOURNAL, 78, 4499 (1956). synthesis of p-hydroxyisovaleric acid (and pre(2) H. R . Skeggs, L. D . Wright, E. L. Cresson, G. D. E. MacRae, sumably cholesterol), a considerable amount of C . H . Hoffman, D. E. Wolf and K . Folkers, J . Bact., in press. HMG undergoes cleavage to acetate and acetoace(3) L. D . Wright, E. L. Cresson, H. R . Skeggs, G. D . E. MacRae, in press. C . H . Hoffman, D. E. Wolf and K. Folkers, THISJOURNAL, tate.l2,l3 On the other hand, the great extent of ( 4 ) N. L. R. Bucher, THISJ O U R N A L , 7 6 , 4 9 8 (1953). conversion of the C14carbon of DVA into cholesterol (5) J . L. Rabinowitz and S. Gurin, J . Bioi. Chem., 208, 307 (195.1). suggests that the major pathway of cholesterol bio(6) Prepared for us by Dr. C . S. Miller of this laboratory. synthesis from this compound is direct, as opposed ( 7 ) K. Bloch, I-. C. Clark and I . Harary, J . Bioi. Chem., 211, 687 (1 954). to cleavage to smaller molecules. (8) H. Rudney, THISJOURNAL, 76, 2595 (1954). It thus appears that P-hydroxy-p-methyl-6-va'
(9) H. Rudney, ibid., 77, 1698 (1955).
(10) We wish t o acknowledge our indebtedness t o Dr partment of Physiological Chemistry, School of Medicine, University of Pennsylvania, Philadelphia, and t o Dr. J . L. Rabinowitz, Radioisotope Unit, Veterans Administration Hospital, Philadelphia, for generous gifts of both natural and radioactive H M G and D M A
(11) L. L. Abell, B. B. Levy, B. B. Brodie and F. E . Kendall, J . Bioi. C k r i n . , 196, 357 (1952).
(12) B. R.Bachhawat, W. G. Robinson and >I. J . Coon, THISJOUR76, 3098 (1954). (13) J. L. Rabinowitz, i b i d . , 77, 1295 (1035).
NAL,
