COMMUNICATIONS TO THE EDITOR
Nov. .5, 1954
5571
This preparation carried out the over-all reaction
buffer, and ATP with rat liver acetone powder extracts. CA was one of the products of the reaction IITIXA DPNH H C C Oz HzO + using whole rat liver acetone powder. It was not Formylaspartic acid D P N f NHI formed when rat liver particulate fraction or superData indicating the stoichiometry are shown in nate acetone powder extracts were used alone. A solution of C14labeled acetyl-compound X was Table I for an experiment in which DPNH was generated in situ from DPN by glucose plus glucose prepared using acetylglutamic acid and C1402by a dehydrogenase. Formylaspartic acid was isolated method which followed that described previously for and identified by melting point: 136-137' (authen- the preparation of carbamyl-compound X from cartic sample 135-137'; mixed m.p. 135-137')) analy- bamylglutamic acid.8 An alcohol fraction (final sisj ((isolated material) : calculated for C6H705N: concentration 40%) of rat liver particulate acetone C , 37.27; H, 4.37; N, 8.70. Found: C, 37.36; powder was used as source of enzyme. The solution was deproteinized with HC104 (during this H, 4.73; N, 8.67) and the infra-red spectrum.6 The mechanism of the intermediate steps in this treatment unreacted CI4O2was removed) and neuover-all reaction is still unclear. The obligatory re- tralized to pH 7.2 with sodium bicarbonate; 6.4 quirement for both DPNH and oxygen may pos- p-moles of labeled acetyl-compound X was incusibly be explained either by the unfavorable equilib- bated with acetone powder or lyophilized powder rium of the primary reaction or by a peroxidative extracts of rat liver supernate (48 and 104 mg. prostep utilizing peroxide formed by the enzymatic tein, respectively) in the presence or absence of oxidation of DPNH. The latter possibility was 100 p-moles aspartic acid. (Compared on a protein rendered less likely by the absence of DPNH oxi- basis, the lyophilized powder possessed about four dase in the purified preparation; furthermore no times the activity of the acetone powder.) After ImhA was utilized when ImAX was incubated with deproteinization, the solutions were chromatothe enzyme in the absence of DPNH, even when graphed on Dowex-2 anion exchange resin (formate hydrogen peroxide was added or generated by the form). The fractions were assayed for radioactivaction of glucose oxidase [notatin] on glucose. ity. The compound from the elution peak which Further purification of the enzymes involved ap- coincided with that of authentic CA was isolated pears to be necessary to elucidate the nature of the by freeze drying the pooled fractions and removing the buffer salt by sublimation. This peak was abvarious steps. sent when aspartic acid was omitted from the incuFROM THE DEPARTMENT OF MICROBIOLOGY bation. A portion of the compound was identified WASHINGTON UNIVERSITYSCHOOL OF MEDICINE by isotope dilution with authentic CA (D, L-form) ST.LOUIS,MISSOURIA N D THE OSAMU HAYAISHI' and conversion t o hydantoin-5-acetic acid? The NATIONAL INSTITUTE OF ARTHRITISA V D METABOLIC DISEASES,NATIONAL INSTITUTES O F HEALTH melting point remained constant (174") and the speU. S. DEPARTMENT OF HEALTH, cific activity was 14,000, 13,370, 13,710 and 13,230 EDUCATION, A N D WELFARE HERBERT TABOR BETHESDA14, MARYLAXD TAKIKO HAYAISHI c.p.m. per p-mole after each of four recrystallizations as CA. The melting point remained conRECEIVED AUGUST9, 1954 stant (212') and the specific activity was 13,490 (6) The elementary analyses were carried out by t h e Microanalytical and 13,490 c.p.m. per p-mole after each of two reLaboratory of t h e N. I. H. under the direction of Dr. W. Alford; crystallizations as hydantoin-5-acetic acid. t h e infrared spectra were obtained by Mr. H. K. Miller and Mrs. The isolated compound was assayed for radioacP. B. Humphries of this institute. (7) With t h e technical assistance of Natalie Ann Fraser. tivity and CA.IO Its specific activity compared with that of labeled acetyl-compound X was: CA, 3.25 X lo5 c.p.m. per p-mole; compound THE FORMATION OF CARBAMYL ASPARTIC ACID X, 3.49 X lo5 c.p.m. per p-mole. (Specific acBY RAT LIVER PREPARATIONS tivity of labeled acetyl-compound X was deterSir: mined on the basis of radioactivity assays and Carbamylaspartic acid (CA) has been shown to enzymatic assay for compound X.) It is unlikely be a precursor of pyrimidines.2-6 We have ob- that the CA was synthesized de novo by a pathway tained evidence that CA is formed by the enzy- not involving compound X, since the specific activmatic transfer of a carbamyl group (-CO,NH2) from ity of unreacted C1402 remaining after the acidificompound X7to aspartic acid. cation step in the preparation of compound X was Preliminary evidence of this reaction was ob- greatly reduced by neutralizing the reaction mixtained by incubating aspartic acid, acetylglutamic ture with NaHCOs. acid, bicarbonate, ammonium chloride, phosphate ADDENDUM.-After the present communication was submitted, two papers bearing on our findings (1) This investigation was supported in part by a research grant A-540 ('23) from t h e National Institute of Arthritis and Metabolic appeared. L. H . Smith and D. Stetten (THIS Diseases of the National Institutes of Health, Public Health Service, JOURNAL, 76,3864 (1954)) reported that C1*labeled and t h e Wisconsin Alumni Research Foundation. citrulline was a precursor of orotic acid in rat liver (2) P. Reichard, and V. Lagerkvist, Acta Chcm. Scond., 7 , 1207 slices. This finding was interpreted as evidence (1 953). (3) I. Lieberman, and A. Kornberg, J. Bioi. Chcm., 207, 911 for the formation of CA as an intermediate. P. (1954). Reichard (Acta Chem. Scqnd., 8 , 795 (1954)) re(4) I. Lieberman, and A. Kornberg, Biochim ct Biophys. Acta, l a , ported that mitochondrial preparations from rat 223 (1954) liver were capable of synthesizing CA from aspartic (5) D. W. Wilson, and C. Cooper, Fcd. Proc., I S , 194 (1954).
+
+
+
+
(6) 1. Lieberman, A. Kornberg, IS. S. Simms and S. R. Kornberg, ibid., 13, 252 (1954). ( 7 ) S. Orisolia and P. P. Cohen, J , Bioi. Chem., 204, 753 (1953).
(8)S. Grisolia and P. P. Cohen, ibid., 193, 561 (1952). (9) J. P Nyc and H. K. Mitchell, TRISJOURNAL, 69, 1382 (1947). (10) S. B. Koritz and P. P. Cohen, J . B i d . Chcm.. 209, 145 (1854).
5572
COMRIUKTC..ZTIONS TO THE EDITOR
acid, ammonia and carbon dioxide provided that glutamate or carbamylglutamate was present.
Vol. 76 TABLE I
Components
fiM. hydroxamate formed
Complete DEPARTMENT O F P H Y S I O L O G I C A L CHEMISTRY USIVERSITYOF WISCO,VSIS J. h9.LOWENSTEIN No succinate MADISON, WISCONSIN P. P. COHEN 90ATP RECEIVED AUGUST19, 1954 No Co A T H E DIRECT FORMATION OF ACETYL-COENZYME A FROM SUCCINATE'
Sir: Although extracts of the ciliated protozoan, Tetrahymena, reduce cytochrome c in the presence of succinate,* the low cytochrome oxidase activity of the extracts3 indicates that the main route of succinate utilization is not through the usual succinic dehydrogenase-cytochrome system path. Recent experiments with ciliate extracts have revealed that in the presence of succinate, ATP4and Co A, there occurs a rapid formation of acetyl-Co A, as measured by the hydroxamate meth0d.j Washed suspensions6 of Tetrahymena pyriformis, strain S , were homogenized by five passages through a Logeman hand mill. After centrifugation for 20 min. at 1350 X g the precipitate was suspended in 1 Jf tris buffer pH 8.4. Most of the succinic dehydrogenase activity, as measured by the reduction of cytochrome c2, is in the supernatant. A slight but measureable activity is retained in the precipitate. I n the presence of ATP, Co A, M g + f a n d succinate, the precipitate forms a hydroxamic acid. Table I shows that there is no significant amount of hydroxamate formation in the absence of these components. On first examination the reaction seems to be similar to the decarboxylation of succinate where succinyl Co A and propionyl Co A are intermediates.I However, the results obtained upon extraction of the formed hydroxamate and chromatography on paper with water saturated butanol as solvent8 excludes this possibility. The developed chromatograms show only a single spot a t RF 0.51 which is identical to that of acetyl hydroxamate prepared from acetyl phosphate. Mixtures of the extracted hydroxamate and acetyl phosphate also yield a single spot. I n addition, no significant amounts of carbon dioxide are formed when the enzyme is incubated under nitrogen with the components listed in Table I. No hydroxamate is recovered when hydroxylamine is added to the mixture (containing 100 &I. of potassium phosphate buffer in place of hydroxylamine) a t the end of the incubation period; this appears to preclude the formation of an intermediate acyl phosphate. Since the extract contains very low succinic dehydrogenase activity, and, as would thus be anticipated, malonate does not inhibit the reaction (Table (1) Aided by grants E-I59 and G-3364 from t h e h-ational Institutes of Health, United States Public Health Service. (2) G. R. Seaman, Avch. Biochrm. B i o p h r s . , 3 5 , I 3 2 (1952). (3)TG. R. Seaman, ibid, 4 8 , 421 (1954). (4) T h e following abbreviations are used: ATP, adenosine triphosphate; A D P , adenosine diphosphate; Co A , coenzyme .4; trii, tris-(hydroxymethy1)-aminomethane; Pi, inorganic phosphate ( 5 ) F. Lipmans and L. C. Tuttle, J . Biol. Chem.. 161, 415 ( l < M 5 ) . (6) G. R. Seaman, J . Gen. Micvobiol., 11,300 (1954). (7) H. R. Whiteley, Proc. Nail. Arod. Sci. Li.S., 39, 779 (1953). (8) E. R. Stadtman, and H. A. Barker, J. Bioi. C h e m , 184, 7139 (lQ50).
2.23 0.64
0.30 0.06 so h l g + + 0.87 Complete 100 pM. malonate 2.73 The complete mixture contained in 1.0 ml.: 200 p A I . Na succinate, 25 units Co A , 10 pM. K-ATP, 50 &I. KaF, 15 p M . MgC12, 400 fiM. hydroxylamine, 20 p h l . glutatris . buffer pH 8.4, and extract containing 22 thione, 100 &I mg. of protein.
+
I), acetyl formation is not achieved through Krebs cycle oxidation of the succinate. Formation of the acetyl group by reversal of the cycle, through aketoglutarate, also seems unlikely, since no trace of a succinyl hydroxamate or phosphate is obtained on the paper chromatograms. In the absence of Co A, succinate and the protozoan enzyme do not replace acetate in the formation of acetyl phosphate by extracts of Streptococcus faecalis which contain acetokinase activity. Xcetyl formation is thus not the combination of a C2Cz split to free acetate and acetate activation to form acetyl-Co A. Equimolar amounts of inorganic phosphate and acetyl groups are formed by the reaction. The ratio is not affected by fluoride. Succinic acid is the only carboxylic acid revealed when ether extracts of the reaction products are chromatographed on paper with n-butanolformic acid-water ( 5 :1 :4) as the solvent system.I0 These observations indicate the direct formation of acetyl-Co X from succinate by extracts of Tetrahymena. ( 9 ) I. Rose, el a i . , F e d . Pvoc., 13, 283 (1954) (10) I%-.H . Lugg and B. T. Overell, Azis. J , S r i . Res., 1, 98 (194(?
CARTER P a ~ s r o t o LABORATORY c ~ GERALD R. SEAMAN UNIVERSITYO F TEXAS M E D I C A L BRASCH
C,ALVESTON, TEXAS MARYDELL SASCHKE RECEIVED AWUST 23, 1Shl T H E ENZYMATJC FORMATION OF RIBULOSE DIPHOSPHATE
Sir: We have recently reported that a soluble extract from spinach leaves is capable of fixing carbon dioxide in the carbonyl group of phosphoglyceric acid in the absence of light.' Ribose 5-phosphate, T P X , ATP, and h l g + + are required in this reaction. Fractionation of this crude extract has now yielded a preparation which in the absence of T P K , catalyzes the formation of ribulose diphosphate (RuDP) from ATP and ribose 5-phosphate. This reaction requires T\Ig++. The activity has been purified about ten-fold by ammonium sulfate precipitation and adsorption and elution on calcium phosphate gel. To isolate the product formed in the reaction, ATP labeled with P3?in the two terminal phosphate groups was incubated with ribose %phosphate and Mg++ in the presence of the partially purified enzyme. Ion exchange chromatography of the (1) A
Weissbach, P
J O U R N A L , 76,3611 (1954).
Z
Smyrnrotia and B
I
Horecker
1 HIS
