Nov. Ei, 1954
COMMUNICATIONS TO THE EDITOR
markedly the activity of the washed sediment but not of the crude preparations (Table 11). TABLE I1 Preparation
Additions
Initial rates of Pi uptake in the light pM. P/hr,/ pM. P/br /unit mg. protein opt. d . a t 800 mp
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(ATP)2while the bacterial degradation of the ureido group of citrulline is coupled to ATP synthesis according to the over-all equation Citrulline
+ Orthophosphate + ADP Ornithine + NHI + COP + ATP3
From knowledge of the fate of phosphate oxygen in the enzymic formation of 3-phosphoglycerate and in phosphate transfer reactions, the intermediate formation of an anhydride compound between carbonic acid or a derivative thereof and Resuspended DPN orthophosphoric acid accompanying COZ release sediment KGA and ATP synthesis would be expected to result in DPN, KGA 5.1 incorporation of phosphate oxygen into the COz. DPN, KGA, 5.1 It appeared feasible t o check this possibility cyt. c with the use of bacterial preparations5 which catT P N , KGA 4.4 19.1 All preparations contained the following additions per alyze the above reaction provided that the exchange of oxygen of carbon dioxide with that of ml.: 12 p M : MgC12, 10 pM. K F , 7 p M . Pi, 10.3 p M . ADP. Other additlons were present as indicated in the following water could be sufficiently minimized to allow concentration per ml.: 0.5 p M . D P N , 0.25 p M . T P N , detection of excess 01*from O1*-labeled ortho0.012 MM.cyt. c, 15 p M . KGA. Resuspended sediment consisted of top half of sediment obtained after centrifuga- phosphate in the COZformed. Using conditions similar to those described in tion of crude sonic preparation for l hour a t 135,000 X gravity. Other experimental conditions the same as Table I, a small but definite incorporation of excess indicated for Table I. 0 I 8 of orthophosphate into the COz formed was Light dependent phosphorylation of ADP did found (mass 46/44 ratio of COz = 0.00431). Innot require oxygen and was not affected by 10-4 creased yield of 0 I 8 in the COZ was obtained with M dinitrophenol or M iodoacetarnide, loF3M change of conditions to reduce the exchange of hydrogen cyanide and M mersalyl4 gave inhi- oxygen of COz with that of water before escape of bitions between 25-5070, M o-phenanthroline COS into the evacuated reaction chamber; i.e., inhibited completely, and 2,6-dichlorophenolindo- decrease in total liquid volume from 30 to 17.5 ml., phenol inhibited phosphorylation from 60 to 100%. increase in flask size from 100 to 250 ml. and with The system was completely inactivated after incu- the reaction mixture strongly swirled instead of bation at 80" for two minutes. Slow freezing and occasionally mixed. Results of such experiments thawing of the preparation also caused extensive are shown in Table I ; they give conclusive proof inactivation. Preparations kept on ice in the dark that oxygen from orthophosphate appears in the for eight hours did not show an appreciable loss of COz formed. The COZproduced from citrulline in presence of unlabeled orthophosphate contained, activity. Similar systems have been described in the case within experimental error, the same amount of 01* as tank COz. The amount of 01*in the COz from of chloroplast preparations from higher In these systems, however, labelled Pi was employed the sample containing orthophosphate-018 was to demonstrate the light induced formation of ATP. approximately 28y0 of that expected if one oxygen I wish to thank Dr. F. Lipmann for his hospital- of phosphate appeared in the COz formed directly ity and advice during this investigation, and Drs. as a gas or 43% of that expected if one oxygen of A. Brodie, M. E. Jones, R. Bandurski and J. A. phosphate appeared in HzC03 which liberated gaseous COz. Under the reaction conditions a t Johnston for their many helpful suggestions. 37" with phosphate present (without consideration (6) W.Vishniac and S. Ochoa, J . Bioi. Chem., 198, 501 (1952). of any possible carbonic anhydrase activity of the (7) D. I. Arnon, M. B. Allen and F. R. Whatley, Nature, 174, 394 bacterial preparation), the half-time for complete (1954). exchange of COZ oxygen with that of water was BIOCHEMICAL RESEARCH LABORATORY estimated from the data of Mills and Urey* to be MASSACHUSETTS GENERAL HOSPITAL BOSTON14, MASSACHUSETTS about 19 seconds. That relatively rapid exchange DEPARTMENT OF BOTANY ALBERTFRENKELwas occurring was also shown by the increased UNIVERSITY OF MINNESOTA yield of 0l8in the COz obtained as a result of reMIXNEAPOLIS 14, MINNESOTA duction in liquid volume, increase of size of reaction RECEIVED SEPTEMBER 13, 1954 flask, and vigorous swirling of the reaction mixture. These considerations together with the known specificity of enzyme reactions make it INCORPORATION O F PHOSPHATE OXYGEN Crude sonic
DPS DPN, KGA
2.0 1.9 1.9 2.1 2.1 4.3
22.6 21.5 21.5 9.1 9.1 18.7 22.1 22.1
INTO CARBON DIOXIDE FORMED BY ENZYMIC DEGRADATION OF CITRULLINE COUPLED W I T H ATP SYNTHESIS1
Sir:
The biosynthesis of citrulline from ornithine by animal tissues requires adenosine triphosphate (1) Supported by a grant from t h e National Science Foundation and grant 1033 from the National Institutes of Health, Public Health Service. Contribution No. 3226, Scientific Journal Series, Minnesota Agricultural Experiment Station.
(2) S. Grisolia and P. P. Cohen, J . B i d . Chem., 204, 753 (1953); 198,561 (1952). (3) H. D. Slade, C. C. Doughty and W. C. Slamp, Arch. Biochem. Bioghrs., 4 8 , 338 (1954); V. A. Knivett, Biochem. J . , 66, 602 (1954); M. Korzenovsky and C. H. Werkman, ibid.,67, 343 (1954). (4) P. D. Boyer and W. H. Harrison in W. D. McElroy and B. Glass, "Mechanism of Enzyme Action," Johns Hopkins Press, Baltimore, Md., p. 658 (1954); M. Cohn, J . Biol. Chcm., 201, 735 (1953). (5) We are indebted to Dr. H. D. Slade for kindly supplying us with
a culture of the Pseudomonas used in his studies. (6) G. A. Mills and H. C . Urey, T X I SJOURNAL. 62, 1019 (1940).
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CORIMUNIC.\TIONS TO TIIE EDITOR
probable that one oxygen of the COz (or H2COa) as given herein may apply to other related CO? as formed enzymically is derived from the ureido fixation and release reactions. group of the citrulline and onc frorn the ortho- DEPARTMENT OF AGRICULTURAL UIOCIICMMISTRY phosphate. I.NIVERSITY OF MINNESOTA 51. P. S I U I . ~ E R G ST.PAUL1, MINN. P. 1) 1?Ok'ER Tnnr,F: i ESZYMATIC FORMATION O F Con I N PRESENCE O F 0
RECEIVED SEPTEMBER 27, 1954 1 8 - L ~ ~ ~ ~ ~ ~
ORTHOPHOSPHATE
-4250-ml. round-bottom flask contained, in 0.2 ill succinate buffer, 60 micromoles of L-citrulline, 100 micromoles of lIgS04, 50 micromoles of adenylic acid, and 2.5 millimoles of potassium phosphate in a total volume of 12.5 ml. a t p H 5 . 3 . A 50-ml. round-bottom flask contained 5 ml. of a Pseudomo?tns extract prepared according to Slade, et al.3 The flasks were arranged so t h a t they could be attached t o a high-vacuum system and the contents intermixed. The contents of both flasks were frozen in Dry Ice and acetone, the flasks evacuated to a pressure of
