COMMUNICATIONS TO THE EDITOR
Aug. 20, 1958
space group P212121with four molecules (K or Rb)CsOeHs in cells of dimensions: Cell volume a
K Rb
(A,)
b
(A,)
c
(A,)
(Aa.)
9.059 12.681 6.640 762.8 9.190 12.639 6.825 792.7
Density (g. cm.-’) obs. calcd.
1.838 2.142
1.848 2.166
The standard error of the cell dimensions is about &0.1570 and that of the observed densities about =k0.5%. The structure was determined from projections on the three principal planes using the method of isomorphous replacement. The three projections for the potassium salt have been refined by difference Fourier maps and then b y least squares using the full matrix to account for the overlap of atoms. At the present stage of refinement the R values for the hk0, Okl, and h0Z projections are 7.6, 11.5, 12.6%, respectively, the unobserved reflections being included a t one-half their estimated upper limit. The structure which we have obtained for the lactone ion confirms the conclusion of Gawron and Glaid that the carboxyl groups are cis with respect t o the lactone ring. We also find that the metal atom is coordinated with eight oxygen atoms. All oxygen atoms including that in the lactone ring take part in this co6rdination. The structure clearly explains the pronounced cleavage of these crystals parallel to the b face. Complete details of the structure analysis and a discussion of the bond lengths and bond angles will be presented elsewhere. We also hope t o determine the absolute configuration of the rubidium salt by the method of Bijvoet and his c o - ~ o r k e r s . ~
4427
The reversibility of Reaction 1 lias been shown prev i ~ u s l y . ~Indirect evidence suggesting reversal of the overall reaction has been reported? Reversal has been followed directly here by measuring the cleavage of isolated amino acid-RNA and incorporation of AMP into ATP. Cleavage of isolated threonine-RNA and leucineRNA, and incorporation of C14-labeled AMP into ATP, dependent on amino acid-RNA, is shown in Table I. No ATP or free amino acid was added in TABLE I CLEAVAGE OF AMINOACID-RNA AND INCORPORATION OF AMP INTO ATP
Constituents
(1) Complete mixture‘ (2) Zero time control (3) As ( l ) , but RNA in
Starting with Starting with threonine-RNA leucine-RNA Counts/ Counts/ Counts/ Counts/ min. of min. of min. of min. of C14Cl4. C14C14AMP threonine- AMP leucine found RNA found RNA in ATP remain- in ATP remaining ing
408 114
168 983
..
526 84
416 860
...
place of amino acid-RNAb 108 . 64 (4) As (l), but boiled enzyme 100 887 .. . ... (5) As (l),but P-Pomitted 128 316 100 820 (6) As (l), but AMP omitted ... 805 ... 804 The complete reaction mixture for AMP incorporation into ATP contained 0.5 ml. of activating enzyme, approximately 0.5 mg. of RNA or Cl*-amino acid-RNA; 100 pmoles of Tris buffer, pH 7.5; 2 pmoles of C14-AMP (Schwarz Laboratories), containing 60,000 counts/min./pmole; 2 pmoles of magnesium chloride; 2 pmoles of P-P; and water to make 1.8 ml. Mixtures were incubated at 37’ for 15 minutes; then 0.7 mg. of casein, 1.8 ml. of 7% TCA (4) J. M. Bijvoet, A. F. Peerdeman and A. J. vau Bommel, Nalurc and 10 pmoles of ATP were added and the ATP isolated 168, 271 (1951). and counted (see Holley‘). Incubation conditions for JENNY PICKWORTH GLUSKER studying cleavage of amino acid-RNA were similar, but A. L. PATTERSONusing CI*-AMP and C14-amino acid-RNA, and counting INSTITUTE POR CANCER RESEARCH WARNER E. LOVE residual amino acid-RNA (precipitated by perchloric acid). PHILADELPHIA 11, PA. MARILYN L. DORNBERG Separate activating enzyme fractions for leucine or threonine RECEIVED JUNE 7, 1958 activation, free of RNA, were prepared from guinea pig liver! Labeled amino acid-RNA compounds were prepared by phenol extraction from the usual reaction mixture for REVERSIBILITY OF AMINO ACID INCORPORATION incorporation,& and contained 1 mpmole of C1*-aminoacid (2500 c.p.m.) per mg. of RNA. bRNA was prepared by INTO RIBONUCLEIC ACID phenol extraction and was active for amino acid incorporaSir: tion.‘ In this experiment 0.5 mpmole of CWhreonine or was added. The dash indicates “experiment The incorporation of CI4-labeledamino acids into leucine omitted.”
ribonucleic acid‘ has been reported2 and confirmed. T h a t a specific amino acid-activating enzyme is required for the incorporation into ribonucleic acid me-amino of the amino acid it activates,savbsuggests these reactions
+ amino acid + ATP [enzyme-amino acyl-AMP] + P-P (Enzy acyl-AMP] + RNA J _ amino acid-RNA -1- enzyme + AMP
Enzyme
(1)
(2)
(1) These abbreviations are used: RNA, ribonucleic acid: AMP, adenosine 5’-monophosphatr; ATP, adenosine 5‘-triphosphate; ADP, adenosine b’-diphosphate: TCA, trichloroacetic acid: GMP, guanosine 5‘-monophosphate: CMP, cytidine 5‘-monophosphate; P-P, pyrophosphate. (2) M. B. Hoagland, P. C. Zamecnik and M. L. Stephenson, Biochim. el Biophys. Acto, ah, 215 (1957). (3) (a) R. S. Schweet, F. C. Bovard, E. H. Allen and E. Glassman, Proc. Nan. Acod. sci., 44, 173 (1958); (h) P. Berg and E. J. Ofengand, ibid., 44, 78 (1958): (c) K. Ogata and H. Nohara, Biochim. el Biophyr. Ada, P I , 650 (1957).
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these experiments. Both amino acid-RNA com pounds were cleaved in the presence of AMP plus P-P (complete mixture), although the quantitative importance of P-P depends on the particular aminoacid-RNA and enzyme fraction used. In other experiments, the amount of threonine-RNA split depended on the amount of AMP added, G M P or CMP did not replace AMP; Mg ion was essential; and leucine-RNA was not split by threonine-activating enzyme. (4) M. B. Hoagland, E. B. Keller and P. C. Zamecnik, 1. Biol. Chem., 218, 345 (1956); J. A. De Moss, S. M. Genuth and G. D. Novelli, Proc. Null. Acad. Sci., 4!2, 325 (1956); P. Berg, Fed. Proc., 16, 152 (1957). ( 5 ) R. W. Holley, THISJOURNAL, 79, 658 (1957); M. B. Hoagland, M. L. Stephenson, J. F. Scott, L. I. Hecht and P. C. Zamecnik, J . Biol. Chcm., 1S1, 241 (1958): J. Mager and F. Lipmann, Proc. Nail. Acad. Sci.. 44, 305 (1958). (6) E. H. Allen, E. Glassman and R. S. Schweet, in preparation, (7) K. S. Kirby, Biochem. J . . 64, 405 (1956).
Incorporation of C'?-labeled *iSlP into =1TP is the most direct evidence for reversibility of amino acid incorporation into RNA. S o incorporation of AMP into ATP occurred when RNA plus free amino acid was used in place of amino acid-RNA, or when P-P was omitted. Calculation of the data shows that the molar rate of AMP incorporation into ATP greatly exceeds the rate of amino acidRNA breakdown, suggesting that reactions which form unlabeled 4 T P occur during the incubation. The approximate equilibrium position of these reactions was near unity, indicating the high energy nature of the amino acid-RNA%linkage.
23.3s nip ( e 1.4,200). -1nal. Found: C , 69.24, H, 7.21. Hydrolysis with potassium hicarbonate gave 1GB-methylprednisone, m .p. 2 10-204 O , [ a ] D 4-190.2", ": :A : 238 n1p ( E 14,'700).A,lnal. Found: C, 71.19; H, 7.37 Reaction of I with methylmagnesium iodide produced 3a-hydroxy-lBa-methylpregnane-l1,20dione,G imp. lG-lXo, [ a ] D +100.5",no selective absorption between 200 and 340 mp. *final. Found: e, 74.lG; H.9.31. This was converted into 1Ba-methylprednisone 21-acetatc (11i.p. 212314' [.ID f13'i.S0, ":A:: 238 lnp ( E 1,SUSSIIAI'R? 74.58; H, 8.55. Reduction with palladium yielded \YILLIAM GEBIXT 3a-acetoxy- 16P-methyl pregnane- 11,20-dione (11) RESEARCH LAROR.ITORII;S f ? n. E r E R S H B E R G m.p. 160-163', [ a ] D +93.G0, no ultraviolet ab- SCHERISG s.T O L K S D O R P CORP. 9. J. MILTOX EISLER sorption between 220-300 mM. Ana,l. Found: C, BLOOMFIELD, P. 1,. P E R L M A Y 74.37; H, 9.06. Enol acetylation with p-toluenesulfonic acid and acetic anhydride, then treatment ~IASSACHr-SETTSGEh ERAI. I ~OSPITAI. 11. I1 PecllFI. with peracetic acid and finally alkaline hydrolysis, I Y E I I J 1 . x ~30, 1938 gave 3 a ,17a-dihydroxy-16P-methylpregnane11,20dione, m.p. 1S2-1SS0, [ a ]f83.6'. ~ A n d . Found: C, 72.82; H, 8.92. Bromination a t (2-21and then THE INTERMEDIATE COBALT HYDROCARBONYLtreatment with potassium acetate gave 3a,17a-21OLEFIN COMPLEX IN THE OXO REACTION' trihydroxy- 16/3-methylpregnane-l1,20-dione 2 1- Sir: acetate, m.p. 200-206', DI.[ +119.Go. Anal. The several m e c h a n i s m ~ ~n-hich , ~ * ~ have been Found: C, 68.79; H, 8.39. Oxidation with N- suggested for the oxo synthesis all involve a ratebromosuccinimide produced the 3-ketone, m.p. determining displacement of a mole of carbon mon198-202", [ a ]$128.0". ~ A n d . Found: c, 69.04; oxide from a carbonyl of cobalt by the attacking oleH, 8.10. Dibromination a t positions 2 and 4, fol- fin. The present study shows that not only does lowed by dehydrobromination with dimethyl- complexing occur between olefin and hydrocarformamide, produced 16P-methylprednisone 21- bonyls under room conditions without the liberaacetate (111) m.p. 232-235', [ a ] ~ +213.6", (1) We wish to thank the Houdry Process Corp. for a generous (1) After submission of this manuscript, a Communication appeared [C. Arth, D. Johnston, J. Fried, W.Spooncer, D. Hoff and L. Sarett, THISJOURNAL, 80, 3160 (1958)l describing the preparation of 16umethylprednisone by essentially the same route. We have tried t o eliminate as much of the common material as possible. (2) E. W. Boland, C O ~M.e d . , 88, 417 (1958). (3) H. Slates and N. Wendler, J . Or& Chem., 22, 498 (1957). (4) Cf,A. Wettstein, Helv. Chim. A d a , 27, 1803 (1944).
A":f
fellowship which made this work possible. (2) H. W,Sternberg, R. hfarkhy a n d I. Wender, THISJOURNAL, 79, 0116 (1957); I. Wender and M.Orchin, in "Catalysis," Vol. V, Reinhold Publishing Corp., New York, N.Y.1957, p. 124. (3) A. R . Martin. C h e w ~ i i Idn d . . 1536 (1954). ( 4 ) G. Natta, R. Ercoli, S. Castellano and P. H. Barbieri, Tms J O U R N A L , 76, 4094 (1954). (6) M Orchin, L Kirch and 1. C:oldfarb, ibid.. 78, 5450 (1956).
