M a y 20, 1957
CONMUNICATIONS TO THE EDITOR
2649
lyzes the incorporation of serine-C14 into sphingo- twice recrystallized sphingosine was found to have sine and to identify some of the cofactors required constant specific radioactivity. for this conversion. The incorporation of radioacAs shown in the table, the conversion of serinetivity from a ~ e t a t e - 1 - C ' ~ and ~-serine-X-C'~ into 3-C14 to sphingosine requires the presence of pysphingosine has been observed in v ~ v o . ~ J ridoxal phosphate, magnesium chloride, triphosBrain tissue obtained from 10-14 day old rats phopyridine nucleotide, diphosphopyridine nucleowas homogenized with 3 volumes of isotonic sucrose tide, and an as yet unidentified substance present in or 0.1 M potassium phosphate buffer (QH 7.8), and a kochsaft obtained from rat liver tissue. The redifferential centrifugation was accomplished ac- covered sphingosine was degraded with sodium cording to Brody and B a i r ~ . ~The enzymes re- p e r i ~ d a t e , ~ and . ~ the radioactivity of the residual quired for the conversion of L-serine to sphingosine aliphatic aldehyde was determined. It is apparent were found in the supernatant solution obtained from these data t h a t the radioactivity from the serafter sedimenting the particulate matter by centri- ine-C14 is preferentially localized in positions 1 and fuging a t 25,000 X g for 30 minutes. The enzyme 2 of sphingosine and t h a t the reaction does not reppreparation was dialyzed for 4 hours against 100 resent random incorporation of radioactivity via volumes of a solution containing 0.01 M potassium an active one or two carbon fragment. These findphosphate buffer (QH 7.0), 0.001 M cysteine hydro- ings have been confirmed with the use of L-serinechloride and 0.001 M disodium ethylenediamine- U-C14as substrate in this system. tetraacetic acid. (5) H. E. Carter, F. J. Glick, W. P. Norris and G. E. Phillips, J . Following incubation, the lipids were quantita- Bioi. Chem., 170, 285 (1947). tively extracted with n-butanol and 18 pmoles of NATIONAL INSTITUTE OF NEUROLOGICAL DISEASES AND sphingosine sulfate were added to each sample as BLINDNESS INSTITUTES OF HEALTH carrier. The mixtures were refluxed for 6 hours in NATIONAL 0. BRADY ROSCOE PUBLICHEALTH SERVICE 85% methanol made 1.2 M with respect to HzSO~, U. S. DEPARTMEXT OF HEALTH, EDUCATION, A N D WELFARE and subsequently extracted with petroleum ether BETHESDA, MARYLAND GEORGE J. KOVAL which was back-washed two times with dilute acid. RECEIVED APRIL 1, 1957 The methanolic solution containing the sphingosine sulfate was concentrated under vacuum, and the sphingosine base was recovered from salt-saturated A NOVEL ORGANO-CHROMIUM COMPOUND alkaline aqueous phase by extracting with 25710 iso- Sir : amyl alcohol in h e ~ t a n e . ~ The solvents were evapThe known organo-chromium compounds, apart orated under reduced pressure and the sphingosine from the anionic cyanide and ethynyl complexes' was recrystallized two times from petroleum ether. of chromium(II1) are compounds of chromium(0), Approximately SOYo of the carrier sphingosine was chromium(1) or chromium(II)2 which generally recovered with the use of this procedure. The do not contain simple carbon-chromium bonds. We wish to report the first preparation (in solution) TABLEI ESZYMATIC C O X V E R S I O S OF sERIXE-3-cl4TO SPHINGOSINE of a simple organo-chromium compound of the Except as noted, the incubation mixtures contained 1.5 ml. type Cr(H20)6R++ where R is benzyl. The comof dialyzed superiiatant solution obtained by centrifuging a pound has not yet been obtained crystalline, but 1:3 ( w . / v . ) cell-free preparation of r a t brain tissue at solutions of the pure perchlorate in dilute per25,000 X g for 30 minutes, 130 pmoles of potassium phos- chloric acid solution have been obtained and are phate buffer (PH SA), 4 pnmolcs of o ~ - s e r i n e - 3 - C l(4~ pC,), 1 pmole of pyridoxal phosphate, 5 pmoles of MgC12, 0.3 fairly stable (half-life in absence of oxygen a t room temperature of about 1.5 days). The structure of pmole of triphosphopyridine nucleotide (TPN), 0.3 pmole of diphosphopyridine nucleotide ( D P N ) , 20 pmoles of nicotinthe compound, benzylpentaaquochromium(II1) amide, Bpmoles of uridine triphosphate (UTP), 0.4 pmole perchlorate, can be regarded as analogous to that of cytidine triphosphate (CTP), 5 pmoles of adenosine triof chrophosphate (dTPj, and 0.1 rnl. of liver kochsaft in a total of the chloride complex, Cr(H*O)&!l+f, mium(II1) ; i.e., the compound is a complex of Crvolume of 2.0 ml. Incubation time, 3 hours at 38' in air. (111) with the benzyl anion. The remarkable staDistribution of radioactivity in recovered sphingosine bility of this organo-metallic compound in acid Residue after periodate solution is undoubtedly related to the inertness of Sphingosine, degradation, Cr(II1) complexes to s ~ b s t i t u t i o n . ~ Reactant omitted c./m./wmole c./m.rmole The benzylchromium compound was prepared None 155 26 by reducing benzyl chloride (or bromide or iodide) None, heated a t 100" for 10 min. 3 .. with aqueous 1 M chromous perchlorate in 1 M Kochsaft 11 .. perchloric acid, either heterogeneously or in presPyridoxal phosphate 35 23 ence of alcohol or other suitable organic solvents. MgClz 71 20 Countercurrent distribution of the product a t 5" TPN 90 20 with the solvent system 0.01 M perchloric acid22 DPX 93 butanol gave, after 70 transfers, a separation of the Sicotinamide 110 25 organo-chromium compound (partition coefficient 26 UTP 140 CTP ATP (1) (2) (3) (4)
165 170
25 28
I. Zabin and J . I'. Mead, J . Bioi. C h e m . . 206, 271 (1953). D. B. Sprinson and A. C o d o n , ibid., 207, 585 (1954). T. hl. Rrody and J. A. Bain, ibid., 196, 885 (1952). R . 0 . Brady and I
