Jan. 5, 1953
249
~ O M M U N I C A T ~ O TO N S THE EDITOR
working with soluble enzyme extracts of rat liver have demonstrated the presence of enzymes capable of converting L-a-glycerophosphate into a lipide product tentatively identified as a phosphatidic acid. These workers4have also described an enzyme system which is capable of converting phosphorylcholine into a lipide product. With TABLE I doubly-labeled phosphorylcholine (P32,C14) the ratio Factor for preferential incorporation of P32to C14in the product approximates that in the Animal Duration of of deuteriuma no. expbdays Liver glycogen Liver fatty acids substrate, suggesting the incorporation of phosphorylcholine as a unit into a phospholipide mole1 3 1.06 =I= 0.02 cule (presumably lecithin). Free choline is de2 4 1.08* .02 1.19 f 0.02 scribed by these authors as being only about one1.17=I= .02 3 2 tenth as active as phosphorylcholine in the forma4 1 1 . 0 9 f .02 1 . 1 9 * .02 tion of phospholipide. This factor is defined as (T/D)water/(T/D)compound It is the purpose of this communication to report where T is proportional to the tritium atom fraction, D is the atom per cent excess of deuterium and the compound the finding in isolated rat liver mitochondria of an refers to glycogen or the fatty acid fraction. Normal deu- enzyme system which incorporates free choline terium abundance was taken to be 0.020 atom per cent. labeled with C14 into the lecithin fraction of the Experimental.-Liver glycogen was prepared and enzyme granules by a pathway which does not inpurified following the procedure of Stetten and volve phosphorylcholine. When mitochondria isoBoxers while the method described by Schoen- lated from sucrose homogenates of rat liver are inheimer and Rittenberg,’ with minor modifizations, cubated with choline-meth~1-C’~ and added cofacwas used to obtain the fatty acid fraction. Iso- tors, the mitochondrial phospholipides rapidly betopic analyses were made on the hydrogen gas ob- come radioactive. When phosphorylcholine-methtained by complete conversion over zinc a t 415’ of yl-C14 of identical specific activity is tested in the the water obtained’ by combustion. Memory ef- same system, no significant incorporation of radiofects were eliminated by measuring the results of a activity into phospholipide is noted. Data from a second and third combustion and conversion after typical experiment are shown in Table I. Simidiscarding the products of a preliminary combustion larly, Ps2-labeled phosphorylcholine is also inactive intended to season the train. Deuterium was as a precursor of radioactive phospholipide. If is tested in the presence of a measured using a dual collector Nier-type hydrogen choline-meth~1-C’~ mass spectrometer while tritium was counted large pool of unlabeled phosphorylcholine, no rein the upper portion of the proportional region.8 duction in the radioactivity of the phospholipide The reproducibility of the deuterium and tritium fraction is observed. The lack of activity of phosanalyses is better than one per cent. The tritium phorylcholine in this system is not the result of the atom fraction in the rat body fluid was approxi- impermeability of the mitochondrial membrane t o mately while the deuterium concentration this substrate, since identical results are obtained was kept below two per cent. in order to minimize with extracts of acetone powder preparations of mitochondria, which have been found to carry out the abundance of DOD molecules.
levels were maintained for a period of two weeks. The above results support the conclusion that a preferential incorporation of protium relative to deuteriuln is occurring simultaneously in these experiments and is probably of a magnitude comparable with the D :T factors reported here.6’
(I
(,5) J. Rixeleiwn. Science, 110, 14 (1949). (6) D. W. Stetten, Jr., a n d G. E. Boxer, J. H i d . C / I P ! ! !166, , , 231 (1944).
(7) R. Schoenheimer and D. Rittenherg, ibid.. 111, 177 (19315). (8) M. L. Eidinoff, J. E. Knoll, D. K.Fukushirna a n d T.P. Gallagher, TRISJOURNAL, 74, 5280 (1952).
DIVISIONOF PHYSICSA N D M. L. EIDINOFF BIOPHYSICS G. C. PERRI SLOAN-KETTERINC INSTITUTE J. E. KNOLL 13. J . MARANO FOR CANCER RESEARCH J. ARNHEIM KEIVYORK, N. Y. RECEIVED DECEMBER 3, 1952
TARLE I Experiment A
Complete system “Zero time” control Adenylic acid omitted 1.0 pM of 2,4-dinitrophenol added Phosphorylcholine-methyl-C14in place of choline-methyl-C14
Sir:
3000 68 383 759 179
Experiment B
Complete system THE SYNTHESIS OF LECITHIN IN ISOLATED MITOCHONDRIA
Total radioactivity of phospholipides, cts./min.
1240
Phosphorylcholine-methyl-C14 in place of choline-methyl-C14
52
In Experiment A, each vessel contained 15 pM of MgCln, Recent reports from this 1aboratory’J have 100 pM of sodium succinate, 3 pM of adenylic acid, 100 pM phosphate buffer, PH 7.4 and 5.0 pM of choline-methylshown that a-glycerophosphate is a n important of C14 or phosphorylcholine-methyl-C1~ of identical specific intermediate in the reaction scheme by which inor- activity (125,000 cts./pM/min.). The final volume was ganic phosphate labeled with P32is incorporated 3.0 ml. Approximately 20 mg. dry weight of freshly preinto the phosphorus-containing lipides of isolated pared rat liver mitochondria were added just prior to incubafor one hour in a Dubnoff apparatus a t 38’ with air as rat liver mitochondria. Romberg and Pricer3l4 tion gas phase. The total phospholipide fraction was isolated and counted by methods closely similar t o those described (1) E. P. Kennedy, Fcderdfion Pmc., 11, 239 (1952). previously.’ The complete system in Experiment B was ex(2) E. P. Kennedy. J. B i d Chcm., in press. actly the same, except that the succinate and adenylic ( 3 ) A . Kornhrrg and W. E . Pricer, J r , , THISJ O U R N A L , 74, 1617 acid were replaced by 5.0 r M of adenosine triphosphate, and (1952). 1.0 ml. of a 10% extract of mitochondria acetone powder (4) A. Kornherg and W. E. Priccr, Jr., Federation Proc., 11, 242 was used as enzyme instead of fresh mitochondria. (1962).
250
VOl. 75
CORlMUIiICATIONS TO TIIE EDITOR
the incorporation of labeled choline into phospholipide when supplenientecl with ATP and other cofactors. Choline-1,2-CL4and choline-niethyl-C14 are incorporated a t identical rates. The incorporation reaction is dependent upon oxidative phosphorylation for the generation of adenosine triphosphate in fresh preparations of mitochondria, being severely inhibited by the addition of dinitrophenol or the omission of adenine nucleotide. I n acetone powder extracts, in which no oxidative phosphorylation occurs, there is an absolute requirement for adenosine triphosphate. -2fter hydrolysis of the phospholipide extracts with N / l KOH a t 37" by the method of Hack5 the radioactivity may be quantitatively recovered as choline reineckate, indicating that it is lecithin rather than sphingomyelin which is labeled. By chromatography of the phospholipides by a variation of the method of Haiiahan et u L . , ~ radioactive lecithin fractions may be isolated with a choline/P ratio close to unity. The curves for the elution of C14 and lipide P from the columns are very nearly identical in these experiments. The nature of the intermediates involved in the incorporation of choline into the lecithin of mitochondria, and in particular the possible role of phosphatidic acids in the process, is not yet known, and is the subject of continuing investigation in this laboratory.
molecules of octene-1 with one of ethanol (4.0 g., b.p. 120-126' a t 1 mm.; n Z o D 1.4479; mol. wt. %Si, and a residue (18 g., mol. wt. 488). In the apparatus described above a reaction mixture containing propanol-2 (356.7 g., 5.95 moles), octene-l (28 g., 2 5 mole) and t-butyl peroxide ( 5 1111.) was held at 120" for 30 hours. Distillation gave t-butyl alcohol and unreacted propanol-2 (342.3 g., b.p. Sl-SZO), and a fraction shown to be 2-iiiethyldecanol-2 (16.5 g., b.1). 49" a t 0.1 mm.; 7L2"D 1,4339). A n d . Calcd. for C11HT40:C , 76.67; H, 14.04; niol. wt., 1'72. Fountl: C, i 6 . 3 3 ; H, l 3 . X O ; 11101. w t . , 178. I t was identical in infrared spectrum and other physical properties with 2-methyl-decanol-2 (b.p. 49..io a t 0.1 mm., T Z ~ O D 1.4358) prepared by the reaction of n-octylmagnesium bromide with acetone. Its allophanate (m.p. 113.5-114'; 11i.p. of mixture with authentic sample, 113-114') was prepared. A n d . Calcd. for C13H26N203: N, 10.8. Found: s,10.5. Further distillation gave a 2 : 1 product (7.4 g., b.p. 120-130" a t 0.1 mm., mol. wt. 263) and a resiclue (7 g., mol. wt. 449). X similar reaction was observed when a mixture of propanol-2 (198.8 g., :?.:31 mole) and octene-1 (19.9 g., 0.178 mole) was illuminated with a quartz mercury resonance lamp for 96 hours. 2-Methyldecanol-2 (6.0 g., b.p. 5032" a t 0.2 mm.; n Z 0 D 1.4369; m.p. of its allo( 5 ) M. H. Hack, J . B i d . Chem., 169, 1 3 7 ( 1 9 4 7 ~ . phanate, 113-114'; m.p. of mixture with authentic (6) D. J. Hanahan, M. H. Turner a n d h l . E. Jayko. ibrd., 192,623 sample, 113-114°), 2 : l product (3.1 g., n z o ~ (1051). 1.4525) and a residue (4.7 g. , mol. wt. 422) were obBEN MAYLABORATORY FOR CANCER RESEARCH AXD tained. DEPARTMENT OF BIOCHEMISTRY UNIV~RSITY OF CHICAGO The reaction of butanol-1. (581 g., 7.85 moles) CHICAGO 37, ILL. with octene-1 (29.1 g., 0.26 mole) and t-butyl perRECEIVED NOVEMBER 17, 1952 oxide ( 3 g., 2 g. added after 18 hours) a t 115-116" for 43 hours gave dodecanol-4 (18 g., b.p. 53-84' PEROXIDE- AND LIGHT-INDUCED REACTIONS OF a t 1 lllltl.; %*OD 1.4409). ALCOHOLS WITH OLEFINS Anal. Calcd. for C12H26O: C , 77.35; H, 14.07; Sir: iiiol. wt., 186. Found: C, 77.64; H, 13.82; mol. Preliminary results of a geiicral prograin of re- mt., 192. search to determine if free-radical, chain addition is This product was identical in infrared spectruiii a property common to substances containing a and other physical properties with dodecanol-4 methylene or methiiie group directly attached to an (b.p. 83-54" a t 1 mm.,a Z o1.4409) ~ prepared by the electronegative atom' have led to the discovery reduction of dodecanone-4 (prepared by the per that primary and secondary alcohols add to olefins oxide induced reaction of n-butyraldehyde with in the presence of peroxides or light. The reac- octene-1) with lithium aluminum hydride. Its tions of ethanol, propanol-2, butanol- 1 and butanol- a-naphthylurethane (1n.p. 57-38' ; n1.p. of niixX with octene-1 in the presence of t-butyl peroxide ture with authentic sample, 57-5S") was prepared. give telemeric products whose chief components are, A n d . Calcd. for C ~ ~ H ~ ~ N C,O'77.70; L : H, 9.36. respectively, decanol-2, 2-methyldecanol-2, dodec- Found: C, 77.53; H, 9.58. anol-4 and 3-methylundecanol-3. : l product (7.5 g., b.p. 120-145" a t 1 niin.; A reaction mixture containing ethanol (454 g., %"OD 2 1.4518, mol. wt. 297) and a residue (15 g., niol. 9.87 moles), octene-1 (35 g., 0.31 mole) and t-butyl wt. 421) were obtained. peroxide (4.4 g., 0.03 mole) was heated in a glassButanol-2 (247 g., 3.33 mole), octene-1 (26.3 g., lined, stainless-steel autoclave a t 115-1 18' for 40 0.24 mole) and t-butyl peroxide (5 ml.) a t 117-118" hours. Distillation, after the removal of t-butyl for 40 hours gave 3-methylundecanol-3 (13.3 g., alcohol and unreacted ethanol, gave decanol-2 h.p. 58-60" a t 0.2 mm.; % z f l ~ 1.4418). (11.6 g., b.p. Fi2-54" :It 1 inm.; n% 1.4357; Antel. Calctl. for C12HL'GO: C, 77.35; H, 14.07; mol. wt. 152; 111.1). of its a--iiaplitliylurethane, Found: C, 77.70; H, 14.18; mol. 69°)233,a product formed by the reaction of two riiol. wl., 1%. Cf. W. 11. Urry, 0 . 0. .luvelmd aird I:. W.Stacey, THISJ O U R . 74, 6155 (1952). (2) D. W. Adnmson and J. Kciiuer, J . C h c i ! ~A. m . , 8.1'2 (1994). (3) R . €1. Pickurd and J. Kenyon, ibid.. 99, 5; (1911). (I) NAL,
rLt., 191.
It
WdS
compared as above with 3-methylundec-
(4) hI. b ~ h d l d Z C l 1 .W II U l l y atid 14, 218 (1343)
B. A I
~ ~ I d L r I l Jd O i g
Chew
