March 20, 1952
COMMUNICATIONS TO THE EDITOR
1G17
counts in Zones 8-10 is unexplained and may represent an additional component.) Indications of two distinct compounds with C14/P32ratios differing by a factor of 2 were also obtained with acetone DEPARTMENT O F PHARMACOLOGY and aqueous n-butanol as developing solvents. WESTERNRESERVEUNIVERSITY THOMAS G. BIDDER Treatment of the reaction products with 0.2 N CLEVELAND 6, OHIO RECEIVED JANUARY 2, 1952 HC1 in ethanol or 0.1 N NaOH in ethanol a t 75’ for one hour resulted in a quantitative conversion of the components in Zones 1-4 to free stearic and glycerophosphoric acids (as judged by paper chroENZYMATIC SYNTHESIS OF PHOSPHORUS-CONTAINING LIPIDES matography with the above-mentioned solvents and also with chloroform). Sir : The formation of phosphorus-containing lipid TABLE I1 substances from L-a-glycerophosphate (a-GP) and OF THE REACTION PRODUCTS PAPERCHROMATOGRAM long-chain fatty acids is catalyzed by a partially Incubation conditions were as in Table I except for the inpurified enzyme preparation from rat liver. The clusion of 0.8 p curie of C14 stearic acid. The ethanol exreaction has been followed by measuring the in- tract (5 ml.) of the acid-washed residue was adjusted to pH 4 corporation of a-GP labeled with P32into a “phos- and concentrated to 0.1 ml. (under a stream of helium a t pholipid fraction” (it?.,an ethanol extract of an room temperature). An aliquot (0.02 ml.) was chromatoon Whatman No. 1 filter paper with diisopropyl acid-washed residue precipitated from the incuba- graphed ether as the solvent. The front advanced 26.5 cm. in two tion mixture by perchloric acid). With enzyme hours. The paper was divided into eleven 2.4-cm. zones purified 4-fold from homogenates by treatment with and analyzed for CI4 and P32counts; zone 1 contains the calcium phosphate gel or by fractionation with point of origin. Zone 1 2 3 4 6 methanol a t low temperature, the system requires 10’ counts per minute adenosine triphosphate (ATP), coenzyme A (CoA) C“ 1.72 0.86 0.79 1.15 0.02 0 06 and stearic acid (Table I). The latter could be PJ2 1.11 .62 .85 1.64 .03 .O1 replaced by fatty acids with chain lengths of 12 0.7 to 18 carbon atoms (including oleate and linoleate). C14/P32 1 . 6 Labeled inorganic orthophosphate was incorporated Zone 7 8 I) 10 11 108 counts per minute only slightly. Addition of choline (0.0025 M ) , 5.66 0.00 0.21 6.47 16.4 phosphorylcholine (0.002 M ) , glycerophosphoryl- C“ 0.11 . 00 0.59 0.64 -02 choline (0.002 M ) or glycerol (0.05 M ) did not in- P32 fluence the incorporation of C ~ - G P ~ ~ . In view of the participation of ATP and Co4 in Two reaction products, tentatively designated as this system, a mechanism analogous to that prophosphatidic acids, were observed on paper chromatographic analysis of the products resulting from posed for the ATP-CoA activation of acetate (i.e., incubation of a-GP32in the presence of stearate- in choline esterification) may be postulated : l-C14 (Table 11). The area occupied by free ATP stearate (Zones 8-10) is widely separated from the (1) Stearate CoA -+ Stearyl-CoA bulk (75%) of the P32 products (Zones 1 4 ) . (2) Stearyl-CoA a-GP + monostearylphosphatidic acid CoA Zones 1 and 4 each represent distinct peaks for both C14 and P32counts. The ratios of C14 to P3* (3) Stearyl-CoA monostearylphosphatidic acid + counts in the peak zones suggest two compounds, distearylphosphatidic acid -t CoA one containing twice as much stearate per mole of phosphate as the other. (The nature of the P32 Resolution of the enzyme system into the postulated components and identification of stearyl-CoA are required to validate this mechanism. The TABLE I INCORPORATION OF a-GP3’ INTO AN ETHANOL FRACTION possible relation of these findings to the mechanism of esterification of other alcohols (;,e., sterols, The complete incubation mixture (1.0 ml.) contained 0.1 ml. of a-GPS2(0.02 M , approximately lo5counts per minute) glycerol) by long-chain fatty acids is apparent. It is pertinent to consider the relation of these 0.2 ml. of ATP (0.03 M ) , 0.05 ml. of CoA (200 units/ml., 50% pure), 0.1 ml. of stearate (0.02 M , adjusted to PH 9 results to lecithin and cephalin biosynthesis, the with “,OH), 0.05 ml. of cysteine (0.2 M ) , 0.1 ml. of phos- nature of which has been obscure. I t now appears phate buffer (0.5 M,pH 7.0), 0.2 ml. of water and 0.2 ml. plausible that phosphatidic acids may serve as preof an enzyme fraction (9 mg. protein/ml.). The latter is prepared from a 0.25 M sucrose homogenate (20 mg. of cursors, reacting with phosphate esters of the protein/ml.) by collecting a fraction precipitated by meth; nitrogenous bases. Preliminary observations2 on anol (between 7 and 20%) a t -5 . Incubation was a t 22 the enzymatic incorporation of phosphorylcholine for 30 min. into a phospholipide fraction are consistent with Ethanol extract such a scheme. (108 c.p.m.)
and Harland G. Wood is gratefully acknowledged. Dr. H. S. Isbell, National Bureau of Standards, kindly supplied the l-C14-glucose.
J
+
+ +
+
’
Complete systcni Enzyme heated 2 min. a t 50’ Without ATP Without CoA Without stearate a-GP in place of a-GP”; 1 0 6 c.p.m. Pa*as inorganic pliosplinte
10.60 0.00 0.24 1.82 2.30 0 02
NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES ARTHUR KORNBERG NATIONAL INSTITUTES OF HEALTH W. E. PRICER,JR. BETHESDA, MARYLAND RECEIVED FEBRUARY 20, 1952 (1) For review and primary references see 11, A. R u r k e r i n “Pliospllorus Mctal,olisiii,” Vol. I , 13;iItiiiiore. Md.. 1!151. (2) A. Koriilrcrg arid \V. E. Pricrr, J r . , P r d r r u l i u n P r o r . . i i i p w s i .
