176 PRELIMINARY NOTES

Synthesis of a new phosphatide from monoglyceride and adenosine triphosphate

It was recently shown that phosphatidic acid is synthesized from diglycerides and adenosine triphosphate in dialyzed deoxycholate extracts of brain microsomes 1. Using the same enzyme preparation but substituting monoglyceride for diglyceride a new radioactive phospholipide spot appeared below the phosphatidic acid in the paper chromatographic system of MARINETTI ct al. 2. This spot had not been seen previously on autoradiograms of chromatograms of total phosphatides labeled with a2p either in vivo or in vitro. In brain preparations the formation of this phosphatide was completely dependent on the presence of a monoglyceride (Table I, Expt. I).

T A B L E I

F O R M A T I O N O F P H O S P H A T I D I ' . ' S IN D E O X Y C H O L A T E E X T R A C T S O F

G U I N E A - P I G B R A I N M 1 C R O S O M E S

The incuba t ion condi t ions and the me thod of i so la t ion of the phospha t i de s were the same as repor ted p rev ious ly 1. The a m o u n t of p h o s p h a t i d e syn thes ized is t h a t ob ta ined from an a l iquo t r ep resen t ing one - th i r t i e th of the to ta l yield of mic rosomal e x t r a c t from the cerebral hemisphe re s of one gu inea pig. o.oo~, M diolein, o.oo2 M mono61ein, o.ooo2 31 sodium oleate, o.oooi M CoA,

o.ooo 5 M g lu ta th ione .

( ~uantity O/phosphatide synthesized

LPA* PA*" m~moles ml~moles

Expt. •

Control ll.d. * * * 0.065 l ) io le in n.d. 0.280 Mono61ein 0.380 0.324 Mono61ein + sodium oleate o.4~o o.2oi Mono61ein + ( 'oA + g lu t a th ione o.61o o.193 Monoi}leiri + sodium oleate + ( 'oA -~ gll t a th ione 0.650 0.254 Sodium oleate n.d. o.067 Sodium olea te + Co-\ + g lu ta th ion~ n.d. 0.024

Expt. 2

Mono61ein + sodium oleate o. I 14 o. 104 Mono61ein -I- sod ium oleate + CoA + g lu t a th ione 0.230 o . i67 Mono61ein + sodium oleate + g lu t a th ione 0.240 o.183

• P h o s p h a t i d e p re sumed to be ly sophospha t id i c acid. • * I ' hospha t id i c acid.

* * * Not de tec ted (the syn thes i s of 0.03 m/ imole would have been de tec ted on the au to rad iograms) .

I)iglyceride or fa t ty acids plus CoA were ineffective as substrates for the formation of the phosphatide. Higher concentratibns of fa t ty acid than those shown in Table I were inhibitor 3 CvA plus glutathione ,:nhanced the synthesis of the phosphatide when monoiilein was incubated with dialyzed deoxycholate extracts of guinea-pig-brain microsomes. No stimulation was observed with similar preparations from frozen calf brain. The .~timulatory effect of CoA plus glutatifione in guinea-pig preparations could be obtained with glutathiolle alone (Table I, Expt. 2), suggesting that the

A b b r e v i a t i o n : ( 'oA, co,enzyme A.

llioctdn~. Biophys. Acta, 37 (~96o) 176--177

PRELIMINARY NOTES 177

synthesis of the phosphatide was not dependent on CoA. The glutathione effect is presumably due to the protection of sulfhydryl groups on the enzyme. The quanti ty of the new phosphatide synthesized in the enzyme preparations was insufficient for characterization by conventional means. However, the following experiments indicate that it is a lysophosphatidic acid. Carrier lysophosphatidic acid was synthesized by the method of KABASHIMA 3 and purified by silicic acid column chromatography according to HANAHAN et al. 4. I t eluted from the column in the chloroform-methanol (3:2, v/v) fraction. I t had a phosphorus-ester ratio of 1:0.9 8. The unknown radio- active phosphatide could be isolated in the same manner by silicic acid column chromatography. When it was mixed with carrier lysophosphatidic acid and the mixture rechromatographed on the column, the phosphorus peak and the radioactive peak coincided. The radioactive spot had the same RF as the stained lysophosphatidic acid spot in the paper-chromatographic systems of MARINETTI et al. 2, BEISS AND ARMBRUSTER 5 (the spots were elongated in this system), and in butanol-ammonia (equal volumes of butanol and 2 N NH~OH were shaken, and the upper phase was used as the solvent with silicic acid impregnated paper).

The synthesis of phosphatidic acid was always increased on adding monoelein, which suggested that lysophosphatidic acid was a precursor for the phosphatidic acid. This would be expected if the acylating enzyme of KORNBERG AND PRICER 6 were present in the enzyme preparation.

KENNEDY and his associates 7 have presented evidence which indicates that phosphatidic acid is an important intermediate in lipid biosynthesis. The studies of HOKIN AND HOKIN 8,9 indicate that phosphatidic acid plays a role in transmembrane transport. I t has already been shown that phosphatidic acid can be formed from a-glycerophosphate and fat ty acyl CoA 6 or from diglyceride and adenosine triphos- phatO. The formation of phosphatidic acid from monoglyceride via lysophosphatidic acid would provide yet another pathway for the synthesis of this important phos- phatide. This would also provide a means for direct utilization of monoglyceride for lipid biosynthesis; this would be consonant with the recent studies of SKIPSKY et al. 1°,

who showed that monoglyceride can be absorbed intact from the intestinal tract. I t would also be of interest to know whether lysophosphatidic acid might be an inter- mediate in the synthesis of phosphatides such as lysolecithin.

Department o~ Physiological Chemistry, University oi lVisconsin, LOWELL E. HOKIN Madison, Wise. (U.S.A.) MABEL R. HOKIN

1 M. R. HOKIN AND L, E. HOKIN, J. Biol. Chem., 234 (1959) I318. ,2 G. V, M~.~INETII, J. ERBLAND AND J. KOCHEN, Federation Proe., 15 (1956) 837. 3 I. ]{ABASHIMA, Ber., 71 (1938) lo73. 4 1). J. HANAHAN, J. C. DITT,'~ER AND E. \VARASHINA, J. Biol. Chem., 228 (1957) 685.

U. BEIsS AND O. ARMBRUSTER, Z. Natur/orsch., 136 (1958) 79. 6 A. KORNBERG AND \V. E. PRICER, J. Biol. Chem., 2o 4 (I953) 329, 345. v E. P, KENNED'~', AnJz. ReiJ. Biochem., 26 (1957) 119. s L. E. HOI<tN AND M. R. FIoKIN, J. Biol. Chem., 234 (1959) 1387 . 9 L. E. HOKIN AND M. t{. HOKIN, Nature, ~84 (1959) lO68.

10 U. P. SKIPSKI, M. G. MOREHOUSE AND H. J. I)EtYEL, JR., Arch. Biochem. Biophys., 8i (I959) 93.

Received October 27th, I959

Biochim. Biophys. . lcla, 37 (19(~o) 176 177