Biosynthesis of Actinomycin Chromophore

substance was also hydrolyzed lvith acid to split vinyl ethers (13). The products were partitioned between ether and water and cr-glycols determined in each phase. Saponification released considerable amounts of glycerol (Table I, 3b). Thus Eluate C1 from the silicic acid column apparently still contained some ester triglycerides. The ether-soluble fraction contained glycols which yielded on acid treatment a watersoluble glycol shown above to be glycerol (Table I, 4b). I f it is assumed that the glycols of the unsaponifiable fraction were originally esterified by two fatty acids and that the glycerol of Table I, 3b was esterified by three fatty acids, one accounts for 91% of the fatty acid ester groups of Eluate C1. From the unsaponifiable fraction, 1.1 moles of glycerol were released by acid hydrolysis per mole of plasmalogen (measured as fatty aldehyde (13)) in Eluate C1 (Table I, 2 and 4). It was reasoned that mild acid hydrolysis of Eluate C1 without prior saponification should release a diglyceride. A sample of Eluate C1 was subjected directly to hydrolysis according to Kiyasu and Kennedy (15), and the products were partitioned between ether and water. The ether-soluble material was rechromatographed on silicic acid as above (Column 2). Three eluates were analyzed. These were Eluates Bz and CZ, defined as for Column I, and Eluate Dz, diethyl ether, which removed diglycerides. The behavior of free fatty aldehydes (stearaldehyde) and diglycerides (diolein) on this column had previously been established with model mixtures. Free fatty aldehyde was found to be present in Eluate Bz. Of the ester fatty acids of Eluate CZ, 94% were accounted for, determined as for Eluate C1. Eluate Dz contained 1.74 pmoles of fatty acid ester groups. An aliquot of Eluate Dz was saponified and 1.71 pmoles of fatty acids were recovered, measured titrimetrically (16). After extraction of fatty acids, 1.42 pmoles of formaldehyde, corresponding to 0.71 pmole of glycerol, were recovered in the aqueous saponification mixture. A fatty acid to glycerol ratio of 2.4 was thus obtained for Eluate Dz. Recovery of glycerol in the manipulations of the minute amounts of substance available might not have been complete, thereby resulting in the high ratio obtained. The position of Eluate Dz on Column 2, and the fact that a sample of this eluate examined by chromatography on siliconed paper (17) moved identically with a sample of authentic diolein (Rp 0.43) further indicate that it is a diglyceride. After this work was completed, nonphosphatide aldehydogenic lipids were reported in the unsaponifiable fractions of milk fat, beef tallow, and oxheart lipids (19). These substances are apparently derived from native neutral plasmalogens. The data concerning aldehydogenic lipids of unsaponifiable fractions in both studies are in agreement. A wide distribution of neutral plasmalogens in nature is suggested by the work so far reported. It appears that there is now evidence for three categories of glycerol-containing lipids which exist as neutral lipids or as phosphatides: (a) conventional neutral triglycerides and glycerophosphatides; (b) glycerol ethers saturated (rp to the oxygen bridge (e.g. batyl alcohol), existing as fatty acid diesters, and the corresponding phosphatides (18) ; (c) fatty acid diesters of glycerolvinyl ether (neutral plasmalogens), and the well known phosphatide plasmalogens. Lipids of the batyl type might well be derived metabolically from the plasmalogens.