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Diacylgalactosylglycerol synthesis was a prerequisite for the incorporation of [1-14C]-acetate into linoleate and alpha-linolenate of isolated spinach (Spinacia oleracea) chloroplasts. Oleate at position 1 of diacylgalactosylglycerol was desaturated to linoleate and alpha-linolenate both in the light and in the dark. Some desaturation of palmitate was also observed after prolonged incubations.

In a horse serum-based medium containing a full complement of fatty acids, cells of Spiroplasma citri were seen to preferentially incorporate palmitic acid. In the same medium, which had a steryl ester-to-sterol ratio of 3.64, a steryl ester-to-sterol ratio of 0.23 was seen in the cells, cholesterol being preferentially incorporated over cholesteryl ester. Like most other mycoplasmas, S. citri was shown to be unable to synthesize fatty acids or esterify cholesterol. The neutral lipids of S. citri grown in a medium containing horse serum consisted of free cholesterol, cholesteryl ester, free fatty acids, triglycerides and diglycerides. All polar lipids were phospholipids, with no glycolipids detected. These phospholipids, which are characteristic of many mycoplasmas, are phosphatidyl glycerol, diphosphatidyl glycerol, and their lyso derivatives. Sphingomyelin was also incorporated when cells were grown on horse serum. A sterol requirement for the growth ofS. citri was confirmed using a serum-free medium supplemented with bovine serum albumin, palmitic acid, and various concentrations of sterols dissolved in Tween 80. The addition of palmitic acid stimulated growth but was not essential for growth. S. citri was shown to grow best on cholesterol and ,Bsitosterol and was able to grow on stigmasterol and ergosterol to a lesser degree. No growth was obtained using mevalonate, deoxycholate, or taurodeoxycholate as an alternative to sterol. S. citri was also able to grow when palmitic acid was replaced with oleic acid, linoleic acid, or linolenic acid. Alterations in the lipid composition of the growth medium and hence in the lipid composition of S. citri induced changes in the characteristic helical morphology of the cells, concurrent with loss of cell viability. Culture, age, and pH were also factors in determining cell morphology and viability.

The metabolism of uridine 5'-pyrophosphate-galactose by spinach (Spinacia oleracea) chloroplast preparations was inhibited by ozone. The formation of digalactosyl diglyceride and trigalactosyl diglyceride was inhibited much more than the formation of monogalactosyl diglyceride, steryl glycoside, and acylated steryl glycoside. Essentially identical results were obtained when glycolipid synthesis was inhibited by N-ethyl maleimide, p-hydroxymercuribenzoate, and CdCL. lodoacetate and iodoacetamide affected neither the total incorporation of sugar from uridine 5'-pyrophosphate-galactose nor distribution of the incorporated sugar in the various glycolipids.Ozone reacted with model membrane systems prepared with egg lecithin. In the absence of reduced glutathione, products included malonaldehyde and hydrogen peroxide. In the presence of glutathione, malonaldehyde was still produced, but the glutathione was oxidized and no peroxide was detected. When these studies were extended to chloroplast preparations, it was also found that malonaldehyde was produced and glutathione was oxidized.It was concluded that ozone inhibits glycolipid biosynthesis in chloroplast preparations by way of oxidation of enzyme sulfhydryl groups but that this reaction may be a secondary effect of oxidation of unsaturated fatty acids.The synthesis of galactosyl diglycerides by cell-free preparations from leaves of higher plants was first reported by Neufeld and Hall (18). Their results have been substantiated and extended by subsequent work. This work has shown that in addition to the predominant naturally occurring monogalactosyl diglyceride and digalactosyl diglyceride, there is at least one additional compound, trigalactosyl diglyceride, which becomes labeled during the incubation of chloroplast preparations with 4C-UDP galactose (11,17,20).The ratio of monogalactosyl diglyceride, digalactosyl diglyceride, and trigalactosyl diglyceride, synthesized by cell-free preparations, can be changed by variations in pH and temperature (17 MATERIALS AND METHODS Chloroplasts. Chloroplasts were prepared from spinach (Spinacia oleracea) purchased at local markets. The leaves were washed in distilled water and the petioles and midribs were removed. The leaves (75 g) were ground in a Waring Blendor (70 v, 5 sec) with an equal weight of ice-cold homogenizing medium (0.5 M sucrose, 10 mm phosphate, pH 7.5). The homogenate was filtered through cheesecloth and the filtrate was centrifuged at 200g for 2 min. The pellet was discarded and the supernatant was centrifuged at 1 OOg for 7 min. The supernatant was discarded and the pellet was resuspended in 0.1 M phosphate, pH 7.5. Chlorophyll concentration was determined by the method of Arnon (2), and the chloroplast suspension was diluted so that addition to the reaction mixtures would give 1 mg of chlorophyll per vessel.Reaction Mixtures. Reaction mixtures for the study of glycolipid biosynthesis consisted of 100 ,umoles of tris-Cl, pH 7.5, or phosphate, pH 7.5; 0.4 ml of the broken chloroplast suspension con...

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