UDP-galactose-'4C, uniformly labeled in the galactose moiety, and UDP-glucose-l4C, uniformly labeled in the glucose moiety, were purchased from New England Nuclear. UDP-galactose-'2C was purchased from Calbiochem. UDP-glucose-12C was purchased from Pabst. Silica Gel G was obtained from Brinkman and silicic acid (Bio-Sil A, 100-200 mesh) was obtained from Calbiochem.Steryl glucosides have been known to exist in plants for some time (21), but the existence of acylated steryl glucosides has been recognized only recently (15). The acylated steryl glucosides have since been studied by Kiribuchi et al. (13,14), and. some aspects of their biosynthesis have been described by Hou et al. (10,11) and Kauss (12). Eichenberger and Menke (5) have made a study of the distribution of free sterols, steryl glycosides, and steryl esters in the subcellular fractions of leaves. They discovered that although half of the leaf lipid was localized in the chloroplast, only one quarter of the total leaf sterol was found in this subcellular organelle. The sugars found in the steryl glycosides were glucose and mannose, and the fatty acid found in the steryl esters was palmitic acid (5). No further information appears to be available concerning the subcellular distribution, and no physiological function has been ascribed to the free sterols and sterol derivatives in plants.In an earlier investigation on the biosynthesis of galactolipids in plants (20)
METHODSPreparation of Pea Root Mitochondrial Fraction. Pea seeds were soaked for 16 hr in water before being planted in trays of wet vermiculite. Germination and growth took place in darkness for 5 to 6 days, and the trays were then transferred to light (14 hr of illumination per day). The plants were normally harvested 10 days after germination. The roots were taken and washed with distilled water and then cut -into small pieces. The roots (e.g., 30 g) were ground with mortar and pestle in cold 0.5 M sucrose which was 0.01 M with respect to phosphate buffer, pH 7.4 (e.g., 30 ml). The homogenate was filtered through cheesecloth and then centrifuged at 1,OOOg for 2 min. The pellet was discarded and the supematant centrifuged at 18,000g for 15 min. The pellet was resuspended in the sucrose-phosphate solution used for homogenization and the pellet was centrifuged down again at 18,000g. This washed pellet was resuspended in 0.01 M tris-HCl buffer, pH 7.4 (e.g., 6 ml), with the aid of a glass Potter-Elvehjem homogenizer.Preparation of Spinach Chloroplast Fraction. Spinach was taken from the greenhouse, and the leaves were washed with distilled water. Petioles and midribs were removed, and the remainder of the leaves (e.g., 30 g) were ground with a mortar and pestle in cold 0.5 M sucrose, 0.01 M with respect to phosphate buffer, pH 7.4 (e.g., 30 ml). The homogenate was filtered through four layers of cheesecloth, and this filtrate was centrifuged at 200g for 2 min. The pellet was discarded, and the supernatant was centrifuged at lOOOg for 7 min. The supernatant was carefully removed, and the pellet ...