The Biosynthesis of Steryl Glucosides in Plants

Abstract

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 ...

Effect of Filipin on Liposomes Prepared with Different Types of Steroids

“…The identification of fractions 3 and 4 were confirmed by hydrolyzing the acyl steryl glycoside fraction for 3 hr at 40 C with 0.5 N NaOH. Mild alkaline hydrolysis removes the fatty acid from the acyl steryl glycoside, converting it to steryl glycoside (22). Rechromatographing on a thin layer plate and on a silicic acid column showed that the acyl steryl glycoside fraction 3 was converted to steryl glycoside, fraction 4 (Fig.…”

Comparative studies of biosynthesis of galactolipids in Euglena gracilis strain Z.

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Biosynthesis of galactolipids in spinach chloroplasts has been reported (2,5). In these studies uridine diphosphate galactose was used as a galactosyl donor to some endogenous acceptor (diglyceride) (Branson Instruments Inc.).

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Free Fatty Acids Regulate Two Galactosyltransferases in Chloroplast Envelope Membranes Isolated from Spinach Leaves

“…This is favorable to the production of 1,2-DG from MGDG, but unfavorable to the reverse production of MGDG from 1 ,2-DG. It has been shown that incorporation of UDP-Gal into MGDG is gradually decreased, but incorporation into oligogalactolipids is steadily increased or accelerated when chloroplasts (9,19) and envelopes (5) are incubated for prolonged periods. This observation may be partly accounted for by the inhibition of UDGT and the stimulation of GGGT activity by FFA, since FFA is known to be readily produced by aging of chloroplasts (13,17,22).…”

“…The former 'Abbreviations: MGDG, DGDG, TGDG, and TTGDG, mono-, di-, tri-, and tetragalactosyldiacylglycerol, respectively; 1,2-DG, 1,2-diacylglycerol; UDGT, UDP-galactose: 1,2-diacylglycerol galactosyltransferase; GGGT, galactolipid:galactolipid galactosyltransferase; TG, triacylglycerol; FFA, free fatty acid; 16:3, hexadecatrienoic acid; 18:3, a-linolenic acid; 16:0, palmitic acid; 16:1, palmitoleic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid. reaction is catalyzed by UDGT (5,11,19) and the latter by GGGT (4,10,26). GGGT can also synthesize TGDG and TTGDG, normally absent in plant leaves in vivo (5, 26).…”

“…The final process ofthe galactolipid synthesis proceeds by two galactosylation steps of 1,2-DG to MGDG and MGDG to DGDG in the envelope membranes (6,14). The former 'Abbreviations: MGDG, DGDG, TGDG, and TTGDG, mono-, di-, tri-, and tetragalactosyldiacylglycerol, respectively; 1,2-DG, 1,2-diacylglycerol; UDGT, UDP-galactose: 1,2-diacylglycerol galactosyltransferase; GGGT reaction is catalyzed by UDGT (5,11,19) and the latter by GGGT (4,10,26). GGGT can also synthesize TGDG and TTGDG, normally absent in plant leaves in vivo (5,26).…”