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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 ...

Stocking, C. Ralph, and Alpaslan Ongun. (U. California, Davis.) The intracellular distribution of some metallic elements in leaves. Amer. Jour. Bot. 49(3): 284–289. Illus. 1962.—A comparison of the potassium, sodium, calcium, magnesium, and nitrogen levels of chloroplasts isolated in an aqueous sucrose medium and by the nonaqueous method reveals that large amounts of these elements are lost from the plastids during their isolation in sucrose solution. Nonaqueously isolated chloroplasts of bean and tobacco leaves contained over 40% of the total leaf potassium, magnesium, and calcium. High levels of nitrogen up to 70% of the total leaf nitrogen were found associated with nonaqueously isolated chloroplasts. As much as 40–50% of this nitrogen is lost when chloroplasts are isolated in a sucrose solution. Electron photomicrographs of nonaqueously isolated chloroplasts reveal only a partial disruption of the internal structure and a loss of the plastid membrane during isolation.

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...

Evidence for the close relationshil) of proteini synthesis in green tissue anld the process of photosynthesis has been accumulating in recent years (1. 3,4,8). Hellebust and Bidwell (5) The complete operation of opening the chamnber, removing the leaf, anid immersing it in liquid N2 could be done in less than 5 seconds. MXethods for nonaqueously isolating the chloroplasts, extraction of the products of photosynthesis, separating the products oni resini exchange coluinis alnd by paper chromatograplhy have been described previously (9).The serine feeding experiments were conducted in the following way. The petiole of the leaf was cut under water. The freshly cut end of the petiole was immlierse(l in 2.25 nil of L-serine-3-C'4 solution (6.6 mc/mM or 50 lic/ml). The leaf with its petiole imirersed in the serine was placed in a vacuuml chamlber and a partial vacuum was pulled for 20 seconds in the dark. The leaf was then kept in the dark at room temperature for 20 minutes to allow conmplete u)take of the serine. After the 20 minutes feeding, 1 lot of leaves was frozen with liquid nitrogen as controls. a seconid lot of leaves was placed in the dark for an additional 20 minutes, and a third lot was placed in the light at 2000 ft-c for 20 minutes. The leaves were frozen xvith liquid nitrogen at the enld of the treatment period and then freeze dried. The chloroplasts were isolated nonaqueously froml the freeze dried tissue and the products of serine metabolislmi were determined (9).

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