The properties of a discrete membranous fraction isolated on sucrose gradients from castor bean endosperm have been examined . This fraction was previously shown to be the exclusive site of phosphorylcholine-glyceride transferase . The distribution of NADPH-cytochrome c reductase and antimycin insensitive NADH-cytochrome c reductase across the gradient followed closely that of the phosphorylcholine-glyceride transferase . This fraction also had NADH diaphorase activity and contained cytochromes b5 and P 450. On sucrose gradients containing 1 mM EDTA this fraction had a mean isopycnic density of 1 .12 g/cma and sedimented separately from the ribosomes ; electron micrographs showed that it was comprised of smooth membranes . When magnesium was included in the gradients to prevent the dissociation of membrane-bound ribosomes, the isopycnic density of the membrane fraction with its associated enzymes was increased to 1 .16 g/cma and under these conditions the electron micrographs showed that the membranes had the typical appearance of rough endoplasmic reticulum . Together these data show that the endoplasmic reticulum is the exclusive site of lecithin formation in the castor bean endosperm and establish a central role for this cytoplasmic component in the biogenesis of cell membranes .
A selection of C4 species was surveyed to determine the relationship between their content of C4 acid decarboxylating enzymes, the activities of several other enzymes implicated in the C4 pathway, and their anatomical and ultrastructural features. The species examined clearly fell into three groups according to whether they contained high levels of either NADP malic enzyme (EC 1.1.1.40), phosphoenolpyruvate carboxykinase (EC 4.1.1.49) or NAD malic enzyme (EC 1.1 .l.39). The occurrence of high NADP malic enzyme activity was always associated with higher NADP malate dehydrogenase activity, while those species distinguished by high activities of either of the other two decarboxylases invariably contained high aspartate aminotransferase and alanine aminotransferase activities. Each of these decarboxylating enzymes was located in bundle sheath cells. NAD malic enzyme, but not phosphoenolpyruvate carboxykinase, was associated with mitochondria.Light and electron micrographs revealed differences between these groups with respect to the intracellular location of chloroplasts and mitochondria in bundle sheath cells, and the content and ultrastructure of mitochondria. The trend was for species with high NAD malic enzyme to contain the most mitochondria in the bundle sheath cells with apparently the most extensively developed cristae membrane systems. However, mitochondria1 respiratory enzyme activities were similar for the three groups of species.The b&c simila&ies and differences between the three groups of C4 plants distinguished by their differing C, acid decarboxylating systems are discussed, and schemes for the probable photosynthetic reactions in bundle sheath cells are presented. A nomenclature to distinguish between these groups is proposed. * Abbreviations used: RuDP, ribulose 1,5-diphosphate; PEP, phosphoenolpyruvate; 3-PGA, 3-phosphoglyceric acid; DTT, dithiothreitol; HEPES, N-2-hydroxyethylpiperazine-Nf-2-ethanesulphonic acid.
The occurrence and subcellular distribution of enzymes of the cytidine diphosphate choline pathway of lecithin synthesis have been examined. Choline kinase (EC 2.7.1.32) was completely soluble, while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine-glyceride transferase (EC 2.7.8.2) were associated with particulate fractions. Although components sedimenting at 10,000 to 100,000 X g contained both enzymes, phosphorylcholine-cytidyl transferase and particularly phosphorylcholine-glyceride transferase were present in the 10,000 X g pellet, which contained the major organelles, mitochondria, and glyoxysomes. When the crude homogenate was centrifuged on a sucrose density gradient, four major bands of particulate protein were recovered. A band at density 1.24 g/cm3 contained the glyoxysomes and was devoid of phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase activity. Enzyme activity was barely detectable in the mitochondria, at density 1.18 g/cm3. Phosphorylcholineglyceride transferase was found almost exclusively in a sharp band at density 1.12 g/cm3, and phosphorylcholinecytidyl transferase was found in the uppermost band at density 1.08 g/cm3. Thus, for the synthesis of lecithin in their membranes, the glyoxysomes and mitochondria depend on enzymes elsewhere in the cell; the final two steps in lecithin formation occur, apparently exclusively, in separate particulate cell components.Recent studies have shown that ['4C]choline is an effective precursor of membrane-bound lecithin in endosperm tissue of germinating seedlings of castor bean (1). Labeled membrane components were separated after application of ['4C]choline by centrifugation of homogenates on linear 30-60% (w/w) sucrose density gradients. Three particulate bands were separated on such gradients: a membranous fraction at a buoyant density of 1.12-1.13 g/cm3, mitochondria (1.18 g/cm3), and glyoxysomes (1.24 g/cm3).The time course of [14C]choline incorporation into these cellular components showed that the membranous fraction acquired [14C]lecithin before the mitochondria and glyoxysomes. Apparently, the final stage of lecithin biosynthesis occurred in components of the membranous fraction, and the preformed phospholipid served as a precursor for the formation of mitochondrial and glyoxysomal membranes. Accordingly, we have investigated the distribution of enzymes involved in lecithin synthesis among cell fractions separated from castor bean endosperm.A major pathway for the synthesis of lecithin from choline has been described by Kennedy (2). The sequence involves three enzyme-catalyzed steps: (a) the ATP-dependent phosphorylation of choline to phosphorylcholine catalyzed by (3)(4)(5), and the importance of the pathway in relation to the synthesis of membranous cell constituents has been discussed (4, 5).In this paper, we report the compartmentation of PCholcytidyl transferase and PChol-glyceride transferase in two distinct components of the light membrane fraction, and we discuss their role i...
The origin and turnover of organelle membranes in castor bean (Ricinus communis L. var. Hale) endosperm was examined using choline-"C as a phospholipid precursor. On sucrose gradients three major particulate fractions were separated; a light membranous fraction (density 1.11-1.13 gram per cm3), the mitochondria (1.18 gram per cm3), and the glyoxysomes (1.24 gram per cm'). Choline-14C was readily incorporated into lecithin in all three particulate fractions, but the light membranous fraction became labeled first. Incorporation continued into all three fractions for 6 hours, at which time the available choline-"4C had been completely used. Subsequently, 14C was lost from the three components at distinctly different rates. When an excess of unlabeled choline was added after 1 hour (pulse-chase experiment), incorporation of choline-14C into glyoxysomes and mitochondria continued for three hours, but at a diminishing rate. This was followed by a period in which the 14C content of the mitochondria declined at a rate expected, if the half life of lecithin in the membrane were about 50 hours and that of the glyoxysomes 10 hours. These values are close to those calculated from the experiments in which no chase was used. The labeling in the light membrane fraction behaved differently from that of the mitochondria and glyoxysomes following the chase of unlabeled choline. Incorporation continued for only 1 additional hour, and then the "C content declined sharply in the subsequent 4 hours. The early kinetics and subsequent interrelationships are those expected if the lecithin in the membranes of mitochondria and glyoxysomes originates in components of the light membrane fraction.A striking increase in organelles housing enzymes of the gluconeogenic pathway occurs during the onset of fat breakdown in seedlings (4, 8, 11-13, 17, 20, 21). In the endosperm of castor bean seeds germinating at 30 C, the numbers of glyoxysomes and mitochondria increase during the first 5 days and decline when fat utilization is complete (8). The mechanism of biogenesis, maintenance, and destruction of these organelles is of interest. One approach is to examine the turnover of proteins and individual enzymes (9, 16). Another consideration is the behavior of the membranes surrounding the organelles. The effectiveness of choline as a precursor of membrane 'This work was supported by National Science Foundation Grant GB 24961. 61 constituents has already been demonstrated in several plant systems (3, 7, 14).Here we examine the origin and turnover of organelle membranes as revealed by the incorporation of choline-"C into lecithin. MATERIALS AND METHODSPlant Material. Seeds of castor bean (Ricinus communis L. var. Hale) were soaked in running water for 1 day and grown in moist vermiculite in darkness at 30 C. Seedlings of particular ages were carefully chosen for uniformity, and the seeds were detached at the hypocotyl. The testae were removed, and the seeds were surface sterilized by immersion for 10 sec in 30% (v/v) Clorox solution and 30...
The intracellular location of several enzymes concerned with phospholipid metabolism was investigated by examining their distribution in organelles separated on sucrose gradients from total homogenates of castor bean (Ricinus communis var. Hale) endosperm. The enzymes phosphatidic acid phosphatase, CDP-diglyceride-inositol transferase, and phosphatidylethanolamine-L-serine phosphatidyl transferase were all primarily or exclusively confined to membranes of the endoplasmic reticulum. These results and those reported previously on lecithin synthesis establish a major role of the endoplasmic reticulum in phospholipid and membrane synthesis in plant tissues.
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