Phosphorylcholine Glyceride Transferase Research Articles

Organelle Membranes from Germinating Castor Bean Endosperm

Glyoxysome ghosts were isolated from germinating castor bean endosperms using established methods. Electron microscopic examination showed that some matrix material was retained within the glyoxysomal membrane. Two cytochrome reductases and phosphorylcholine glyceride transferase co-sedimented with the alkaline lipase, a known component of the glyoxysome membrane, in sucrose gradient centrifugation of osmotically shocked glyoxysomes. The activities of these enzymes in the glyoxysome membranes were compared to those in the endoplasmic reticulum relative to phospholipid content. On this basis, the phosphorylcholine glyceride transferase was 10-fold more active in the endoplasmic reticulum, whereas the lipase was 50-fold more active in the glyoxysome membrane. The cytochrome reductases were only 2-fold more active in the endoplasmic reticulum, indicating that they are components of the two membranes. Difference spectroscopy of the glyoxysome membrane suspension revealed the presence of a b5-type cytochrome similar to that found in the endoplasmic reticulum. Since the glyoxysome membrane is apparently derived from the endoplasmic reticulum, components of the endoplasmic reticulum such as these are likely to be incorporated into the glyoxysome membrane during biogenesis.Enzyme activities involving the cofactors NADH or CoA were measurable in broken, but not in intact, glyoxysomes. Thus, it appears that cofactors for enzymes within the organelle cannot pass through the membrane.

Rapid Degradation and Limited Synthesis of Phospholipids in the Cotyledons of Mung Bean Seedlings

Seedling growth of mung bean is accompanied by the rapid catabolism of the three major phospholipids in the cotyledons (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). The decline starts 24 hours after the beginning of imbibition and by the 4th day of growth more than 50% of the phospholipids have been catabolized. Extracts of cotyledons of 24-hour-imbibed beans contain enzymes capable of degrading membrane-associated phospholipids in vitro. This degradation involves phospholipase D and phosphatase activity.Studies with radioactive acetate, glycerol, and orthophosphate indicate that the three major phospholipids are also synthesized in the cotyledons. Incorporation of glycerol and acetate into phospholipids of cotyledons is relatively constant throughout seedling growth, while the incorporation of [(32)P]orthophosphate steadily declines from a high value 24 hours after the start of imbibition. The newly synthesized phospholipids become associated with membranous organelles, especially the endoplasmic reticulum, and have an in situ half-life of 2 to 2.5 days.Determination of the activities of two enzymes involved in phospholipid biosynthesis (phosphorylcholine-glyceride transferase and CDP-diglyceride-inositol transferase) shows that the enzymes have their highest activities 12 hours after the start of imbibition. High activities for both enzymes were found in cotyledons of beans incubated at 1 C, indicating that the enzymes may preexist in the dry seeds.The experiments demonstrate that cotyledons start synthesizing new phospholipids immediately after imbibition, but that the rate of phospholipid catabolism far exceeds the rate of synthesis long before the cotyledons start to senesce.

Organelle membranes from germinating castor bean endosperm

Endoplasmic reticulum, mitochondria, and glyoxysomes were obtained from germinating castor bean endosperm,Ricinus communis, by sucrose gradient centrifugation. When each of the three organelle preparations was diluted in 150 mM KCl and centrifuged, all of the component membrane material, measured as phospholipid, was sedimented. Also, the respective membrane enzymes, phosphorylcholine-glyceride transferase, cytochrome c oxidase and alkaline lipase were recovered. The endoplasmic reticulum retained most (60%) of its protein. The mitochondria lost almost no protein while the glyoxysomes lost much of their soluble contents.

Effect of Gibberellin A3 on the Endoplasmic Reticulum and on the Formation of Glyoxysomes in the Endosperm of Germinating Castor Bean

Seedlings of castor bean (Ricinus communis cv. Hale) were exposed to a range of concentrations of gibberellin A(3) (GA(3)). Treatments for 20 hours with GA(3) concentrations of 0.5 muM or higher resulted in increased levels of NADH-cytochrome c reductase, phosphorylcholine glyceride transferase, and malate synthase in endoplasmic reticulum (ER) isolated from endosperm on linear sucrose gradients. GA(3) treatment also resulted in increased RNA associated with ER. Malate synthase and catalase in crude homogenates were enhanced by 1 to 100 muM GA(3) concentrations. Isocitrate lyase, citrate synthase, malate synthase, catalase, and glycolate oxidase in isolated glyoxysomes were enhanced by 60, 20, 18, 40, and 28%, respectively, over controls. Treatment with abscisic acid led to decreased levels of glyoxysomal enzymes and reduced glyoxysomal protein. The effect of GA(3) and abscisic acid on the specific activities of glyoxysomes of different densities suggests that GA(3) influences enzyme levels and glyoxysome assembly.

Development and intracellular localization of lipase activity in rapessed (Brassica napus L.) cotyledons

In homogenates of resting rapeseeds no lipase activity (glycerolester hydrolase, EC 3.1.1.3) could be detected using a titrimetric assay procedure. Following a 30-h lag-phase after imbibition, lipase activity increased sharply, reaching its maximum at day 4 after sowing. Simultaneously triglyceride content of the cotyledons decreased sharply. At any time during the 11-day period of seedling growth examined, only an alkaline lipase activity with a pH optimum around 9 was present. White light had essentially no effect on the development of lipase activity. However, the disappearance of lipase activity from the cotyledons after fat utilization was found to depend on nitrogen nutrition of the seedlings. The activities of the glyoxysomal enzymes catalase and malate synthetase showed the usual rise and fall patterns with peak activities at day 4 after sowing, independently of the mineral nutrition of the seedlings.About 90% of the lipase activity was associated with a microsomal membrane fraction. Resolution of this fraction by sucrose density gradient centrifugation (62,000 g for 14 h) yielded three distinct membrane fractions. Maximum activities of membrane marker enzymes were recovered from the gradients at following densities: The major portion of microsomal protein and lipase activity at 1.085 kg/l; microsomal malate synthetase and phosphorylcholineglyceride transferase at 1.116 kg/l; NADH-cytochrome c reductase and phosphorylcholinecytidyl transferase at 1.133 kg/l. Evidently in rapeseed cotyledons lipase activity is associated only with a discrete microsomal membrane fraction which sediments differently from membrane fractions of the endoplasmic reticulum.

Effect of maternal isoxsuprine on fetal rabbit lung biochemical maturation

Isoxsuprine was given intramuscularly for 24 hours to pregnant rabbits at 26 to 27 days' gestation. There was no effect on fetal weight within one day of the last injection. Fetuses of isoxsuprine-treated mothers had higher lung lecithin (6.67 μm per gram ± 2.81) and higher traceal wash lecithin/sphingomyelir (LS) ratios (5.85 ± 4.5) than did untreated fetuses—lung lecithin (5.35 μm per gram ± 1.23) and tracheal wash LS (4.08 ± 2.3). In contrast, phosphorylcholine glyceride transferase activity of the fetal lung was less in isoxsuprine-treated fetal lung tissue.

Changes in pulmonary surfactant and phosphatidylcholine metabolism in rats exposed to chrysotile asbestos dust

1. Pulmonary surfactant was isolated from rats that had been exposed to chrysotile asbestos dust for from 3 days to 15 weeks. 2. Asbestos-treated rats showed a progressive increase in amounts of surfactant. After 15 weeks, treated animals contained 4 times as much as non-treated. 3. No significant change was seen in the total protein or total fatty acid composition of surfactant with exposure. 4. The increase in surfactant phosphatidylcholine normally seen on maturation of rat lung was accelerated by exposure of animals to asbestos. 5. An increase in the activity of phosphorylcholine glyceride transferase in lung homogenates and free cell populations was found. 6. Lysosomal phospholipase A was relatively unaffected by dust exposure. 7. It is suggested that the increase in surfactant amounts could be due to an increase in its synthesis without a corresponding alteration in its degradation.

Phosphatidylcholine- Lysophosphatidylcholine Cycle Pathway Enzymes in Rabbit Lung. II. Marked Differences in the Effect of Gestational Age on Activity Compared to the CDP-Choline Pathway

Lysophosphatidylcholine (lysoPC), a breakdown product of phosphatidylcholine (PC), might be important in pulmonary PC synthesis through fatty acid exchange reactions. This study defines the levels of three of the enzymes of the PC-lysoPC cycle pathway (lysophospholipase (LPL) (EC. 3.1.1.5), lysophosphatidylcholine-lysophosphatidylcholine acyltransferase (LAT), and acyl-CoA lysophosphatidylcholine acyltransferase (acryl-CoA LAT) (EC. 2.3.1.23)) in developing fetal rabbit lung and compared them with the enzymes of the CPD-choline synthetic pathway (choline kinase (CK) (EC. 2.7.1.32), phosphorylcholine cytidyl transferase (CyT) (EC. 2.7.7.15), and phosphorylcholine glyceride transferase (PCGT) (EC. 2.7.8.2). Lung homogenates of fetal rabbits of known gestation, newborn, and adult rabbits were used for the enzyme, protein, and phospholipid analyses. Total lung phospholipid, PC, and protein increased with gestational age. Thirty days' gestation, newborn, and maternal lung activities of CK, CyT, and PCGT had decreased to only 50% of their activities at 22-26 days' gestation. In contrast, LPL and LAT activities increased 4-5-fold from 22-26 days to 30 days' gestation, and increased further in the newborn lung, finally to a level matching maternal lung (about 8-10-fold higher than the 22-26 days' gestation activities). The microsomal acyl-CoA LAT also showed a similar increasing activity with gestational age. In fetal lung, enzymic activities for the apparent major PC synthetic pathway decreased. In contrast, the marked increases in LPL, LAT, and acyl-CoA LAT activities with increasing gestational age and at birth suggests importance of the PC lysoPC cycle pathway in regulating synthesis and turnover with maturation.

Dependence of Wound-induced Respiration in Potato Slices on the Time-restricted Actinomycin-sensitive Biosynthesis of Phospholipid

Actinomycin D prevents the full development in a 24-hour period of both wound respiration and cyanide resistance only when given in the first 10 to 12 hours following the cutting of potato tuber (Solanum tuberosum var. Russet) slices. The capacity for choline incorporation into phosphatidylcholine increases with slice aging and is inhibited by actinomycin D in the same time-restricted way. The time-restricted effectiveness of actinomycin D applies to the cutting-elicited enhanced synthesis of three critical enzymes of phosphatidylcholine synthesis, namely phosphorylcholine-glyceride transferase, phosphorylcholine-cytidyl transferase, and phosphatidylphosphatase. By contrast, actinomycim D given at any time is without effect on the measurable levels after 24 hours of a selection of glycolytic and mitochondrial respiratory enzymes. Neither succinic dehydrogenase nor cytochrome oxidase activity increases with time in aging potato slices in the presence or absence of chloramphenicol. The foregoing observations emphasize the central role of phospholipid, and ultimately membrane biosynthesis, in the development of wound-induced respiration.

Phosphatidylcholine Synthesis in Castor Bean Endosperm

Three pathways for phosphatidylcholine synthesis were assayed in castor bean (Ricinus communis var. Hale) endosperm. Phosphatidylethanolamine: S-adenosylmethionine methyl transferase occurred predominantly in the endoplasmic reticulum fraction, but some activity appeared in the mitochondria. Phosphorylcholine glyceride transferase occurred exclusively in the endoplasmic reticulum. The phosphorylcholine glyceride transferase activity was approximately 20-fold greater than the methylation pathway in the endoplasmic reticulum. No exchange activity was found. The Michaelis constant for the methylation was 31 mum for S-adenosylmethionine; phosphatidylethanolamine promoted the reaction slightly while other intermediates stimulated it by about 50%. The pH optimum was 9. Phosphorylcholine glyceride transferase had a Michaelis constant of 9.7 mum for cytidine diphosphate choline but variable results were obtained from diglycerides. The pH optimum was 7.5 and a divalent cation was required, Mg(2+) giving the greatest stimulation.

The Role of Membrane-Bound Enzymes in an Early Response of Aleurone Tissue to Gibberellic Acid

Treatment of aleurone layers of barley seed with gibberellic acid increases the observable phosphorylcholine glyceride transferase activity in a membrane fraction prepared from extracts of the aleurone cells. This gibberellic acid-dependent increase in glyceride transferase activity requires neither RNA synthesis nor protein synthesis. Membrane fractions prepared from mixtures of extracts of gibberellic acid-treated layers and control layers have a specific activity of glyceride transferase higher than expected on the basis of simple addition of the activities from the two sources. Therefore, some kind of activation is occurring.

Glycerolipid metabolism in cellular and subcellular fractions of fasted rat liver.

The effect of overnight fasting on glycerolipids of the rat liver organelles was studied. The concentration of microsomal phospholipids was markedly decreased by fasting, whereas that of mitochondria and whole liver increased significantly. The significant increase due to fasting in the activity of microsomal phosphorylcholine-glyceride transferase appeared to be responsible for the increased concentration of hepatic phospholipid. The incorporation by the liver slice of [1(-14)C] glycerol into PC3 in mitochondria was increased by fasting, while there was no difference in the microsomal fraction containing supernatant. The significant decrease in the relative proportions of oleic and linoleic acids and a significant increase in stearic and arachidonic acids in phosphatidylcholine were observed in the intact liver as well as its subcellular fractions. The possible relationship of these changes after fasting to the similarity in the incorporation pattern of [1(-14)C] glycerol into the molecular species of these glycerolipids was demonstrated.

An Early Response to Gibberellic Acid Not Requiring Protein Synthesis

Cell-free extracts from gibberellic acid-treated barley (Hordeum vulgare L. cv. Himalaya) aleurone layers show phosphorylcholine glyceride transferase activity greater than that from control layers. The increase in activity is not prevented by a mixture of amino acid analogs nor by cordycepin under conditions in which it is demonstrated that the analogs and the cordycepin are entering the cells in effective concentrations. We conclude therefore that the GA(3)-dependent increase in phosphorylcholine glyceride transferase activity (which occurs within the first 4 hours of GA(3) treatment) does not require RNA synthesis or protein synthesis.

Stimulation of phosphorylcholine-glyceride transferase activity by unsaturated fatty acids.

A number of unsaturated fatty acids markedly stimulate chicken liver microsomal phosphorylcholine–glyceride transferase (CDP-choline:1,2-diglyceride cholinephosphotransferase, EC 2.7.8.2). Oleate (0.8 mM) activates this enzyme severalfold only if a mixed diglyceride such as 1-palmitoyl-2-oleoyl-sn-glycerol is used as a substrate for the reaction. Dipalmitin and diolein incorporation into lecithin is not stimulated to any significant extent by unsaturated fatty acids. Fatty acid esters and numerous detergents either inhibit the enzyme or have no effect. Phosphorylethanolamine–glyceride transferase (CDP-ethanolamine:1,2-diglyceride ethanolaminephosphotransferase, EC 2.7.8.1) activity is only very slightly stimulated while diglyceride O-acyltransferase activity is inhibited to a small extent.

The enzymes of lecithin biosynthesis in human newborn lungs. 3. Phosphorylcholine glyceride transferase.

Extract: Phosphorylcholine glyceride transferase (EC. 2.7.8.2), the enzyme responsible for the final step of lecithin biosynthesis via the cytidine diphosphorylcholine (CDP-choline) pathway, is active in human neonatal lung tissue. A crude homogenate enzyme preparation in phosphate buffer was made from lung samples obtained at autopsy. The synthesis of product was linearly dependent upon protein concentration and linear with time for 30 min. The Michaelis constant for CDP-choline was 1.4 × 10-5 m. Optimal activity was attained at pH 7.5–8.0, at 35–39°, in the presence of Mg. Whole homogenate was 20–50 times as active as supernatant. No effect of oxygen or cysteine could be demonstrated. Triton and calcium inhibited the enzyme. The difference in enzyme activity between premature infants (3.53 × 10-7 mM lecithin/mg protein) and infants of longer (>32 weeks) gestation periods (1.76 × 10-7 mM lecithin/mg protein) is statistically significant at the 95% confidence limit. Speculation: Phosphorylcholine glyceride transferase could play a significant role in the relation between lung lecithin biosynthesis and respiratory distress syndrome of the human neonate. Characterization and study of the enzyme in human lung may lead to definition of its role as the possible regulator of pulmonary surfactant biosynthesis. There may be a relation between the level of enzyme activity and gestational age.

Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm.

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 b(5) and P 450. On sucrose gradients containing 1 mM EDTA this fraction had a mean isopycnic density of 1.12 g/cm(3) 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/cm(3) 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.

Intracellular distribution of enzymes of the cytidine diphosphate choline pathway in castor bean endosperm.

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/cm(3) 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/cm(2). Phosphorylcholine-glyceride transferase was found almost exclusively in a sharp band at density 1.12 g/cm(3), and phosphorylcholinecytidyl transferase was found in the uppermost band at density 1.08 g/cm(3). 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.

Effect of bile salts on hepatic phosphatidylcholine synthesis and transport into rat bile

The effect of transport of micelle-forming and non-micelle-forming conjugated bile salts on phosphatidylcholine synthesis and transport into bile was studied in the ex vivo perfused rat liver. Single additions of sodium taurocholate, a good micelle-forming conjugated bile salt, caused an increase in bile flow associated with increased phosphatidylcholine and taurocholate concentration. The specific activity of phosphatidylcholine with respect to incorporated [1,2-(14)C]choline and [(3)H]methyl of l-[Me-(3)H]methionine was not significantly altered by the increased transport of phosphatidylcholine. The data suggested that bile phosphatidylcholine is synthesized to a great extent, although not exclusively, by phosphorylcholine glyceride transferase. Single additions of the glycine conjugate of dehydrocholate, a poor micelle-forming bile salt, caused an increase in bile flow comparable to that seen with sodium taurocholate administration. However, the concentrations of phosphatidylcholine in bile decreased. Thin-layer and gas-liquid chromatographic analyses of bile secreted before and after glycodehydrocholate administration revealed no significant increase in bile salt secretion other than the administered glycodehydrocholate. Investigations utilizing radiochemically pure [(14)C]glycine dehydrocholate revealed that increased bile flow after [(14)C]-glycine dehydrocholate administration occurs concomitantly with the secretion of 75-95% of the administered [(14)C]glycine dehydrocholate as a single peak into bile. Thus the increased bile flow without increased phosphatidylcholine concentration noted after glycodehydrocholate administration is due to transport of an intact, nonmetabolized, conjugated bile salt with poor micelle-forming properties. The data indicate that the formation of a bile salt-phosphatidylcholine micelle is responsible for phosphatidylcholine transport into bile.

Estradiol- and testosterone-induced alterations in phosphatidylcholine and triglyceride synthesis in hepatic endoplasmic reticulum

Pathways of phosphatidylcholine and triglyceride biosynthesis were studied in hepatic endoplasmic reticulum from castrated and noncastrated male rats pretreated with estradiol or testosterone. In vitro measurements of hepatic microsomal enzymes which catalyze phosphatidylcholine biosynthesis revealed a significant increase in the specific activity of the enzyme governing phosphatidylcholine biosynthesis by the sequential methylation of phosphatidylethanolamine in the estradiol-treated castrate animals. The specific activity of phosphorylcholine-glyceride transferase was decreased by estradiol treatment in both castrate and noncastrate animals. The specific activity of diglyceride acyltransferase, which catalyzes triglyceride biosynthesis, was decreased by estradiol pretreatment in both castrate and noncastrate animals and was increased by testosterone in the castrate animals. The changes in specific activity of the enzymes governing phosphatidylcholine biosynthesis may account for the previously noted increased in vivo incorporation of methyl groups of l-methionine into hepatic phosphatidylcholine in female and estradiol-treated animals; the data suggest that in female and estradiol-treated rats a greater proportion of hepatic phosphatidylcholine is synthesized by the stepwise methylation of phosphatidylethanolamine. The decrease in diglyceride acyltransferase specific activity seen after estradiol administration may account for the lipotropic-like effect of estradiol.

Lecithin Biosynthetic Enzymes of Onion Stem and the Distribution of Phosphorylcholine-Cytidyl Transferase among Cell Fractions

Enzymatic activities of the cytidine 5'-diphosphate choline pathway for lecithin biosynthesis were demonstrated in homogenates of onion stem (Allium cepa). Choline kinase activity was present in the postmicrosomal supernatant, with less than 3% sedimenting with the particulate fractions. Phosphorylcholine-cytidyl transferase was distributed among all fractions, and phosphorylcholine-glyceride transferase was predominantly found in the particulate fraction.The phosphorylcholine-cytidyl transferase activity of onion stem required a divalent ion (Mg(2+) or Mn(2+)) for activity, was inhibited by Ca(2+), and was specific for cytidine triphosphate, with optimal activity in the range pH 6 to 7. To evaluate the distribution among cell fractions, conditions of pH, cofactors, substrate, and assay were optimized for each fraction. One-third of the transferase activity sedimented with the mitochondria-proplastids fraction, and one-third was in the microsomal supernatant. The dictyosome fraction contained about 10% of the total activity but showed a greater specific activity than the other fractions. Similar results were obtained with homogenates from rat liver, in that purified Golgi apparatus fractions contained the highest phosphorylcholine-cytidyl transferase activity on a protein basis when compared with other cell fractions at pH 7.2.