Enzymes Of Phospholipid Synthesis Research Articles

Fatty-acid synthase activity and mRNA level in hypertrophic type II cells from silica-treated rats.

Silica instillation causes a massive increase in lung surfactant. Two populations of type II pneumocytes can be isolated from rats administered silica by intratracheal injection: type IIA cells similar to type II cells from normal rats and type IIB cells, which are larger and contain elevated levels of surfactant protein A and phospholipid. Activities of choline-phosphate cytidylyltransferase, a rate-regulatory enzyme in phosphatidylcholine biosynthesis, and fatty-acid synthase (FAS) are increased in type IIB cells isolated from rats 14 days after silica injection. In the present study, we examined the increase in FAS and cytidylyltransferase activities in type IIB cells as a function of time after silica administration. FAS activity increased rapidly, was approximately threefold elevated 1 day after silica administration and has reached close to the maximum increase by 3 days. Cytidylyltransferase activity was not increased on day 1, was significantly increased on day 3 but was not maximally increased until day 7. Inhibition of de novo fatty-acid biosynthesis, by in vivo injection of hydroxycitric acid and inclusion of agaric acid in the type II cell culture medium, abolished the increase in cytidylyltransferase activity on day 3 but not FAS and had no effect on activities of two other enzymes of phospholipid synthesis. FAS mRNA levels were not increased in type IIB cells isolated 1-14 days after silica injection. These data show that the increase in FAS activity in type IIB cells is an early response to silica, that it mediates the increase in cytidylyltransferase activity, and that it is not due to enhanced FAS gene expression.

42] Phosphatidylserine decarboxylase from rat liver

Publisher Summary Phosphatidylserine decarboxylase is identified as a protein associated with the inner mitochondrial membrane. The enzyme decarboxylates phosphatidylserine to form phosphatidylethanolamine. Although once thought to be a minor enzyme in phospholipid synthesis in animal cells, recent evidence suggests that this enzyme can contribute significantly to both membrane biogenesis in cultured cells and lipoprotein maturation in hepatic cells. Additional interest in this enzyme has been generated with the realization that the phosphatidylserine substrate for the decarboxylase must be imported into the mitochondria before it is available for catalysis. Consequently, the action of the decarboxylase can be used as a discrete chemical signal for monitoring phosphatidylserine transport. The enzyme has proved quite resistant to purification. This chapter describes the approaches used to obtain partially purified preparations of the enzyme.

27] Purification of CDPdiacylglycerol synthase from Escherichia coli

This chapter discusses the purification of CDPdiacylglycerol synthase from Escherichia coli (E.coli). CDPdiacylglycerol synthase catalyzes the activation of phosphatidic acid by CTP to form CDPdiacylglycerol and inorganic pyrophosphate. This activity is essential for all phospholipid biosynthesis in E.coli and for the biosynthesis of acidic phospholipids in mammalian cells. The enzymes of phospholipid synthesis are usually membrane bound and present in relatively low levels, making purification difficult. The purification of E. coli CDPdiacylglycerol synthase is greatly aided by enzyme overproduction driven by an expression plasmid. Starting with cells that overproduce the enzyme 50-fold, essentially homogeneous enzyme can be obtained after chromatographic procedures that yield 160-fold purification. The enzyme can be partially purified from wild-type cells using the same procedures. The assay follows the conversion of [α- 32 P]dCTP to chloroform-soluble material, dependent on phosphatidic acid.

Release of fatty acid-binding protein from ischemic-reperfused rat heart and its prevention by mepacrine

In an attempt to resolve the issue of whether there is a loss of fatty acid binding protein (H-FABP) from heart during ischemia and reperfusion, and to further examine the role of this protein in ischemic-reperfusion injury, the amount of H-FABP of heart was monitored during ischemia and reperfusion. Excellent correlation was obtained between the loss of H-FABP from heart and its appearance in the perfusate buffer when examined by Western blot using the specific antibody to H-FABP. Further quantitation was achieved by densitometric scanning of the Western blot and rocket electrophoresis. Maximum release of H-FABP was observed within 20 min of reperfusion, the total release being 10% of the H-FABP content of the heart. Mepacrine, a membrane stabilizer and a phospholipase inhibitor, reduced the release of H-FABP from the heart and prevented the accumulation of nonesterified fatty acids in the tissue during ischemia and reperfusion. In view of the established role of H-FABP in the preservation of membrane phospholipids by either scavenging free radicals during ischemia and reperfusion or by modulating the enzymes of phospholipid synthesis, it seems likely that the loss of H-FABP may have some contribution towards the ischemic-reperfusion injury.

Developmental profiles of protective mechanisms of heart against peroxidative injury.

The developmental profiles of the protective mechanisms of heart against peroxidative injury during neonatal growth was examined in the pigs of three different age groups. Lipid peroxidation expressed in terms of malonaldehyde formation was considerably higher in the pig hearts of the 8-10 day age group compared to that either by newborn or adult age groups. The four principal antioxidative enzymes, superoxide dismutase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase (G6PD), were enhanced during early neonatal growth and, with the exception of G6PD, all other enzymes were further enhanced during further growth to adulthood. G6PD activity dropped significantly in adult heart. The phospholipid contents of myocardial membrane between newborn and week-old pigs did not vary significantly. Total phospholipids and phosphatidylcholine contents were significantly higher in adult heart compared to those in neonatal heart. The enzymes of phospholipid synthesis and degradation, fatty acyl CoA synthetase (FACS), phospholipase A2 (PLA2), lysophospholipase (LPL), and lysophophatidylcholine acyltransferase (LPCAT) increased during early neonatal growth. During further growth to adulthood, FACS decreased, PLA2 did not change, whereas both LPL and LPCAT increased significantly. Analysis of free fatty acids showed that palmitic and stearic acids decreased during the first week of growth, but increased during further growth to adulthood. Oleic acid did not change with aging, but arachidonic acid dropped in adult heart compared to that in neonatal heart. Linoleic, palmitoleic and free fatty acids increased dramatically during the first week of neonatal growth, but dropped thereafter. These results suggest that the unusual peroxidative status of the week-old pig heart is related to the presence of high concentrations of polyunsaturated fatty acids in the membrane phospholipids and not with the antioxidative defense system.

Regulation of phosphatidylcholine synthesis and the activity of CTP-cholinephosphate cytidylytransferase in myoblasts by 12-O-tetradecanoylphorbol-13-acetate.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increases rate of incorporation of 32P into phosphatidylcholine and phosphatidylethanolamine about twofold in a line of rat skeletal myoblasts (L6). This effect is specific for TPA and is not caused by the nontumor-promoting analogue 4 alpha-phorbol didecanoate (alpha-PDD). Cycloheximide and actinomycin D have no effect on the TPA-dependent increase in labelling. There is no significant difference in the rate of disappearance of [14C]glycerol from phospholipids in cells exposed to TPA. Several enzymes of phospholipid synthesis in myoblasts exposed to TPA are unaltered in activity, except CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) whose activity is enhanced 1.5- to 2-fold compared with cells exposed to alpha-PDD. Exposure of cells for only 15 min to a concentration of 1 X 10(-6)M TPA is sufficient to cause maximum activation of the enzyme. The cytidylyltransferase activity is associated largely with the particulate fraction in the cell-free extracts of myoblasts. Following gel filtration on Bio-Gel A-1.5m, the enzyme activity appears in the void volume and smaller molecular weight forms are not discernible. The kinetic parameters of cytidylyltransferase in preparations from treated cells are unaltered except for an increased Vmax. The apparent Km value for CTP for the enzyme from treated and untreated cells is about 0.9mM. Both enzyme preparations are weakly inhibited by lysophosphatidylcholine, but are unaffected by several other phospholipids.

Lung surfactant.

Aspects of pulmonary surfactant are reviewed from a biochemical perspective. The major emphasis is on the lipid components of surfactant. Topics reviewed include surfactant composition, cellular and subcellular sites as well as pathways of biosynthesis of phosphatidylcholine, disaturated phosphatidylcholine and phosphatidylglycerol. The surfactant system in the developing fetus and neonate is considered in terms of phospholipid content and composition, rates of precursor incorporation, activities of individual enzymes of phospholipid synthesis and glycogen content and metabolism. The influence of the following hormones and other factors on lung maturation and surfactant production is discussed: glucocorticoids, thyroid hormone, estrogen, prolactin, cyclic AMP, beta-adrenergic and cholinergic agonists, prostaglandins and growth factors. The influence of maternal diabetes, fetal sex, stress and labor are also considered. Nonphysiologic and toxic agents which influence surfactant in the fetus, newborn and adult are reviewed.

Glucocorticoid-Thyroid Hormone Interactions in Fetal Rat Lung

Previous studies have shown that triiodothyronine (T3) enhances the effect of dexamethasone on phosphatidylcholine (PC) synthesis in organ cultures of fetal rat lung. The aim of this study was to investigate whether similar interactions occurred in vivo and to explore possible mechanisms for this phenomenon. Injection of 7.0 mg/kg T3 into pregnant rats on d 18 and 19 of gestation resulted in a mean fetal serum T3 level of 2380 ng/dl on d 20 (control, 84 ng/dl) and in maximal (34%) stimulation of choline incorporation into PC. Injection of 1.0 mg/kg betamethasone using the same protocol as for T3 resulted in maximal stimulation of 33% and administration of both hormones together produced a 69% increase, an additive affect. The percentage of PC that was disaturated was increased with betamethasone, but decreased with T3. Betamethasone treatment resulted in an increase in the whole lung disaturated PC content, but treatment with T3 did not. Betamethasone administration also increased fetal serum T3 levels, but T3 injection did not produce elevated fetal serum corticosterone levels. Injection of T3 in vivo, or exposure of explants of 18-d fetal lung to 100 nm T3 for up to 48 h did not result in an increase in cytoplasmic glucocorticoid binding or nuclear translocation of the receptor steroid complex. Exposure of explants to glucocorticoid or T3 in vivo or in culture (dexamethasone, 100 nM and T3, 100 nM; for 48 h) resulted in a significant increase in the activity of cholinephosphate cytidylyltransferase, an enzyme in the choline incorporation pathway of PC synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of type II alveolar epithelial cells using low protease concentrations.

The proteolytic digestion of lung tissue is a necessary prerequisite for the isolation of relatively pure populations of type II alveolar cells. The choice of digestion conditions can have a profound effect on the yield and metabolic integrity of such cells. The specific effects of various proteases on isolation of type II cells from adult rabbits were investigated in a systematic way; maximal numbers of cells were obtained using a mixture of purified elastase (0.3 mg/ml), purified trypsin (0.025 mg/ml) and inflation of intact lung with the protease mixture. Under such conditions, the average yield of cells from an adult rabbit was 10±2.1×107, of which 80–90% were type II cells. Lower yields were obtained by exposing lung tissue to protease after mincing, even if protease concentrations were increased 2–4 fold. By virtue of the lowered protease concentrations, cellular damage as measured by effects on cellular enzyme activities was minimal. NADPH cytochromec reductase activity was not reduced by isolation conditions. In addition, no significant enhanced release of activity into the 150,000 supernatant was observed. The isolated cells were enriched 2–3 fold for the enzymes of phospholipid synthesis compared to whole lung and alveolar macrophages.

Enzymatic control of membrane lipids inMycobacterium smegmatis ATCC 607 grown at low temperature

Enzymes of phospholipid synthesis were studied inMycobacterium smegmatis ATCC 607 grown at 37 and 27°C. The synthesis seemed to be regulated, at least partly, at the phosphatidic acid level by the specificity of the acyltransferases. The individual phospholipid species were found to be regulated at the enzymatic level in the maintenance of membrane fluidity.

The enzymes of phospholipid synthesis in Clostridium butyricum

We have examined extracts of Clostridium butyricum for several enzymes of phospholipid synthesis. Membrane particles were shown to catalyze the formation of CDP-diglyceride from [3H]CTP and phosphatidic acid. The reaction was dependent on Mg2+ and stimulated by monovalent cations. CDP-diglyceride formed in vitro was found to be a substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. The formation of phosphatidylglycerophosphate from added CDP-diglyceride and [U-14C]sn-glycerol-3-phosphate was dependent on Mg2+ and Triton X-100. The dephosphorylation of endogenously-generated phosphatidylglycerophosphate to yield phosphatidylglycerol was observed to be pH-dependent. The formation of phosphatidylserine from CDP-diglyceride and L-[3-14C]serine was stimulated by Mg2+ and Triton X-100. dCDP-diglyceride was a suitable substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. Phosphatidylserine decarboxylase activity was barely detectable in membrane particles from C. butyricum. The addition of E. coli membrane particles provided efficient phosphatidylserine decarboxylase activity in this system. Although plasmalogens are the principal lipids of C. butyricum, none of the products of phospholipid synthesis formed in vitro contained measurable amounts of plasmalogens. The subcellular distribution of both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase in C. butyricum was also studied. Both were found to be membrane-associated.

The influence of hormones on the biochemical development of fetal rat lung in organ culture. II. Insulin.

Summary: The influence of insulin on the biochemical and morphologic maturation of 19-day fetal rat lung (term is 22 days) was studied in an organ culture system. Exposure to insulin for 24 hr resulted in a significant increase in the glycogen content and the rate of glucose oxidation to CO2. In association with this, there was morphologic evidence of delayed lung maturation as evidenced by a significant decrease in the number of lamellar bodies and type II cells in the explants.Insulin treatment had no effect on the rate of choline incorporation into phosphatidylcholine (PC) or disaturated PC and did not antagonize the dexamethasone-induced stimulation of choline incorporation into PC. When the incorporation of [3H]acetate into the various phospholipid fractions was examined, however, it was found that exposure to insulin resulted in a significant decrease in the percentage of phospholipid radioactivity in the disaturated PC fraction and an increase in the percentage of radioactivity in the “membrane” phospholipids, phosphatidylethanolamine, phosphatidylinositol plus phosphatidylserine, and sphingomyelin. There was no significant change in the unsaturated PC and phosphatidylglycerol fractions. Treatment with dexamethasone generally had the opposite effect to insulin with regard to acetate incorporation into the various phospholipid fractions, and when the two hormones were combined, this effect was diminished or abolished. The effects of insulin could not be accounted for on the basis of a change in activity of any of the enzymes of phospholipid synthesis that were examined.These findings suggest that insulin stimulates the synthesis of the cell membrane phospholipids while decreasing that of the surfactant phospholipid, disaturated PC.Speculation: Insulin stimulates lung cell growth while delaying lung cell differentiation.

Fetal lung in organ culture. III. Comparison of dexamethasone, thyroxine, and methylxanthines.

Exposure of explants of fetal rat lung to dexamethasone, thyroxine, or the methylxanthines, aminophylline and caffeine, resulted in a significant increase in the rate of choline incorporation into all the choline-containing phospholipids. Dexamethasone, aminophylline, or caffeine treatment also resulted in an increase in the percentage of radioactivity from [3H]acetate in the surfactant-associated phospholipids, disaturated phosphatidylcholine, and phosphatidylglycerol and a corresponding decrease in the membrane phospholipids. Exposure to thyroxine had different effects. Only aminophylline and caffeine produced an increase in the rate of incorporation of acetate into phospholipid. Differences were also observed in the activities of enzymes of phospholipid synthesis. The activity of cholinephosphate cytidylyltransferase was increased by dexamethasone and that of choline kinase and lysolecithin acyltransferase by aminophylline. Thyroxine had no effect on any of the enzymes examined. All these agents produced a significant decrease in lung glycogen content and a small decrease in the protein-to-DNA ratio. These data indicate that corticosteroids, thyroxine, and the methylxanthines act directly on the fetal lung, but produce different effects and presumably act via different mechanisms.

Enzymes of phospholipid synthesis: Axonal versus schwann cell distribution

Using quantitative EM autoradiography to localize sites of incorporation of tritiated inositol and choline into mouse sciatic nerve, we observed a substantial axon-based phosphatidylinositol synthesis 13, but no axonal phosphatidylcholine synthesis 13,14. In the present communication we provide biochemical evidence for the axonal transport of CDP-diglyceride:inositol transferase (EC 2.7.8.11), the terminal enzyme in de novo phosphatidylinositol biosynthesis. Axonal transport of 1,2-diacylglycerol: CDP-choline choline phosphotransferase (EC 2.7.8.2),. required for de novo phosphatidylcholine synthesis, was not apparent in these studies. During subcellular fractionation activities for the synthesis of phosphatidylinositol by inositol transferase (IT) and phosphatidylcholine by choline phosphotransferase (CPT) were recovered in crude microsomal fraction of rat sciatic nerve. However, CPT was much more highly enriched in the microsome fraction than IT, which may be an indication of the different subcellular localizations of these enzymes. Following ligation, we detected localized increases in the activities of both enzymes in 5 (and 3) mm segments taken immediately proximal and distal to the ligature. Both activities increased in a linear fashion in the proximal segments over the ensuing 72 h period. It took about 40 h (IT) and 56 h (CPT) for the activities in the segments proximal to the ligature to double compared to unligated contralateral (control) nerves. The time-dependent accumulation of IT was primarily due to axonal transport, while that of CPT was largely a result of increased enzyme activity in local Schwann cells. Evidence came from double ligation studies, where a proximal ligature, acting to restrict orthograde axonal transport, reduced accumulation in a distal ligature by 80% for IT, but only 28% for CPT. Conversely, blockage of the Schwann cell response with actinomycin D, reduced accumulation of CPT by 83% and IT by only 36%. Finally, light microscopic autoradiography was used to show that in the segment proximal to the ligature, tritiated inositol incorporation into lipid was primarily axonal, whereas that of tritiated choline remained primarily associated with Schwann cells.

The influence of hormones on the biochemical development of fetal rat lung in organ culture: I. Estrogen

It was recently demonstrated that 17β-estradiol enhances phosphatidylcholine synthesis in fetal lung in vivo. In order to determine whether estrogen acts directly on the lung, we examined the influence of 17β-estradiol on phospholipid and glycogen metabolism in explants of 18 day fetal rat lung in organ culture. Exposure of the explants to 17β-estradiol resulted in significant stimulation of [Me- 3H]choline incorporation into phosphatidylcholine, disaturated phosphatidylcholine and sphingomyelin. Incorporation of [ 3H]acetate into total phospholipid was increased by 63% ( P < 0.001). Incorporation into individual phospholipids was similarly increased, except for that into phosphatidyl-glycerol which was enhanced by 111% ( P < 0.005). When the data were expressed as the percentage of radioactivity from [ 3H] acetate in the various phospholipid fractions, it was found that there was a 29% increase in the phos-phatidylglycerol fraction ( P < 0.005) and a 12% reduction in phosphatidyl-inositol plus phosphatidylserine combined ( P < 0.005). There was no significant change in the percentage of radioactivity in any of the other phospholipid fractions, or in the activity of any of the enzymes of phospholipid synthesis examined. Estrogen treatment also resulted in a decrease in the glycogen content ( P < 0.05), but no change in the protein/DNA ratio. These data indicate that estrogen acts directly on the fetal lung and stimulates the incorporation of choline and acetate into phospholipids. It may specifically enhance incorporation into phosphatidylglycerol, the second most abundant phospholipid in pulmonary surfactant.

Enzymes of phospholipid synthesis in bacillus Calmette-Guerin induced rabbit alveolar macrophage: Characterization and localization of cytidine diphosphocholine phosphotransferase and monoacylphospholipid acyltransferase

The rabbit alveolar macrophage is capable of renewing its plasma membrane by at least two metabolic pathways. It contains (1) a monoacylphospholipid acyltransferase, which catalyzes the synthesis of diacylphospholipids by recycling monoacylphospholipids produced by the action of phospholipases and (2) a cytidine diphosphocholine phosphotransferase (CDPcholine phosphotransferase), which catalyzes the last step in the synthesis de novo of diacylglycerophosphocholine. These activities have been characterized in the cell homogenate with respect to time, protein, pH optimum (for CDPcholine phosphotransferase), substrate specificity (for monoacylphospholipid acyltransferase) and cation requirement (for CDPcholine phosphotransferase). Monoacylphospholipid acyltransferase activity is localized solely in the endoplasmic reticulum. On the other hand, the CDPcholine phosphotransferase activity can be measured in the endoplasmic reticulum and in the plasma membrane, characterized by both differential and gradient sedimentation techniques. In addition to the normal route of phospholipid synthesis in the endoplasmic reticulum, the rabbit alveolar macrophage may thus possess the capacity for in situ synthesis of phospholipids of plasma membrane as a mechanism for membrane renewal following phagocytosis.

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Effect of cessation of net phospholipid synthesis on cytoplasmic and outer membranes

The effect of cessation of net phospholipid synthesis on the cytoplasmic and outer membranes of Escherichia coli was investigated in a mutant strain defective in the first enzyme of phospholipid synthesis, the sn-glycerol-3-phosphate (glycerol-P) acyltransferase. The glycerol-P (glycerol) auxotropic phenotype of this strain resulted from an altered membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of the parental activity. When net phospholipid synthesis was halted during glycerol deprivation, both soluble and cell envelope protein synthesis continued. Fractionation of the membranes derived from glycerol-supplemented and glycerol-deprived cultures by isopycnic banding in sucrose gradients revealed that both the cytoplasmic and outer membranes of the deprived culture banded at higher buoyant densities. The protein/phospholipid ratio of both the cytoplasmic and outer membranes increased approximately 60% during the period of glycerol deprivation. The distribution of two cytoplasmic membrane activities, NADH oxidase and 1-acylglycerol-P acyltransferase, and an outer membrane activity, phospholipase A1, showed that the total membranes derived from glycerol-deprived cultures were separated cleanly into cytoplasmic and outer membrane fractions. Both cytoplasmic and outer membrane proteins were synthesized and integrated into their respective membranous structures when net phospholipid synthesis was halted. Hence, the biosynthesis of membrane phospholipid and membrane protein are not tightly coupled. Further, these data suggest that cellular control mechanisms exist which maintain the protein content of both membranous structures below the point where they are saturated with protein.

Studies on the Membranes of Bacilli: II. CHARACTERIZATION OF A NOVEL PROTEIN OF A BACILLUS SPECIES

Abstract During an earlier study on the localization of the enzymes of phospholipid synthesis in the ghosts of an unidentified Bacillus species (designated Bacillus PP) the presence of fibers in ghost preparations was noted. More extensive electron microscopic study has revealed the presence of large quantities of these fibers at the periphery of intact cells. The majority of the fibers were found to remain associated with the ghosts isolated from Bacillus PP. Treatment of the ghosts at pH 11 resulted in release of the fibers, which were isolated and analyzed by chemical and physical methods. Disc gel electrophoresis in two systems and gel filtration indicate that the fibers are composed of a single polypeptide of molecular weight 31,000 to 33,000. The purity of the fibers was confirmed by amino acid analysis and by fingerprint analysis after tryptic digestion. Electron microscopic examination, physical and chemical analysis, and immunological studies showed that these fibers are unrelated to the flagella of Bacillus PP. Detailed electron microscopic studies revealed that the fibers were composed of bundles of fibrils having a diameter of 30 to 40 A and a regular wavelike structure. Under certain conditions these fibrils dissociated into single filaments 12 to 15 A in diameter. The function of these fibers and the nature of their association with the cytoplasmic membrane is unknown.

Distribution of phospholipid-synthesizing enzymes in the wall and membrane subfractions of the envelope of Escherichia coli

The distribution of the enzymes of phospholipid synthesis in cell wall and membrane subfractions of the cell envelope of Escherichia coli, isolated by a procedure involving particle electrophoresis and sucrose gradient density centrifugation, has been studied. The results show that, with the possible exception of CDP-diglyceride:L-serine phosphatidyltransferase all of the enzymes involved in the synthesis of phosphatidic acid, CDP-diglyceride, phosphatidylglycerol, and phosphatidylethanolamine, are localized in the inner cytoplasmic membrane.