Rita Roberti

THE SYNTHESIS OF CHOLINE AND ETHANOLAMINE PHOSPHOGLYCERIDES IN NEURONAL AND GLIAL CELLS OF RABBIT IN VITRO

Abstract— The de novo synthesis of phosphatidylcholine and phosphatidylethanolamine in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labelled phosphorylcholine (phosphorylethanolamine) or cytidine‐5′‐phosphate choline (cytidine‐5′‐phosphate ethanolamine), as lipid precursors. Synthesis of phospholipid from phosphorylcholine and phosphorylethanolamine in both fractions was extremely low when compared to that derived from the corresponding cytidine nucleotides. The neuronal cell‐enriched fraction was found to possess a much higher rate of synthesis of both lipids from all precursors. Neuronal/glial ratios of about 5–9 were found for the synthesis of phosphatidylcholine and phosphatidylethanolamine from cytidine‐5′‐phosphate choline and cytidine‐5′‐phosphate ethanolamine, respectively. Several kinetic properties of the choline‐phosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found to be similar both in neurons and in glia (e.g. Km of cytidine‐5′‐phosphate ethanolamine, Km of diacyl glycerol, pH optimum, need for divalent cations), but the Km value for cytidine‐5′‐phosphate choline in glial cells was much lower (2.3 × 10−4m) than in neurons (1 × 10−3m). The Kmfor cytidine‐5′‐phosphate ethanolamine in both cells was much lower than in whole brain microsomes. It is concluded that the cytidine‐dependent enzymic system for phosphatidylcholine and phosphatidylethanolamine synthesis is concentrated mostly in the neuronal cells, as compared to glia.

Exogenous phospholipids specifically affect transmembrane potential of brain mitochondria and cytochrome C release.

Release of cytochrome c, a decrease of membrane potential (Δψm), and a reduction of cardiolipin (CL) of rat brain mitochondria occurred upon incubation in the absence of respiratory substrates. Since CL is critical for mitochondrial functioning, CL enrichment of mitochondria was achieved by fusion with CL liposomes. Fusion was triggered by potassium phosphate at concentrations producing mitochondrial permeability transition pore opening but not cytochrome c release, which was observed only at >10 mm. Cyclosporin A inhibited phosphate-induced CL fusion, whereas Pronase pretreatment of mitochondria abolished it, suggesting that mitochondrial permeability transition pore and protein(s) are involved in the fusion process. Phosphate-dependent fusion was enhanced in respiratory state 3 and influenced by phospholipid classes in the order CL > phosphatidylglycerol (PG) > phosphatidylserine. The probe 10-nonylacridine orange indicated that fused CL had migrated to the inner mitochondrial membrane. In state 3, CL enrichment of mitochondria resulted in a pH decrease in the intermembrane space. Cytofluorimetric analysis of mitochondria stained with 3,3′-diexyloxacarbocyanine iodide and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzymidazolylcarbocyanine iodide showed Δψm increase upon fusion with CL or PG. In contrast, phosphatidylserine fusion required Δψmconsumption, suggesting that Δψm is the driving force in mitochondrial phospholipid importation. Moreover, enrichment with CL and PG brought the low energy mitochondrial population to high Δψm values and prevented phosphate-dependent cytochrome c release.

The metabolism of phosphoric esters and of cytidine-diphosphate esters of choline and ethanolamine in the liver

Abstract 1. 1. The labelled phosphate and cyddine-diphosphate esters of choline and ethanol-amine, together with their bases, were incubated with rat liver homogcnate and microsomal membranes, and the transfer of radioactivity into water-soluble and lipid components examined. 2. 2. Very little conversion of choline into betaine and phosphorylcholine, and of ethanol-amine into phosphorylethanolamine, as well as into their intermediates and lipid material, was observed in both preparations. 3. 3. Small transfer of phosphorylcholine to cytidine-diphosphate choline and to lecithin, and of phospnorylcthanolaminc to cytidine-diphosphate cthanolamine and phosphatidylethanolaminc was observed. 4. 4. Despite a considerable breakdown to hydrosoluble products, the two cyddine-diphosphate esters were noticeably converted into corresponding phosphotipid with both homogcnate and microsomes.

Disruption of the gene encoding 3β‐hydroxysterol Δ14‐reductase (Tm7sf2) in mice does not impair cholesterol biosynthesis

Tm7sf2 gene encodes 3β‐hydroxysterol Δ14‐reductase (C14SR, DHCR14), an endoplasmic reticulum enzyme acting on Δ14‐unsaturated sterol intermediates during the conversion of lanosterol to cholesterol. The C‐terminal domain of lamin B receptor, a protein of the inner nuclear membrane mainly involved in heterochromatin organization, also possesses sterol Δ14‐reductase activity. The subcellular localization suggests a primary role of C14SR in cholesterol biosynthesis. To investigate the role of C14SR and lamin B receptor as 3β‐hydroxysterol Δ14‐reductases, Tm7sf2 knockout mice were generated and their biochemical characterization was performed. No Tm7sf2 mRNA was detected in the liver of knockout mice. Neither C14SR protein nor 3β‐hydroxysterol Δ14‐reductase activity were detectable in liver microsomes of Tm7sf2(−/−) mice, confirming the effectiveness of gene inactivation. C14SR protein and its enzymatic activity were about half of control levels in the liver of heterozygous mice. Normal cholesterol levels in liver membranes and in plasma indicated that, despite the lack of C14SR, Tm7sf2(−/−) mice are able to perform cholesterol biosynthesis. Lamin B receptor 3β‐hydroxysterol Δ14‐reductase activity determined in liver nuclei showed comparable values in wild‐type and knockout mice. These results suggest that lamin B receptor, although residing in nuclear membranes, may contribute to cholesterol biosynthesis in Tm7sf2(−/−) mice. Affymetrix microarray analysis of gene expression revealed that several genes involved in cell‐cycle progression are downregulated in the liver of Tm7sf2(−/−) mice, whereas genes involved in xenobiotic metabolism are upregulated.

Binding and release of cytochrome c in brain mitochondria is influenced by membrane potential and hydrophobic interactions with cardiolipin.

Factors influencing the release and anchorage of cytochrome c to the inner membrane of brain mitochondria have been investigated. Metabolic activity of mitochondria caused a decrease in the membrane potential Δψm, accompanied by detachment of the protein from the inner membrane. In a model system of cytochrome c reconstituted in cardiolipin (CL) liposomes, phosphate was used to breach the hydrophilic lipid-protein interactions. About 44% cytochrome c was removable when heart CL (80% 18:2n-6) was employed, whereas the remaining protein accounted for the tightly bound conformation characterized by hydrophobic lipid-protein interactions. Cytochrome c release from brain CL liposomes was higher compared to heart CL, consistent with lower polyunsaturated fatty acid content. The release was even higher with CL extracted from metabolically stressed mitochondria, exhibiting more saturated fatty acid profile compared to control (30% vs.17%). Therefore, weakening of the hydrophobic interactions due to saturation of CL may account for the observed cytochrome c release from mitochondria following metabolic stress. Moreover, mitochondria enriched with polyunsaturated CL exhibited higher Δψm, compared to less unsaturated species, suggesting that CL fatty acid composition influences Δψm. Mitochondria incorporated exogenous cytochrome c without protease-sensitive factors or Δψm. The internalized protein anchored to the inner membrane without producing swelling, as monitored by forward and side light scattering, but produced Δψm consumption, suggesting recovery of respiratory activity. The Δψm decrease is ascribed to a selected mitochondrial population containing the incorporated cytochrome c.

Enzymic synthesis of ethanolamine plasmalogens through ethanolaminephosphotransferase activity in neurons and glial cells of rabbit in vitro

The de novo synthesis of ethanolamine plasmalogen in isolated neuronal and glial cells from adult rabbit brain cortex was investigated in vitro, using labeled cytidine-5′-diphosphate ethanolamine as lipid precursor. The neuronal cell enriched fraction was found to possess a twofold ethanolaminephosphotransferase activity (EC 2.7.8.1), as compared to the glial fraction. The neuronal/glial ratio was similar both in the absence and in the presence of saturating alkenylacyl glycerol. Under the most favorable conditions, rates of 31 nmoles and 16 nmoles ethanolamine plasmalogen/mg protein/30 min were obtained for neurons and glia, respectively. Several kinetic properties of the phosphotransferase were found to be similar both in neurons and glia, e.g., Km of cytidine-5′-diphosphate ethanolamine, pH optimum, need for divalent cations; the Km value for alkenylacyl glycerol was twofold higher in glia (4 mM) than in neurons (2 mM). The neuronal/glial ratio for the phosphatidylethanolamine synthesizing activity was 2, 4.5, and 6 on using diacyl glycerols prepared from ox heart, ox brain, and soybean, respectively. It is concluded that the cytidine-dependent system for ethanolamine plasmalogen and phosphatidylethanolamine synthesis is concentrated prevalently in the neuronal cells, as compared to glia.

Determination of phosphatidylcholine in a flow injection system using immobilized enzyme reactors.

Two alternative procedures are described for the quantitative determination of phosphatidylcholine in a flow-injection system utilizing immobilized enzymes. Phospholipase C from Bacillus cereus and phospholipase D from cabbage were covalently bound to the surface of controlled-pore glass beads and the enzyme-derivatized beads were packed in small columns. In the first procedure, the phospholipase C column was connected with a second column containing coimmobilized alkaline phosphatase and choline oxidase. In the alternative procedure, the column packed with immobilized phospholipase D was connected with a column packed with immobilized choline oxidase. The hydrogen peroxide produced through the action of choline oxidase in both flow-injection systems was detected amperometrically. Both procedures are suitable for an accurate and rapid quantitation of phosphatidylcholine. The sensitivity of the method based on phospholipase C and alkaline phosphatase is higher than that using phospholipase D. Quantitation of phosphatidylcholine at the nanomole level can be easily obtained using the first method.

Enzymic synthesis of 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamine through ethanolaminephosphotransferase activity in the neuronal and glial cells of rabbit in vitro

The transfer of radioactivity from cytidine-5′-diphosphate ethanolamine into 1-alkyl-2-acyl-sn-glycerophosphorylethanolamine of neuronal and glial cells from adult rabbit brain cortex has been investigated in vitro. The synthesis of 1-alkyl-2-acyl-sn-glycerophosphorylethanolamine in both cell populations was stimulated 23–25-fold by the addition of 6 mM alkylacylglycerol. The neuronal cell-enriched fraction was found to possess/unit protein a 1.7–1.8-fold ethanolaminephosphotransferase activity (EC 2.7.8.1), as compared to the glial fraction, when saturating concentrations (6 mM) of alkylacylglycerols were added in the incubation system. The neuronal/glial ratio was 2.6–2.8 in the absence of lipid acceptor or with low concentrations of alkylacylglycerol. Under most favorable conditions, 6.4 and 3.3. nmoles 1-alkyl-2-acyl-sn-glycerophosphorylethanol-amine/mg protein/30 min was obtained for neurons and glia, respectively. Various kinetic properties of the 1-alkyl-2-acyl-sn-glycerophosphorylethanolamine synthesizing phosphotransferase activity were found to be similar both in neurons and glia.

Activation of TM7SF2 promoter by SREBP-2 depends on a new sterol regulatory element, a GC-box, and an inverted CCAAT-box

TM7SF2 gene encodes 3beta-hydroxysterol Delta(14)-reductase, responsible for the reduction of C14-unsaturated sterols in cholesterol biosynthesis. TM7SF2 gene expression is controlled by cell sterol levels through the SREBP-2. The motifs of TM7SF2 promoter responsible for activation by SREBP-2 have not been characterized. Using electrophoretic mobility shift assays and mutation analysis, we identified a new SRE motif, 60% identical to an inverted SRE-3, able to bind SREBP-2 in vitro and in vivo. Co-transfection of promoter-luciferase reporter constructs in HepG2 cells showed that the binding of SREBP-2 to SRE produced approximately 26-fold promoter activation, whereas mutation of the SRE motif caused a dramatic decrease of transactivation by SREBP-2. The function of additional motifs that bind transcription factors cooperating with SREBP-2 was investigated. An inverted CCAAT-box, that binds nuclear factor Y (NF-Y), cooperates with SREBP-2 in TM7SF2 promoter activation. Deletion of this motif resulted in the loss of promoter induction by sterol starvation in HepG2 cells, as well as a decrease in fold activation by SREBP-2 in co-transfection experiments. Moreover, co-transfection of the promoter with a plasmid expressing dominant negative NF-YA did not permit full activation by SREBP-2. Three GC-boxes (1, 2, 3), known to bind Sp1 transcription factor, were also investigated. The mutagenesis of each of them produced a decrease in SREBP-2-dependent activation, the most powerful being GC-box2. A triple mutagenized promoter construct did not have an additive effect. We conclude that, besides the SRE motif, both the inverted CCAAT-box and GC-box2 are essential for full promoter activation by SREBP-2.

Purification of ethanolaminephosphotransferase from bovine liver microsomes.

CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (EC 2. 7.8.1) has been purified to electrophoretic homogeneity and in a catalytically active form from bovine liver microsomes. The purification method is based on the high hydrophobicity of the protein whose charged sites appear to be masked from the interaction with the chromatographic stationary phases when membranes are solubilized with an excess of non-ionic detergent. The isolated protein has a molecular mass of about 38 kDa, as estimated by SDS-PAGE mobility, and exhibits both ethanolaminephosphotransferase and cholinephosphotransferase activities. Evidence is given that both activities are Mn2+-dependent and that the same catalytic site is involved in cholinephosphotransferase and ethanolaminephosphotransferase reactions. Mg2+-dependent CDP-choline:diacylglycerol cholinephosphotransferase (EC 2.7.8.2) is completely inactivated during the solubilization and purification steps.