Serine Base Exchange Enzyme Research Articles

Presence of Phosphatidylserine Synthesizing Enzymes in Triton Insoluble Floating Fractions from Cerebrocortical Plasma Membranes

Mammals synthesize phosphatidylserine (PS), a binding PKC molecule, by exchanging the nitrogen base of phosphatidylethanolamine or phosphatidylcholine with free serine. Serine base exchange enzyme (SBEE) was found in Triton insoluble floating fractions (TIFFs) from rat cerebellum which contained PKC. Consequently, SBEE might modulate PS levels in the PKC binding area (Buratta et al., J Neurochem 103:942-951, 2007). In the present study, we determined whether SBEE and PKC were localised in rat cerebral cortex TIFFs (cx-TIFFs) and in rat cerebrocortical plasma membrane-TIFFs (PM-TIFFs) which are more directly involved in signal transduction than intracellular membranes. Cx-and PM-TIFFs expressed SBEE activity and contained PKC. SBEE used ethanolamine as free exchanging base which may modulate PS level in the PKC binding area, transforming PS into PE and vice versa. The slight decrease in [(14)C]serine incorporation in the presence of choline indicated the existence of a SBEE isoform which may play a peculiar role in this brain area.

Synthesis of phosphatidylserine by base exchange in Triton‐insoluble floating fractions from rat cerebellum

Phosphatidylserine (PS), which is synthesized in mammalian tissues by the exchange between free serine and the nitrogen bases present in membrane glycerophospholipids, is strictly required for protein kinase C (PKC) activity. PKC, as other molecules involved in signal transduction, is present in lipid rafts, considered as a platform for molecular signaling. Membrane microdomains enriched in components of rafts can be isolated on the basis of their insolubility in Triton X-100 at 4 degrees C and their low density in sucrose density gradient. This study demonstrates the existence of serine base exchange enzyme (SBEE) in Triton-insoluble floating fractions containing associated PKC. Using two fractions of detergent-resistant membranes from rat cerebellum, we observed a correlation between the level of SBEE activity and that of membrane-associated PKC. This suggests that SBEE, synthesizing PS in the binding area for PKC, participates to signal transduction. The capability of SBEE to utilize not only serine but also ethanolamine, as free exchanging base, suggests a mechanism for modulating in loco PS concentration.

Cerebrocortical plasma membrane serine base exchange enzyme activity in hypoxia

Phosphatidylserine (PtdSer) is involved in signal transduction and in apoptosis. Peculiarities of its metabolism (1) are relevant for this study. N2 treatment stimulates the incorporation of [14C]serine (Ser) into PtdSer of cerebrocortical slices and in cerebrocortical plasma membranes (PM) serine base exchange enzyme (SBEE) activity is lowered by G protein activator. PtdSer radioactivity of PM from slices incubated with [14C]Ser was greater in hypoxia than in controls. SBEE activity of PM from slices, incubated in a similar manner and assayed at pH 8.2 and pH 7.4 based on a preliminary study, was lower in hypoxia. The hypoxia induced incorporation of [14C]Ser into slice PtdSer was due to stimulation of the SBEE isoform that exchanges Ser and ethanolamine and that in brain membrane is activated by fatty acids. We suggest the presence of different SBEE isoforms in PM differently influenced by hypoxia.

Serine base-exchange in rat liver nuclei

It has been shown that the incorporation of [ 14C]serine into phosphatidylserine (PS) in isolated rat liver nuclei is intrinsic to this organelle as attested by marker enzyme activity. Serine incorporation into PS was the highest in nuclei depleted of the outer membrane of the nuclear envelope (nucleoplasts) and negligible in the outer membrane. Trypsin treatment of nucleoplasts caused a strong inactivation of PS synthesis and only a moderate one of the NAD pyrophosphorylase activity, the marker enzyme of the inner nuclear membrane. We suggest that the serine base-exchange enzyme is located in the inner membrane of the nuclear envelope and accessible from the periplasmic surface of this membrane.

Regulation of phosphatidylserine exposure at the cell surface by the serine–base exchange enzyme system during CD95-induced apoptosis

Early in the apoptotic process, CD95 induces a translocation of phosphatidylserine (PtdSer) from the inner to the outer leaflet of the cellular plasma membrane. In mammalian cells, PtdSer is only synthesized through a calcium-dependent exchange of the polar head group of pre-existing phospholipids, either phosphatidylcholine or phosphatidylethanolamine, by a serine. Using a pharmacological approach, we examined the influence of PtdSer synthesis on CD95-induced PtdSer exposure at the surface of Jurkat cells. We found that CD3/TCR triggering or thapsigargin treatment of Jurkat cells was accompanied both by a decreased PtdSer synthesis and by a strong reduction of CD95-induced PtdSer at the cell surface, as monitored by fluorescence-activated cell sorting (FACS) analysis of annexin V–fluorescein isothiocyanate (FITC)-labeled cells. PtdSer synthesis through the serine–base exchange enzyme system thus appeared as one of the mechanisms implicated in the recently discovered CD3/TCR-induced down-regulation of CD95-induced apoptosis. Conversely, increasing the activity of the serine–base exchange enzyme system with different drugs, either the K + channel blocker quinine, the cationic amphiphil stearylamine, or three different calmodulin antagonists, chlorpromazine, trifluoperazine, and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), resulted in an increased appearance of PtdSer at the surface of CD95-treated cells. Both PtdSer synthesis and CD95-induced annexin V–FITC reactivity were abrogated in ATP-depleted cells. Also, modifying the membrane potential with valinomycin (hyperpolarization) or either gramicidin or KCl (depolarization) demonstrated a strong relationship between PtdSer synthesis and annexin V–FITC reactivity in CD95-treated cells. Together, our results indicate that CD95-induced exposure of PtdSer at the cell surface could be regulated by the activity of the serine–base exchange enzyme system.

Serine base exchange enzyme in porcine lyophilised platelets: enzyme properties and modulation by AlF4- and different types of heparin.

Phosphatidylserine is one of the PKC modulators and thus it may play an important role in signal transduction. Regulation of the synthesis of this phospholipid is not yet clarified. The contrasting reports are possibly related to the existence of different enzymes which, in mammalian tissues, catalyse the exchange between free serine and the nitrogen base of a membrane phospholipid. This study demonstrates that serine base exchange reactions of commercially available lyophilised porcine platelets exhibit similar pH optima, temperature and Ca2+ dependence as observed in fresh tissues. Analysis of fatty acids composition of the three phospholipid classes involved in base exchange reactions also demonstrated a similarity with fresh platelets. Serine and ethanolamine base exchange enzyme activities were assayed in parallel in platelet lysate subjected to preincubation at various temperatures (30-60 degrees C). When dithioerithrol was omitted from the incubation medium, the two base exchange reactions were inhibited with a similar temperature-dependent pattern. Addition of the reducing agent enhanced the sensitivity to preincubation only for the serine base exchange reaction which was inhibited by 80% after preincubation at 45 degrees C. With respect to its regulation, porcine platelet serine base exchange enzyme(s) was inhibited by fluoroalluminate, a widely used G-protein activator, and stimulated by unfractionated heparin. Low mol. wt. heparin did not influence enzyme activity. Unfractionated heparin greatly stimulated SBEE activity assayed at pH 7.4, a pH value far from the optimal pH.

Dexamethasone increases the incorporation of [3H]serine into phosphatidylserine and the activity of serine base exchange enzyme in mouse thymocytes: a possible relation between serine base exchange enzyme and apoptosis.

The exposure of phosphatidylserine toward the external surface of the membrane is a well-established event of programmed cell death. The possibility that an apoptotic stimulus influences the metabolism of this phospholipid could be relevant not only in relation to the previously mentioned event but also in relation to the capability of membrane phosphatidylserine to influence PKC activity. The present investigation demonstrates that treatment of mouse thymocytes with the apoptotic stimulus dexamethasone, enhances the incorporation of [3H]serine into phosphatidylserine. Cell treatment with dexamethasone also enhanced the activity of serine base exchange enzyme, assayed in thymocyte lysate. Both the effects were observed at periods of treatment preceding DNA fragmentation. The addition of unlabelled ethanolamine, together with [3H]serine to the medium containing dexamethasone-treated thymocytes lowered the radioactivity into phosphatidylserine. Serine base exchange enzyme activity was influenced by the procedure used to prepare thymocyte lysate and was lowered by the addition of fluoroaluminate, that is widely used as a G-protein activator. The increase of serine base exchange enzyme activity induced by dexamethasone treatment was observed independently by the procedure used to prepare cell lysate and by the presence or absence of fluoroaluminate.

Serine Base Exchange Enzyme Activity Is Modulated by Sphingosine and Other Amphiphilic Compounds: Possible Role of Positive Charge in Increasing the Synthesis of Phosphatidylserine

It has been found that sphingosine and sphingosylphosphorylcholine (amphiphilic cations) have a stimulatory, and cholesterol 3-sulfate (an amphiphilic anion), an inhibitory, effect on [14C]serine incorporation into phosphatidylserine in glioma C6 and rat liver microsomes. In glioma intact cells sphingosine stimulates phosphatidylserine synthesis in a process independent of protein kinase C, but suppressed by thapsigargin. We suggest that the stimulation of the enzyme occurs by the interaction of amphiphilic cations with the membrane cosubstrate phospholipids, leading to a charge redistribution on their phosphate groups, and hence facilitating the enzyme action. A new hypothesis concerning the mechanism of the serine base exchange reaction is discussed.

Different mechanisms regulate phosphatidylserine synthesis in rat cerebral cortex.

Transduction of extracellular signals through the membrane involves both the lipid and protein moiety. Phosphatidylserine participates to these processes as a cofactor for protein kinase C activity and thus the existence of a regulatory mechanism for its synthesis ought to be expected. In plasma membranes from rat cerebral cortex, the activity of serine base exchange enzyme, that is mainly responsible for phosphatidylserine synthesis in mammalian tissues, was reduced by the addition to the incubation mixture of AlF4- or GTP-gamma-S, known activators of G proteins, whereas ATP was almost uneffective. GTP-gamma-S inhibited the enzyme activity only at relatively high concentration (> 0.5 mM). When the synthesis of phosphatidylserine in the same cerebral area was investigated by measuring the incorporation of labelled serine into the phospholipid in the homogenate buffered at pH 7.6, ATP had an inhibitory effect as GTP-gamma-S and AlF4-. Heparin activated both serine base exchange enzyme in plasma membranes and phosphatidylserine synthesis in the homogenate. The preincubation of plasma membranes in the buffer without any other addition at 37 degrees C for 15 min reduced by 30% serine base exchange enzyme activity. The remaining activity responded to the addition of GTP-gamma-S but was insensitive to 5 mM AlF-4, a concentration that inhibited by 60% the enzyme assayed without preincubation. These results indicate the existence of different regulatory mechanisms, involving ATP and G proteins, possibly acting on different enzymes responsible for the synthesis of phosphatidylserine. Since previous studies have shown that hypoxia increases the synthesis of this phospholipid in brain slices or homogenate (Mozzi et al. Mol Cell Biochem 126: 101-107, 1993), it is possible that hypoxia may interfere with at least one of these mechanisms. This hypothesis is supported by the observation that in hypoxic homogenate 20 mM AlF-4 was not able to reduce the synthesis of phosphatidylserine as in normoxic samples. A similar difference between oxygenated and hypoxic samples, concerning their response to AlF4-, was observed when the incorporation of ethanolamine into phosphatidylethanolamine was studied. The incorporation of choline into phosphatidilcholine was, on the contrary, inhibited at a similar extent in both experimental conditions.

Phosphatidylserine Synthesis in Glioma C6 Cells Is Inhibited by Ca2+Depletion from the Endoplasmic Reticulum: Effects of 2,5-Di-tert-butylhydroquinone and Thimerosal

The effects of 2,5-di-tertbutylhydroquinone (DBHQ) and thimerosal on phosphatidylserine synthesis by the base exchange reaction and on calcium mobilization in intact glioma C6 cells were compared with that of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+-ATPase. It has been found that all these agents inhibit phosphatidylserine synthesis by 70%, but their effectiveness are different. The data show that this inhibition is caused by Ca2+depletion of the endoplasmic reticulum, indicating that phosphatidylserine synthesis requires high concentration of Ca2+within this structure. On this basis and on literature data, a new model for the localization of the serine base exchange enzyme in the endoplasmic reticulum membrane is proposed.

Nitrogen Dioxide-Induced Phosphatidylserine Biosynthesis and Subcellular Translocation in Cultured Pulmonary Artery Endothelial Cells

Exposure to nitrogen dioxide (NO2) increases phosphatidylserine (PS) content in the plasma membranes of pulmonary artery endothelial cells (PAEC). We examined whether the increased PS content is associated with increased uptake of L-serine and/or biosynthesis of PS. Exposure to 5 ppm NO2 increased uptake and incorporation of exogenous L-[14C]serine into whole cells, total cellular lipids, phospholipids, and phospholipid subclasses compared to control. Incorporation of L[14C]serine into the total lipid extracts from isolated plasma membranes, mitochondria, and microsomes from NO2-exposed cells was increased by 45, 32, and 31%, respectively (p < 0.05 for all membranes). Increased incorporation of L-[14C]serine into the total phospholipids of plasma membranes, mitochondria, and microsomes of NO2-exposed cells was increased by 31, 48, and 33%, respectively (p < 0.05 for all membranes). Incorporation of L-[14C]serine into the PS of plasma membranes and microsomes from NO2-exposed cells was increased by 63 and 89%, respectively (p < 0.05 for both membranes). The incorporation of radioactivity from L-[14C]serine into the phosphatidylethanolamine and phosphatidylcholine contents of plasma membranes, mitochondria, and microsomes from NO2-exposed cells was also observed. Exposure of PAEC to NO2 resulted in a significant (p < 0.01) increase in the activity of PS synthase, the serine base-exchange enzyme located in the microsomes of these cells. When L-[14C]serine-prelabeled microsomes were incubated with unlabeled mitochondria from control and NO2-exposed cells, transfer of PS-derived radioactivity from microsomes to mitochondrial phospholipids was observed. These results demonstrate that exposure to NO2 increases uptake and incorporation of exogenous serine as well as intracellular biosynthesis of PS, resulting in increases in the PS content of PAEC and their plasma membranes.

Modulation of the serine base exchange enzyme activity of rat brain membranes by amphiphilic cations and amphiphilic anions.

The biosynthesis of phosphatidylserine in mammalian tissues is catalyzed by the serine base exchange enzyme. The activity of this membrane-bound enzyme can be manipulated by amphiphiles. Amphiphilic cations, such as oleylamine, W-7, chlorpromazine, and didodecyldimethylamine, stimulate the serine base exchange activity. Amphiphilic anions, such as bis(2-ethylhexyl) hydrogen phosphate and cholesterol sulfate, inhibit the serine base exchange activity. These effects are more pronounced at pH 7.0 than at the pH optimum of 8.5 for this enzyme. Both the stimulators and the inhibitors alter the Vmax values without changing the Km value for serine, suggesting that their mechanism of action is related to interactions of the membrane-bound cosubstrate, phosphatidylethanolamine, with the membrane-bound enzyme. The optimal concentration of stimulator varies with the amount of membrane protein present; however, supraoptimal concentrations cause inhibitions. It is proposed that the amphiphilic cations enhance the interaction of the phosphorylethanolamine moiety of the membrane-bound cosubstrate with the enzyme and the amphiphilic anions interfere with such an interaction. Some of the pharmacological properties of these amphiphilic cations, employed clinically as antidepressants, may be mediated by modulation of the serine base exchange enzyme activity.

Possible involvement of the base exchange enzymes in the phospholipid metabolism in LAN-2 cells

The incorporation of [14C] choline, [14C] ethanolamine, and [14C] serine by LAN-2 cells into their corresponding phospholipids was investigated in the presence or absence of TPA. The presence of TPA increased the amount of radioactivity incorporated into the phospholipids with a corresponding decrease in the amount of radioactivity in the cytosolic compartment compared to control cultures. There were no differences between TPA-exposed and control cells in the distribution of radioactivity in free choline, phosphorylcholine or CDP-choline of [14C] choline labeled cells. This indicates that the increased lipid labeling was not accompanied by enhanced labeling of the intermediates of the de novo pathway. These results suggest that a choline base exchange enzyme was stimulated in TPA exposed cells. In addition, the enhanced incorporation of serine by TPA into its corresponding phospholipid implies the stimulation of the serine base exchange enzyme which is responsible for phosphatidylserine synthesis in mammals. These observations suggest a

Effects of chlorpromazine on phosphatidylserine biosynthesis in rat PUP brain exposed to ethanol in utero

Phosphatidylserine biosynthesis in rat pup brain was examined by assaying the serine base-exchange enzyme activity in the microsomal and plasma membrane fractions, and by measuring the incorporation of [ 3H]serine into phosphatidylserine in brain slices and in the intact brain. Chlorpromazine, either added in vitro into the incubation system or administered to animals via i.p. injection or feeding a liquid diet, gave rise to an increase in the phosphatidylserine biosynthesis activity. Ethanol administered in the form of a liquid diet to pregnant rats (day 11-birth) resulted in a decrease in phosphatidylserine biosynthesis in the newborn and developing brain. The ethanol-induced decrease in phosphatidylserine biosynthetic activity could be reversed by adding chlorpromazine to the ethanol diet. Results demonstrate that phosphatidylserine biosynthesis in the neonatal brain is affected in opposite directions by chlorpromazine and ethanol. This poses the possibility that chlorpromazine administration may be effective in alleviating the deleterious effects caused by the decreased phosphatidylserine biosynthesis in brain due to in utero ethanol exposure.

Agonist-induced inhibition of phosphatidylserine synthesis is secondary to the emptying of intracellular Ca2+ stores in Jurkat T-cells.

The biosynthesis of phosphatidylserine (PtdSer) by the serine base-exchange enzyme system, in Jurkat T-lymphocytes, was inhibited in intact cells maintained in low-Ca(2+)-containing buffer (< 10 microM-Ca2+) by using Ca2+ ionophores (A23187 or ionomycin). The rise in cytosolic Ca2+ concentration under these experimental conditions was only due to the release of Ca2+ from intracellular compartments, suggesting that the inhibition of PtdSer synthesis was correlated with the emptying of intracellular Ca2+ pools. This was further studied in saponin-permeabilized cells, in which PtdSer synthesis was found to be inhibited by EGTA, Ca2+ ionophores (A23187 or ionomycin) and Ca(2+)-ATPase inhibitors [thapsigargin or 2,5-di-(t-butyl)-benzohydroquinone]. Since Ca(2+)-ATPase inhibitors impaired refilling of the Ca2+ stores with Ca2+, and since in CD3-activated Jurkat T-cells the Ca2+ stores remained empty after 1 h of treatment with anti-CD3 monoclonal antibodies, we suggest that PtdSer synthesis is mainly regulated by the level of Ca2+ in the intracellular compartments and that the Ca(2+)-dependent serine base-exchange system responsible for PtdSer synthesis is probably located within or close to a Ca(2+)-storage organelle.

In utero ethanol exposure decreases the biosynthesis of phosphatidylserine in rat pup cerebrum.

Phosphatidylserine is enriched in the brain and has been implicated to play a role in regulating neuronal membrane functions. In this study, three experimental protocols were used to examine the effects of in utero ethanol exposure on phosphatidylserine biosynthesis in rat pup brain, namely, (1) assay of the serine base-exchange enzyme activity in brain microsomes, (2) incubation of brain slices with [3H] serine, and (3) incorporation in vivo of [3H]serine into phosphatidylserine as well as serine-related phospholipids in brain. Results from all three protocols point to a decrease in phosphatidylserine biosynthesis in newborn rat pup cerebrum on exposure to ethanol in utero compared with the pair-fed controls. When in utero ethanol-exposed pups were nursed by mothers given a chow diet, the differences gradually returned to control levels by 17 days of age. The decrease in phosphatidylserine biosynthesis may be important in explaining some of the neuronal deficits associated with in utero ethanol exposure.

40] Serine-ethanolamine base-exchange enzyme from rat brain

Publisher Summary This chapter discusses the serine–ethanolamine base-exchange enzyme from rat brain. The base-exchange enzyme activities are widely distributed in mammalian cell membranes. These base-exchange activities do not result in the net synthesis of a phospholipid; they merely catalyze an exchange of the polar amino alcohol or L-serine of preexisting phospholipids. The de novo pathway of phospholipid biosynthesis involving phosphorylated and cytidine nucleotide derivatives are the principal route for formation of the phospholipids possessing choline or ethanolamine or mono-methylethanolamine or dimethylethanolamine as the characteristic polar head groups. However, the serine base-exchange enzyme is the sole mechanism available for phosphatidylserine formation by mammalian tissues. The assays for the base-exchange activities depend on solubility differences of the precursor amino alcohol employed as substrate and the product phospholipid corresponding to the given amino alcohol. The most frequently used approach based on this physical difference is a partitioning of an aqueous and an organic solution. Utilizing a radioactive water-soluble precursor, which is quantitatively retained in the aqueous phase, the amount of radioactivity appearing in the organic phase is an accurate reflection of the base-exchange enzyme activity of biological sample. Confirmation of the reliability of this approach is obtained from thin-layer chromatographic techniques demonstrating cochromatography of the expected product with authentic standards. Reliable results can also be obtained by estimating the quantity of radioactivity present in a trichloroacetic acid (TCA) precipitate of an incubation mixture.

Regulation of the choline, ethanolamine and serine base exchange enzyme activities of rat brain microsomes by phosphorylation and dephosphorylation

The base exchange enzyme activities of rat brain microsomes were estimated subsequent to preincubations under conditions for either protein phosphorylation or dephosphorylation. Quantitatively the choline base exchange activity was most affected by these treatments. Exposure of the microsomes to alkaline phosphatase resulted in a decrease of all three base exchange activities. Pretreatment with a cAMP-dependent protein kinase resulted in increases of all 3 enzyme activities. Conditions favoring protein kinase C phosphorylation resulted in stimulation of the choline base exchange activity.

The base-exchange enzyme activities of sarcolemma and sarcoplasmic reticulum from rat heart

The Ca 2+ dependent incorporation of [ 14C]ethanolamine, l-[ 14C]serine and [ 14C]choline into phosphatidyl-ethanolamine, phosphatidylserine and phosphatidylcholine, respectively, were investigated in membrane preparations from rat heart. The ethanolamine and serine base-exchange enzyme-catalyzed reactions were associated with the sarcolemma and sarcoplasmic reticulum. There was a 17.2-fold and 6.8-fold enrichment, respectively, of the serine and the ethanolamine base-exchange enzyme activities in the sarcolemma compared to the starting whole homogenate. The sarcoplasmic reticulum was enriched in the ethanolamine and serine base-exchange enzyme activities. The choline base-exchange enzyme activity of all membranes fractions was negligible compared to the ethanolamine or serine base-exchange enzyme activities. The apparent K m for the ethanolamine and serine base-exchange enzyme in sarcolemma was 14 μM and 25 μM. respectively. The pH optimum for these base-exchange activities was 7.5–8.0. There was a dependence upon Ca 2+ for these reactions with a 1 or 4 mM concentration required for maximal activity. The properties of the sarcoplasmic reticulum base-exchange enzymes were similar to the sarcolemmal base-exchange enzymes.

Purification and properties of an ethanolamine-serine base exchange enzyme of rat brain microsomes.

The activity of an ethanolamine and serine base exchange enzyme of rat brain microsomes was copurified to near homogeneity. The purification sequence involved detergent solubilization, Sepharose 4B column chromatography, phenyl-Sepharose 4B column chromatography, glycerol gradient sedimentation, and agarose-polyacrylamide gel electrophoresis under non-denaturing conditions. The ratio of the ethanolamine and serine base exchange activities remained almost constant during purification, and both enzyme activities were enriched 25-fold over the initial microsomal suspension. The final enzyme preparation which contained both enzyme activities showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel, having an apparent molecular mass of about 100 kDa. Serine inhibited the ethanolamine incorporation by this preparation and ethanolamine inhibited the serine incorporation. The competitive nature of this inhibition was apparent from Lineweaver-Burk plots, suggesting that the enzyme catalyzes the incorporation of both ethanolamine and serine into their corresponding phospholipids. The Km and Ki values for ethanolamine were quite similar, being 0.02 and 0.025 mM, respectively. The Km and Ki values for serine were also quite similar being 0.11 and 0.12 mM, respectively. The pH optimum was the same at 7.0 with both substrates. The optimum Ca2+ concentration was 8 mM for serine incorporation.