Phosphatidylethanol Formation via Transphosphatidylation by Rat Brain Synaptosomal Phospholipase D

Phosphatidylethanol (Peth) formation catalyzed by the transphosphatidylation activity of phospholipase D was demonstrated to occur in a rat brain synaptosomal enriched preparation. The optimal pH was determined to be 6.5, and the optimal ethanol concentration was determined to be 0.3-0.4 M with an apparent Km of 0.2 M. Peth formation was barely detectable in the absence of an appropriate activator and several unsaturated fatty acids were found to be effective activators. The concentrations of oleic acid required for maximum activation varied with the concentration of exogenous phosphatidylcholine present in the incubation mixtures. All detergents tested were significantly less active than the unsaturated fatty acids and divalent ions were not required for Peth formation. Phosphatidylcholine was the most effective phosphatidyl donor of the phospholipids tested. Peth forming activity was greatest in the synaptic membrane fraction of the various brain subfractions examined. The 12,000 g-100,000 g particulate fraction of lung, heart, and adipose tissue had activities similar to that of brain.     

Phospholipase D Activity of Rat Brain Neuronal Nuclei

Abstract: Phospholipase D activity of rat brain neuronal nuclei, measured with exogenous phosphatidylcholine as substrate, was characterized. The measured activity of neuronal nuclei was at least 36-fold greater than the activity in glia nuclei. The pH optimum was 6.5, and unsaturated but not saturated fatty acids stimulated the enzyme. The optimal concentration of sodium oleate for stimulation of the enzyme activity was 1.2 m M in the presence of 0.75 m M phosphatidylcholine. This phospholipase D activity was cation independent. In the absence of NaF, used as a phosphatidic acid phosphatase inhibitor, the principal product was diglyceride; whereas in the presence of NaF, the principal product was phosphatidic acid. The phospholipase D, in addition to having hydrolytic activity, was able to catalyze a transphosphatidylation reaction. Maximum phosphatidylethanol formation was seen with 0.2–0.3 M ethanol. GTPγS, ATPγS, BeF₂, AIF₃, phosphatidic acid, and phosphatidylethanol inhibited the neuronal nuclei phospholipase D activity. The addition of the cytosolic fraction of brain, liver, kidney, spleen, and heart to the incubation mixtures resulted in inhibition of the phospholipase D activity. Phospholipase D activity was detectable in nuclei prepared from rat kidney, spleen, heart, and liver.     

重要生化反应“转移磷脂酰反应”的研究进展

转移磷脂酰反应是在磷脂酶D的催化作用下,甘油磷脂和含羟基化合物发生碱基交换生成新的磷脂的反应。该反应为磷脂酶D所特有,被广泛的应用于动物、植物和微生物的脂类代谢、脂类信号研究以及重要生化制剂磷脂的合成工艺中。本文综述了转移磷脂酰反应的反应机制、影响因素、生物学作用及应用现状,讨论了深入研究这一反应所有待揭示的问题,并展望了今后的发展方向。     

Kinetic Analysis in Mixed Micelles of Partially Purified Rat Brain Phospholipase D Activity and its Activation by Phosphatidylinositol 4,5-Bisphosphate

A partially purified rat brain membrane phospholipase D (PLD) activity was characterized in a mixed micellar system consisting of l-palmitoyl-2-[6-N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)-amino]caproyl-phosphatidylcholine (NBD-PC) and Triton X-100, under conditions where Triton X-100 has a surface dilution effect on PLD activity and the catalytic rate is dependent on the surface concentration (expressed in terms of molar ratio) of NBD-PC. PLD activity was specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP₂), and the curve of activation versus PIP₂ molar ratio fitted a Michaelis-Menten equation with a K_act value between molar ratios of 0.001–0.002. Maximal activation was observed at a PIP₂ molar ratio of 0.01. Similar values were obtained when activities of partially purified PLD as well as membrane-bound PLD were determined towards pure NBD-PC micelles. In the mixed micellar system PIP₂ was shown to elevate by 6–22 fold the specificity constant of PLD towards NBD-PC (K_A, which is proportional to V_max/K_m). Kinetic analysis of PLD trans-phosphatidylation activity towards ethanol, 1-propanol and 1-butanol revealed a Michaelis-Menten type dependence on alcohol concentration up to 1000, 200 and 80 mM, respectively. While V_max values were similar towards all three alcohols, enzyme affinity increased as the alcohol was longer, and K_m values for ethanol, 1-propanol and 1-butanol were 291, 75 and 16 mM (respectively). PLD specificity constants (K_A) towards ethanol, 1-propanol and 1-butanol were shown to be respectively 260, 940 and 5,920 times higher than to water, the competing substrate. 1-Propanol and 1-butanol inhibited PLD activity above 400 and 100 mM, respectively. The present results indicate that partially purified PLD obeys surface dilution kinetics with regard to its phospholipid substrate PC and its cofactor PIP₂, and that in the presence of alcohols, its transphosphatidylation activity may be analyzed as a competitive reaction to the hydrolysis reaction.     

Phospholipase D from Streptoverticillium cinnamoneum: protein engineering and application for phospholipid production

This review is focusing on an industrially important enzyme, phospholipase D (PLD), exhibiting both transphosphatidylation and hydrolytic activities for various phospholipids. The transphosphatidylation activity of PLD is particularly useful for converting phosphatidylcholine (PC) into other phospholipids. During the last decade, the genes coding for PLD have been identified from various species including mammals, plants, yeast, and bacteria. However, detailed basic and applied enzymological studies on PLD have been hampered by the low productivity in these organisms. Efficient production of a recombinant PLD has also been unsuccessful so far. We recently isolated and characterized the PLD gene from Streptoverticillium cinnamoneum, producing a secretory PLD. Furthermore, we constructed an overexpression system for the secretory enzyme in an active and soluble form using Streptomyces lividans as a host for transformation of the PLD gene. The Stv. cinnamoneum PLD was proven to be useful for the continuous and efficient production of phosphatidylethanolamine (PE) from phosphatidylcholine. Thus, the secretory PLD is a promising catalyst for synthesizing new phospholipids possessing various polar head groups that show versatile physiological functions and may be utilized in food and pharmaceutical industries.     

重要生化反应“转移磷脂酰反应”的研究进展

转移磷脂酰反应是在磷脂酶D的催化作用下,甘油磷脂和含羟基化合物发生碱基交换生成新的磷脂的反应。该反应为磷脂酶D所特有,被广泛的应用于动物、植物和微生物的脂类代谢、脂类信号研究以及重要生化制剂磷脂的合成工艺中。本文综述了转移磷脂酰反应的反应机制、影响因素、生物学作用及应用现状,讨论了深入研究这一反应所有待揭示的问题,并展望了今后的发展方向。     

Membrane-Associated Phospholipase D Activity in Rat Sciatic Nerve

Rat sciatic nerve contains a membrane-bound phospholipase D that catalyzes the hydrolysis of exogenous phosphatidylcholine (PC) to phosphatidic acid (PA) and choline. The enzyme is associated with a particulate fraction consisting primarily of microsomes and myelin. This fraction also contains phosphatidate phosphohydrolase activity leading to the production of diacylglycerols (DAG). The phosphohydrolase activity can be completely inhibited by NaF. Hydrolysis of exogenous PC requires detergent and is linear up to about 40 micrograms of protein at a pH optimum of 6.5. In the absence of NaF, the sum of PA and DAG increases linearly for 40 min, whereas in its presence, PA production is linear for only 15 min. At optimum conditions, PC hydrolysis proceeds at 15 nmol/h/mg of protein. Addition of increasing amounts of ethanol to the incubation system leads to the generation of increasing amounts of phosphatidylethanol, indicating transphosphatidylation activity. At an ethanol concentration of 0.4 M, phosphatidylethanol represents about one-half of the reaction products generated at approximately the same rate of enzymic activity observed in the absence of ethanol. Higher ethanol concentrations are inhibitory.     

The Presence of Phospholipase D In Rat Central Nervous System Axolemma

An axolemma-enriched fraction prepared from a purified myelinated axon fraction isolated from rat CNS was found to contain phospholipase D at a specific activity similar to that of a microsomal fraction isolated from whole brain. There was a concomitant threefold enrichment in the specific activity of phospholipase D and acetylcholinesterase in the axolemma-enriched fraction compared with the specific activities of these enzymes in the starting white matter whole homogenate. This axonal phospholipase D may be involved in remodeling of phospholipid, which in turn may affect axonal functions such as ion translocation.     

Histamine H1 and Endothelin ETB Receptors Mediate Phospholipase D Stimulation in Rat Brain Hippocampal Slices

Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans -(1 S ,3 R )-aminocyclopentyl-1,3-dicarboxylic acid ( trans -ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [³²P]P_i-prelabeled rat brain cortical and hippocampal slices. The accumulation of [³²P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans -ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC₅₀ 18 µ M ) was inhibited by the H₁ receptor antagonist mepyramine with a K _i constant of 0.7 n M and was resistant to H₂ and H₃ receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC₅₀ 44 n M ) was not blocked by BQ-123, a specific antagonist of the ET_A receptor. Endothelin-3 and the specific ET_B receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC₅₀ values of 16 and 3 n M , respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H₁ and ET_B receptors, respectively.     

Metabotropic Excitatory Amino Acid Receptor Activation Stimulates Phospholipase D in Hippocampal Slices

Metabotropic excitatory amino acid (EAA) receptors are coupled to effector systems through G proteins. Because various G protein-coupled receptors stimulate the hydrolysis of phosphatidylcholine by phospholipase D (PLD), we examined the possibility that metabotropic EAA receptors exist that are coupled to the activation of PLD. We found that the selective metabotropic glutamate receptor (mGluR) agonists 1S,3R-amino-1,3-cyclopentanedicarboxylic acid (ACPD) and 1S,3S-ACPD, but not the inactive isomer, 1R,3S-ACPD, induce a concentration-dependent increase in PLD activity in hippocampal slices. Selective ionotropic glutamate receptor (iGluR) antagonists did not block 1S,3R-ACPD-induced PLD stimulation. Furthermore, although selective iGluR agonists did not activate this response, the nonselective mGluR-iGluR agonists, ibotenate and quisqualate, caused significant increases in PLD activity (all in the presence of iGluR antagonists). L-2-Amino-3-phosphonopropionic acid, which blocks the mGluR that is coupled to phosphoinositide hydrolysis in various brain regions, activates PLD to the same extent as the active isomers of ACPD. These data suggest that metabotropic EAA receptors exist in hippocampus that are coupled to PLD activation and are pharmacologically distinct from phosphoinositide hydrolysis-coupled mGluRs.     

Effects of Temperature on Arachidonic Acid-Induced Cellular Edema and Membrane Perturbation in Rat Brain Cortical Slices

The effects of temperature on arachidonic acid-induced cellular edema in the first cortical brain slices of rats were studied. Incubation of the cortical slice in arachidonic acid at 37 degrees C induced cellular swelling, and increased intracellular Na+ and lactic acid contents concomitant with decreased intracellular K+. When the incubation temperature was reduced these changes were reduced in severity. The uptake of [3H]arachidonic acid in cortical slices was temperature-dependent. The incorporation of [3H]arachidonic acid into various lipid fractions was further studied by HPLC. The majority of [3H]arachidonic acid was incorporated into triacylglycerol and phosphatidylinositol (PI), but the incorporation of [3H]arachidonic acid into PI was temperature-dependent, unlike that into other phospholipids and neutrolipids. Further, cortical (Na+ + K+)-ATPase activity was inhibited whereas its subunit K+-activated p-nitrophenyl-phosphatase was activated by arachidonic acid at various incubation temperatures. The effects of arachidonic acid on these enzymes is similar to that of thimerosal, a lipid removal agent. These data suggest that both temperature and arachidonic acid play an important role in the development of cellular edema associated with membrane perturbation and inactivation of (Na+ + K+)-ATPase activity.     

Guanine Nucleotide-Binding Protein Regulation of Microsomal Phospholipase D Activity of Canine Cerebral Cortex

The hydrolytic activity of microsomal phospholipase D from canine cerebral cortex was measured by a radiochemical assay using 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl[3H]choline and 1-palmitoyl-2-[9,10(n)-3H]palmitoyl-sn-glycerol-3-phosphorylcholine as the exogenous substrates. Of several detergents tested, Triton X-100 was found to be the most effective in allowing expression of phospholipase D hydrolytic activity. The microsomal phospholipase D does not require any metal ion for its hydrolytic activity. Calcium and magnesium were slightly inhibitory between concentrations of 1 and 4 mM, but zinc was greatly inhibitory, causing a loss of greater than 90% activity at the 4 mM concentration. Non-hydrolyzable guanine nucleotide analogues such as guanosine 5'-(3-O-thio)triphosphate and guanyl-5'-yl-(beta, gamma-methylene)diphosphonate but not guanosine 5'-(2-thio)diphosphate were able persistently to stimulate phospholipase D hydrolytic activity at micromolar concentrations. Guanosine 5'-(2-thio)diphosphate was capable of partially blocking guanosine 5'-(3-O-thio)triphosphate stimulation of phospholipase D. Aluminum fluoride was able to cause a two- to threefold increase in hydrolytic activity of the phospholipase D. Cholera toxin had a stimulatory effect on the hydrolytic activity of phospholipase D, whereas islet-activating protein pertussis toxin had no effect. These results indicate that regulation of microsomal phosphatidylcholine phospholipase D activity by the guanine nucleotide-binding protein(s) in canine cerebral cortex may play an important role in signal transduction processes as well as in brain choline metabolism.     

Phospholipase D-catalyzed synthesis of new phospholipids with polar head groups

A series of new phospholipids with polar head groups have been synthesized by enzymatic transphosphatidylation of 1,2-dioleoyl-sn-glycerophosphocholine and identified by 1H NMR and MALDI-TOF-MS. The acceptor alcohols were N- or C2-substituted derivatives of ethanolamine (diethanolamine, triethanolamine, serinol, Tris, BisTris). Phospholipases D from cabbage (PLDcab) and Streptomyces sp. (PLDStr) were compared with respect to product yield and purity as well as the initial rates in transphosphatidylation and competing hydrolysis. In all reactions, PLDStr showed a remarkably higher transphosphatidylation activity than PLDcab. However, higher yields of the phospholipids with diethanolamine, triethanolamine, and serinol were obtained by PLDcab because PLDStr resulted in the additional formation of diphosphatidyl derivatives. In the synthesis of the Tris and BisTris derivatives, PLD(Str) was much more appropriate because voluminous head group alcohols (>129A3) are poorly converted by PLDcab. With BisTris as acceptor alcohol two regioisomeric forms of phosphatidyl-BisTris were obtained.     

Pertussis Toxin-Insensitive G Protein Mediates Carbachol Activation of Phospholipase D in Rat Pheochromocytoma PC12 Cells

In the present study, an activation mechanism for phospholipase D (PLD) in [3H]palmitic acid-labeled pheochromocytoma PC12 cells in response to carbachol (CCh) was investigated. PLD activity was assessed by measuring the formation of [3H]phosphatidylethanol ([3H]PEt), the specific marker of PLD activity, in the presence of 0.5% (vol/vol) ethanol. CCh caused a rapid accumulation of [3H]-PEt, which reached a plateau within 1 min, in a concentration-dependent manner. The [3H]PEt formation by CCh was completely antagonized by atropine, demonstrating that the CCh effect was mediated by the muscarinic acetylcholine receptor (mAChR). A tumor promoter, phorbol 12-myristate 13-acetate (PMA), also caused an increase in [3H]-PEt content, which reached a plateau at 30-60 min after exposure, but an inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 microM), blocked PMA-induced [3H]PEt formation by 77%, it had no effect on the CCh-induced formation. These results suggest that mAChR-induced PLD activation is independent of PKC, whereas PLD activation by PMA is mediated by PKC. NaF, a common GTP-binding protein (G protein) activator, and a stable analogue of GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), also stimulated [3H]PEt formation in intact and digitonin-permeabilized cells, respectively. GTP, UTP, and CTP were without effect. Furthermore, guanosine 5'-O-(2-thiodiphosphate) significantly inhibited CCh- and GTP gamma S-induced [3H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 microM) had no effect on [3H]PEt formation by GTP gamma S.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamatergic Activation of Hippocampal Phospholipase D: Postnatal Fading and Receptor Desensitization

Abstract: Phospholipase D (PLD) activity was determined in rat hippocampal slices between postnatal days 3 and 35. After birth, basal PLD activity was low and, within 2 weeks, increased to reach a plateau that was maintained up to the adult age. Likewise the response to glutamate developed postnatally to reach a maximum at day 8, but then faded rapidly and was almost absent at day 35. Activation of PLD by 4β-phorbol 12β,13α-dibutyrate (PDB) was independent of age, whereas the effect of aluminum fluoride (AlF₄⁻) increased to a plateau within the first week. At day 8, PLD stimulation by glutamate via metabotropic receptors involved protein kinase C activation, but was independent of Ca²⁺ influx; the time course of PLD activation by PDB or AlF₄⁻ was linear throughout the experiment, whereas the response to glutamate or 1-aminocyclopentane-1,3-dicarboxylic acid followed a biphasic pattern: the rapid "first phase activation" desensitized within a few minutes and disclosed a small, but maintained "second phase." Pretreatment experiments confirmed desensitization of PLD activation by glutamate, but not by AlF₄⁻ or PDB. The biphasic pattern of glutamatergic PLD activation changed during development, i.e., the first phase activation faded and the second phase remained. These results were fully confirmed by the time courses of the PLD-mediated efflux of choline evoked by glutamate. In conclusion, postnatal glutamatergic activation of hippocampal PLD is composed of a pronounced and desensitizing first phase activation and a small, but nondesensitizing second phase. The first, but not the second, phase activation fades rapidly during development. The hypothesis is discussed that the glutamatergic activation of PLD occurs along different pathways in neonate and adult tissue.     

Functional Changes of Rat Brain Microsomal Membrane Surface After Learning Task Depending on Dietary Fatty Acids

Abstract: Biochemical characteristics of brain microsomal membranes were examined before and after the brightness-discrimination learning tasks in rats that were fed either safflower oil (α-linolenate-deficient) or perilla oil (α-linolenate-sufficient) diets. We detected small changes in the chain elongation system for polyunsaturated fatty acids in microsomes, whereas no significant difference was detected in the inositol trisphosphate-induced calcium release and ATP-induced calcium uptake profiles of microsomes between the two dietary groups. The calcium ion-induced aggregation rate of microsomes was determined in both groups. We found that the aggregation rate of microsomes in the safflower oil group was significantly greater than that in the perilla oil group. The difference in susceptibility of microsomal membrane phospholipids to phospholipase A₂ between the groups was obvious, and the amount of released fatty acids by phospholipase A₂ from the perilla oil group microsomes was nearly half of that from the safflower oil group microsomes after the learning task. Susceptibility of sialic acids on the brain microsomal membranes to exogenous sialidase was different only after the learning task in the safflower and perilla oil groups. These results suggest that the biochemical characteristics of membrane surfaces of brain microsomes are affected significantly by the learning task itself in a dietary oil-dependent manner.     

磷脂酶D和炎症的关系

磷脂酶Ｄ（ＰＬＤ）广泛存在于动物组织细胞中,并受各种胞外信号调节。其主要底物为磷脂酰胆碱（ＰＣ）。ＰＬＣ引起的ＰＣ水解是细胞内重要的信号转导途径。越来越多的证据表明ＰＬＤ和炎症有密切的关系。本文主要介绍ＰＬＤ在呼吸爆发、脱颗粒及花生四烯酸（ＡＡ）释放等方面的研究进展。     

Lack of Phospholipase D Activity in Chromaffin Cells: Bradykinin-Stimulated Phosphatidic Acid Formation Involves Phospholipase C in Chromaffin Cells but Phospholipase D in PC12 Cells

The role of lipid-bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor-linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase (DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of chromaffin cells prelabelled (24 h) with 32Pi leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [gamma-32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin.     

磷脂酶D与病原微生物感染

磷脂酶D(phospholipase D,PLD)普遍存在于细菌,真菌以及哺乳动物中.在病原微生物中,PLD作为毒力决定因子在减数分裂、孢子形成等过程中起作用;在哺乳动物细胞中,PLD主要在胞膜转运、调节有丝分裂和细胞肌动蛋白骨架等一些信号转导中起作用.在病原菌感染宿主细胞的过程中,病原体和宿主细胞的PLD都被激活并发生级联反应,病原菌PLD可调节自身肌动蛋白丝的聚合和重排,并引起宿主细胞局部肌动蛋白丝的集聚,诱导宿主细胞对其吞噬.深入探讨PLD激活对感染发生的调控作用对透彻理解病原菌感染宿主细胞的分子机制具有重要意义.     

Effect of Dietary Lipids on Phospholipase D Activity in Rat Brain

Phospholipase D (PLD) is emerging as a major player in many novel signaling pathways. Based on recent studies correlating membrane composition with enzyme function, we speculated that feeding of dietary lipids to the newborns has a major impact on brain PLD activity. To test this hypothesis, the rat dams were fed fat-free powder containing either safflower oil or fish oil, and a control powdered chow. The pups were weaned onto the diet and sacrificed at 30 days of age. PLD activity was measured by transphosphatidylation assays using rat brain membranes. This study shows that microsome GTPS-dependent PLD activity in rats fed safflower oil or fish oil was significantly reduced by 38% and 30% respectively compared to controls. Oleate-dependent PLD activity in the safflower oil group, however, was significantly increased by 38%. In contrast, synaptosome membrane (P2) GTPS-dependent PLD activity in rats consuming safflower oil was significantly increased by 29%, but there was no difference in oleate-dependent PLD activity. Likewise, no difference was observed in microsome oleate-dependent PLD and P2 GTPS-dependent PLD activity between the fish oil and the control groups. These results indicate that dietary lipid intake appears to modulate phospholipid metabolism and differential expression of PLD isozymes in the brain.