Stimulation of Phospholipase D Activity by Phorbol Esters in Cultured Astrocytes

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D-catalyzed transphosphatidylation, was formed. The half-maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol-treated cells was accompanied by an inhibition of the TPA-induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoyl-glycerol (100 microM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol-treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.     

Phospholipase D-catalyzed transphosphatidylation in anhydrous organic solvents

A new reaction system suitable for phospholipase D (PLD)-catalyzed transphosphatidylation of alcohols with phosphatidylcholine under anhydrous conditions is reported. The key innovation of the reaction system is a cation-exchange resin serving as a scavenger for choline that forms as a byproduct in the transphosphatidylation reaction. Due to the absence of water in this system, the reaction path dramatically shifts in favor of the target transphosphatidylated product, whereas the undesirable side hydrolysis of phosphatidylcholine is completely suppressed, in contrast to commonly used biphasic water-organic systems. In addition, a salt activation technique is successfully applied to increase the catalytic activity of PLD in this anhydrous system. The new reaction system is successfully used for transphosphatidylation of a wide range of primary, secondary, and aromatic alcohols catalyzed by PLD from Streptomyces sp.     

Enzymatic synthesis of phosphatidylserine on small scale by use of a one-phase system

A modification of the phospholipase D-catalyzed synthesis of phosphatidylserine is described, which allows the handling of small quantities of lipid without the need for an ether-water system. By using octylglucoside to disperse the lipid during the enzymatic conversion, it was possible to reduce the volume of the reaction mixture to 50-100 microliters. The amount of lipid that can be handled in such small volumes is in the order of micrograms. This facilitates the synthesis of phosphatidylserine from rare or expensive phosphatidylcholine species. The yield of phosphatidylserine is increased by replacing phospholipase D from cabbage by the enzyme from Streptomyces species.     

Phosphatidylethanol formation in rat organs after ethanol treatment

An abnormal acidic phospholipid was found in high concentration in kidney and brain, and also in other organs of rats exposed to ethanol by i.p. injection or by a liquid diet. The compound could be identified as phosphatidylethanol. Phosphatidylethanol is probably formed in cell membranes by a phospholipase D-catalyzed transphosphatidylation reaction.     

Transphosphatidylation activity of Streptomyces chromofuscus phospholipase D in biomimetic membranes.

The phospholipase D (PLD) from Streptomyces chromofuscus belongs to the superfamily of PLDs. All the enzymes included in this superfamily are able to catalyze both hydrolysis and transphosphatidylation activities. However, S. chromofuscus PLD is calcium dependent and is often described as an enzyme with weak transphosphatidylation activity. S. chromofuscus PLD-catalyzed hydrolysis of phospholipids in aqueous medium leads to the formation of phosphatidic acid. Previous studies have shown that phosphatidic acid-calcium complexes are activators for the hydrolysis activity of this bacterial PLD. In this work, we investigated the influence of diacylglycerols (naturally occurring alcohols) as candidates for the transphosphatidylation reaction. Our results indicate that the transphosphatidylation reaction may occur using diacylglycerols as a substrate and that the phosphatidylalcohol produced can be directly hydrolyzed by PLD. We also focused on the surface pressure dependency of PLD-catalyzed hydrolysis of phospholipids. These experiments provided new information about PLD activity at a water-lipid interface. Our findings showed that classical phospholipid hydrolysis is influenced by surface pressure. In contrast, phosphatidylalcohol hydrolysis was found to be independent of surface pressure. This latter result was thought to be related to headgroup hydrophobicity. This work also highlights the physiological significance of phosphatidylalcohol production for bacterial infection of eukaryotic cells.     

Epidermal growth factor-induced hydrolysis of phosphatidylcholine by phospholipase D and phospholipase C in human dermal fibroblasts

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining 20% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phosphatidylcholine.     

C-terminal loop of Streptomyces phospholipase D has multiple functional roles

We have recently shown that two flexible loops of Streptomyces phospholipase D (PLD) affect the catalytic reaction of the enzyme by a comparative study of chimeric PLDs. Gly188 and Asp191 of PLD from Streptomyces septatus TH-2 (TH-2PLD) were identified as the key amino acid residues involved in the recognition of phospholipids. In the present study, we further investigated the relationship between a C-terminal loop of TH-2PLD and PLD activities to elucidate the reaction mechanism and the recognition of the substrate. By analyzing chimeras and mutants in terms of hydrolytic and transphosphatidylation activities, Ala426 and Lys438 of TH-2PLD were identified as the residues associated with the activities. We found that Gly188 and Asp191 recognized substrate forms, whereas residues Ala426 and Lys438 enhanced transphosphatidylation and hydrolysis activities regardless of the substrate form. By substituting Ala426 and Lys438 with Phe and His, respectively, the mutant showed not only higher activities but also higher thermostability and tolerance against organic solvents. Furthermore, the mutant also improved the selectivity of the transphosphatidylation activity. The residues Ala426 and Lys438 were located in the C-terminal flexible loop of Streptomyces PLD separate from the highly conserved catalytic HxKxxxxD motifs. We demonstrated that this C-terminal loop, which formed the entrance of the active well, has multiple functional roles in Streptomyces PLD.     

Purification and applications of a phospholipase D from a new strain of Streptomyces

An enzyme with phospholipase D activity was purified to homogeneity from a new strain of Streptomyces. The molecular mass, assessed by electrospray mass spectrometry, was 52672 Da and the isoelectric point 9.2. The enzyme, which had pH optimum between 4 and 7, showed satisfactory stability and transphosphatidylation activity.     

Recognition of phospholipids in Streptomyces phospholipase D

To investigate the contribution of amino acid residues to the enzyme reaction of Streptomyces phospholipase D (PLD), we constructed a chimeric gene library between two highly homologous plds, which indicated different activity in transphosphatidylation, using RIBS (repeat-length independent and broad spectrum) in vivo DNA shuffling. By comparing the activities of chimeras, six candidate residues related to transphosphatidylation activity were shown. Based on the above result, we constructed several mutants to identify the key residues involved in the recognition of phospholipids. By kinetic analysis, we identified that Gly188 and Asp191 of PLD from Streptomyces septatus TH-2, which are not present in the highly conserved catalytic HXKXXXXD (HKD) motifs, are key amino acid residues related to the transphosphatidylation activity. To investigate the role of two residues in the recognition of phospholipids, the effects of these residues on binding to substrates were analyzed by surface plasmon spectroscopy. The result suggests that Gly188 and Asp191 are involved in the recognition of phospholipids in correlation with the N-terminal HKD motif. Furthermore, this study also provides experimental evidence that the N-terminal HKD motif contains the catalytic nucleophile, which attacks the phosphatidyl group of the substrate.     

A novel low molecular weight phospholipase D from Streptomyces sp. CS684

With the aim of isolating economically viable enzymes from a microbial source, a novel phospholipase D (PLD) was purified from Streptomyces sp. CS684 (PLD(684)). PLD(684) had molecular weight of 29 kDa, which makes it the second smallest PLD reported so far. The enzyme activity was optimum at pH 6 and 45 degrees C, and enhanced by various detergents. It was stable from pH 7 to 9 and at or below 45 degrees C when assayed after 40 h and 2h, respectively. The K(m) and V(max) values for phosphatidylcholine were 1.16 mM and 1453.74 micromol min(-1)mg(-1), respectively. It catalyzed the transphosphatidylation of glycerol, but not that of l-serine, myo-inositol or ethanolamine. Low molecular weight PLD(684) with transphosphatidylation activity may be utilized in the industrial production of rare and commercially important phospholipids.     

A new method for determining phospholipase D activity using the monomolecular film technique

A versatile and continuous assay for phospholipase D (PL D) activity was developed using the monomolecular film technique. For this purpose, a two-step enzymatic reaction was used. First, PL D hydrolysis of stable 1,2-diacyl-sn-glycero-3-phosphocholine (PC) films by PL D generated a stable 1,2-diacyl-sn-glycero-3-phosphate (PA) film and water-soluble choline. Secondly, the latter acidic phospholipid, in contrast to the initial PC molecule, was further hydrolysed under the action of porcine pancreatic lipase (PPL) in order to give rise to lysophosphatidic acid and fatty acid, which were rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the PL D-catalyzed reaction with phospholipid monolayers as substrates. The PLD kinetics were linear with time and the velocities recorded were directly dependent upon the amount of PL D used. In a preliminary study, we investigated the effects of the surface pressure on the PL D activity.     

Isolation of phospholipase d producing microorganisms with high transphosphatidylation activity

The transphosphatidylation and hydrolytic activities of phospholipase d in culture supernatants of soil isolates were evaluated by a specific spectrophotometric method for quantitative determination using an artifical substrate, phosphatidyl-p-nitrophenol. Phospholipase d from strain TH-2 showed the highest specific activity and ratio of transphosphatidylation activity to hydrolytic activity among those from the eight soil isolates and commercial Actinomycetes phospholipase d.     

The determination of phospholipase D activity in emulsion systems

Although phospholipase D (PLD) is often used in emulsion systems consisting of buffer and a nonpolar organic solvent, most activity assays have been designed to work in purely aqueous milieu. Here a method is described for the determination of PLD activity in emulsion systems. The assay is based on the transphosphatidylation of phosphatidylcholine with 1-butanol in dichloromethane/buffer with the subsequent densitometric quantification of the products after their separation by HPTLC and staining with a CuSO4/H3PO4 reagent. The method is particularly appropriate for the determination of enzymes such as PLD from Streptomyces sp. that prefer the exchange of the head group in glycerophospholipids to their hydrolysis. Since the application of an organic solvent in the PLD assay allows the determination of the enzyme in analytes insoluble in aqueous media, the method can also be used to determine PLD activity in the presence of high concentrations of phospholipids.     

A facile transphosphatidylation reaction using a culture supernatant of actinomycetes directly as a phospholipase D catalyst with a chelating agent

An attempt was made to use the phospholipase D (PLD)- containing culture supernatants of actinomycetes directly as catalysts for the transphosphatidylation reaction of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) in a biphasic system. Of the five actinomycetes (three Streptomyces sp. and two Streptoverticillium sp.) examined, three (St. mediocidicus, Stv. cinnamoneum and Stv. hachijoense) exhibited good PLD production performance, but the selectivity (ratio of transphosphatidylation to hydrolysis) of the PLDs in the culture supernatant of all three actinomycetes were significantly low. However, the addition of EDTA to the reaction mixture as a chelating agent remarkably improved the selectivity of the PLDs, which approached 100% in all the culture supernatants. Commercially available PLDs were also investigated and classified into two types. The PLDs of one type had high selectivity and no metal was required for the enzyme activity, while those of the other type showed low selectivity and a metal was necessary for the enzyme to be activated. From this finding, it was considered that the culture supernatants used in this study contained several PLDs of both types. When the chelating agent was added to the reaction mixture, the hydrolysis due to PLDs with low selectivity was suppressed by removal of the essential metal, resulting in an increased in the overall selectivity of the PLDs in the culture supernatant. Repeated batch transphosphatidylation reactions were performed 20 times, reusing the PLDs in the aqueous phase by centrifugation; the reaction rate gradually decreased to 60% of that of batch 1 by batch 20. This suggests that the transphosphatidylation reaction using a culture supernatant has potential for industrial application. (c) 1994 John Wiley & Sons, Inc.     

The transphosphatidylation activity of phospholipase D

Transphosphatidylation activity is a characteristic and remarkable property of phospholipase D (PLD) and has been studied in plants and mammalian tissues. This reaction is often used to confirm the properties and/or abnormalities of PLD activity. The mechanism for activating PLD transphosphatidylation seems multiple. Although significant changes of transphosphatidylation activity have been found in some pathological animal models, the biological significance of PLD transphosphatidylation remains largely unknown.     

A spectrophotometric method for the assay of phospholipase D activity

Phosphatidylcholine phosphatidohydrolase (EC 3.1.4.4, phospholipase D) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. We have developed a spectrophotometric assay for phospholipase D using choline kinase, pyruvate kinase, and lactate dehydrogenase to couple the release of choline with the oxidation of NADH. The assay was linear both with time and with enzyme concentration. The assay should prove useful for continuous monitoring of enzyme activity, determination of initial rates of reaction, and detailed kinetic studies of phospholipase D. The method is limited to analysis of purified preparations of phospholipase D lacking competing activities to the coupled system.     

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.     

Phorbol ester activation of phospholipase D in human monocytes but not peripheral blood lymphocytes

Previous reports have shown that 12-0-tetradecanoylphorbol-13-acetate can activate phospholipase D in human peripheral blood mononuclear cells as measured by an enzyme-catalyzed transphosphatidylation reaction (phosphatidylethanol formation). In the present study, the mononuclear cells were fractionated by two procedures to identify the responsive cells. Contrary to earlier suggestions, the results indicate that phorbol ester does not stimulate phospholipase D activity in normal lymphocytes. Thus, phosphatidylethanol was not produced by T lymphocytes (isolated by sheep erythrocyte rosette formation) or by a mixture of T and B lymphocytes (isolated by centrifugal elutriation). Under the same conditions, phorbol ester was able to activate phospholipse D in fractions that contained predominately monocytes. Preliminary experiments have further shown that phorbol ester does not induce phospholipase D activity in human T cell leukemic lines (MOLT-3, CEM, JURKAT, PEER) but can do so in some, but not all, B cell lines that have been infected with Epstein-Barr virus.     

Activation of phospholipase D by chemotactic peptide in HL-60 granulocytes

Activation of phospholipase D (PLD) has been investigated in dimethylsulfoxide differentiated HL-60 granulocytes labeled in endogenous 1-0-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) by incubation with [3H]alkyl-lysoPC. Stimulation of these labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), induces rapid generation of [3H]phosphatidic acid (PA) and slower formation of [3H]diglyceride, suggesting hydrolysis of alkyl-PC by PLD. A unique feature of PLD is its ability to transfer the phosphatidyl moiety of phospholipids to alcohols (transphosphatidylation). This characteristic has been exploited to identify PLD activity. For example, when ethanol is present during stimulation of the HL-60 cells, [3H]phosphatidylethanol (PEt) is formed with a concomitant decrease in [3H]PA. Cells incubated with [32P]orthophosphate to label the terminal phosphate of ATP do not incorporate 32P into PEt, consistent with the [3H]PEt not being synthesized from [3H]diglyceride. In contrast, [3H]PA arises from both PLD and diglyceride kinase activities. Furthermore, PEt synthesis closely parallels PA formation and both are inhibited by an fMLP receptor antagonist, suggesting that both PA and PEt are derived from agonist-stimulated PLD action. These observations are consistent with phospholipase D-catalyzed breakdown of alkyl-PC in fMLP- stimulated granulocytes.     

In vitro synthesis of phosphatidylinositol and phosphatidylcholine by phospholipase D

Phosphatidylinositol (PI) was prepared from egg lecithin by a one-step transphosphatidylation reaction catalysed by phospholipase D in the presence of myo-inositol. Similarly phosphatidylcholine (PC) has been synthesized by the same technique from egg phosphatidylethanolamine using phospholipase D and choline chloride.The yield of PI was ca 25 % and that of PC ca 28 %. The transphosphatidylase function of phospholipase D offers a useful route for the synthesis of different classes of phospholipids.