Phospholipase D and phosphatidate phosphohydrolase activities in rat cerebellum during aging

Aging is a process that affects different organs, of which the brain is particularly susceptible. PA and DAG are central intermediates in the phosphoglyceride as well as in the neutral lipid biosynthetic pathway, and they have also been implicated in signal transduction. Phospholipase D (PLD) and phosphatidate phosphohydrolase (PAP) are the enzymes that generate PA and DAG. The latter can be transformed into MAG by diacylglycerol lipase (DGL). In the present study, we examine how aging modulates the PLD, PAP, and DGL isoforms in cerebellar subcellular fractions from 4-(adult),28-, and 33-mon-old (aged) rats. Pl-4,5-bisphosphonate (PIP₂)-dependent PLD, PAP1, and DGL1 were distributed in different percentages in all cerebellum subcellular fractions. On the other hand, PAP2 and DGL2 activities were observed in all subcellular fractions except in the cytosolic fraction. Aging modified the enzyme distribution pattern. In addition, aging decreased nuclear (45%), mitochondrial-synaptosomal (55%), and cytosolic (71%) PAP1 activity and increased (28%) microsomal PAP1 activity. DGL1 activity was decreased in nuclear (85%) and mitochondrial-synaptosomal (63%) fractions by aging. On the other hand, PIP₂-dependent PLD activities were increased in the mitochondrial-synaptosomal fraction. PAP2 and DGL2 were increased in the microsomal fraction by 87 and 114%, respectively, and they were decreased in the nuclear fraction. The changes observed in cerebellum PAP1 and DGL1 activities from aged rats with respect to adult rats could be related to modifications in lipid metabolism. Differential PA metabolization during aging through PIP₂-dependent PLD/PAP2/DGL2 activities could be related to alterations in the neural signal transduction mechanisms.     

Characterization of phospholipase D activity in bovine photoreceptor membranes

Phospholipase D (E.C. 3.1.4.4.) was detected in isolated bovine rod outer segments (ROS) and its properties determined. The enzyme activity was assayed using either a sonicated microdispersion of 1,2-diacyl-sn-[2³H]glycerol-3-phosphocholine (PC), or [¹⁴C]ethanol. Using [³H]PC and ethanol as a substrate, we were able to detect the hydrolytic properties as well as the transphosphatidylation reaction catalyzed by phospholipase D (PLD): formation of [³H]phosphatidic acid and phosphatidylethanol [³H]PtdEt; whereas with [¹⁴C]ethanol or [³H]glycerol in the absence of exogenous PC, only transphosphatidylation reactions were detected (formation of [¹⁴C]PtdEt or [³H]phosphatidylglycerol, respectively). The use of varying concentrations of [³H]PC and 400 mM of ethanol gave an apparent K _m value for PC of 0.51 mM and a V _max value of 111 nmol × h⁻¹ × (mg protein)⁻¹. The activity was linear up to 60 min of incubation and up to 0.2 mg of protein. The optimal ethanol concentration was determined to be 400 mM, with an apparent K _m of 202 mM and a V _max value for ethanol of 125 nmol × h⁻¹ × (mg protein)⁻¹. A clear pH optimum was observed around 7. PLD activity was increased in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate or sodium deoxycholate and inhibited with Triton X-100. The enzyme activity was also activated in the presence of Ca²⁺ or Mg²⁺ (1 mM) although these ions were not required for measuring PLD activity. The high specific activity of PLD found in purified ROS compared to the activity found in other subcellular fractions of the bovine retina suggests that this enzymatic activity is native to ROS. The present report is the first evidence of PLD activity associated with photoreceptor ROS.     

Protein kinase C-dependent stimulation of phospholipase D in phospholipase C-treated fibroblasts

Treatment of [¹⁴C]choline- or [¹⁴C]ethanolamine-labeled NIH 3T3 fibroblasts withBacillus cereus phosphatidylcholine-specific phospholipase C (PLC) enhanced phospholipase D (PLD)-mediated hydrolysis of the respective¹⁴C-labeled phospholipids. PLD activity was stimulated by 1.5 U/mL of POLC and by 100 nM of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) to similar extents. Treatment of¹⁴C]palmitic acid-labeled fibroblasts with PLC in the presence of ethanol also enhanced PLD-mediated formation of phosphatidylethanol; the effects of PLC and PMA were nonadditive. PLC had no effect on PLD activity in fibroblasts in which PKC was down-regulated by prolonged (24 h) treatment with 300 nM PMA. These data indicate that treatment of fibroblasts with exogenous PLC results in PKC-dependent activation of PLD.     

Characterization of a PLDζ2 Homology Gene from Developing Castor Bean Endosperm

Castor oil contains approximately 90% ricinoleic acid (RA) which is stored mainly in the form of tri-ricinoleic acid containing triacylglycerols (TAG). Ricinoleate is synthesized from oleate (18:1n-9) esterified to the sn-2 position of phosphatidylcholine (PtdCho) catalyzed by oleoyl-12-hydroxylase. PtdCho-derived diacylglycerol (DAG) is an important substrate pool for TAG synthesis, and the interconversion between PtdCho and DAG has been shown to play a critical role in channeling hydroxy fatty acids (HFA) to TAG. Although phospholipase D (PLD) has been reported to catalyze the hydrolysis of PtdCho to produce phosphatidic acid which can then be converted to DAG, its potential functions in the channeling of RA from PtdCho to DAG and the assembly of RA on TAG is largely unknown. In the present study, 11 PLD genes were identified from the Castor Bean Genome Database. Gene expression analysis indicated that RcPLD9 is expressed at relatively high levels in developing seeds compared to other plant tissues. Sequence and phylogenetic analyses revealed that RcPLD9 is a homolog of Arabidopsis PLDζ2. Overexpression of RcPLD9 in the Arabidopsis CL7 line producing C18-HFA resulted in RA content reductions in the polar lipid fraction (mainly PtdCho) and mono-HFA-TAG, but increased RA content in di-HFA-TAG. Since part of RA in di-HFA-TAG is derived from HFA-DAG, the results indicated that RcPLD9 facilitates the channeling of RA from PtdCho to DAG for its assembly on TAG in developing seeds.     

Vasopressin stimulates phospholipase D activity against phosphatidylcholine in vascular smooth muscle cells

It is now clear that various hormones and agonists can stimulate the production of lipid mediators from nonphosphoinositide phospholipids. We have investigated the production of diacylglycerol from nonphosphoinositide sources, and we demonstrated that vasopressin and other vasoactive agents stimulate hydrolysis of phosphatidylcholine in a variety of cultured vascular smooth muscle cells of rat and human origin. We used vasopressin to characterize this response and found that vasopressin stimulates phospholipase D activity against phosphatidylcholine in A-10 vascular smooth muscle cells. The vasopressin-stimulated phosphatidylcholine hydrolysis is both time- and concentration-dependent. The half-maximal dose of vasopressin required for phosphatidylcholine hydrolysis (ED₅₀∼1 nM) correlates well with vasopressin binding to A-10 cells (K_d∼2 nM). The phosphatidylcholine in A-10 cells can be preferentially radiolabeled with [³H]myristic acid; subsequent treatment with vasopressin stimulates a rapid increase in³H-labeled phosphatidate (∼4×control values at 3 min), and after a short lag,³H-labeled diacylglycerol rises and reaches maximal levels at 10 min (∼2×control values). Similar temporal elevations of phosphatidate and diacylglycerol occur in A-10 cells labeled with [³H]glycerol. In A-10 cells radiolabeled with [³H]choline the elevation of cellular phosphatidate and diacylglycerol is concomitant with the release of [³H]choline metabolites (predominately choline) to the culture medium. The temporal production of phosphatidate and diacylglycerol as well as the release of choline to the culture medium are consistent with vasopressin activating phospholipase D. In addition, vasopressin stimulates a transphosphatidylation reaction that is characteristic of phospholipase D. The transphosphatidylation reaction is detected by the production of phosphatidylethanol that occurs when A-10 cells are incubated with ethanol and stimulated with vasopressin. The phospholipase D is active in the absence of extracellular Ca⁺⁺ whereas the vasopressin-stimulated mobilization of arachidonic acid is dependent on extracellular Ca⁺⁺. The data indicate that vasopressin stimulates phospholipase D which hydrolyzes phosphatidylcholine to phosphatidate. The phosphatidate is then metabolized, presumably by a phosphatidate phosphohydrolase, to produce sustained levels of cellular diacylglycerol. These sustained levels of diacylglycerol may activate protein kinase C and theraby function in the “sustained phase” of cellular responses. Portions of this work were presented in poster form (Abstract No. 6229) at the FASEB meeting in Las Vegas, NV May 1988. The abbreviations for DAG, PA, PEt and PC are used to designate “glycerides” and as such do not differentiate between species containing ether- and/or ester-linked moieties.     

Fatty acid mobilized by the vascular endothelial growth factor in human endothelial cells

Release of FFA from membrane phospholipids was observed after incubation of umbilical cord vein-derived endothelial cells (HUVEC) with vascular endothelial growth factor (VEGF). In particular, we found an increase of arachidonate, stearate, and palmitate in a time-dependent manner with a peak at 30 min. The maximum increase was reached by arachidonate (4.4-fold), followed by stearate (2.2-fold) and palmitate (1.3-fold). The arachidonate increase can be ascribed to the activation of phospholipase A₂ (PLA₂). In fact, cells preincubated with arachidonyl trifluoromethyl ketone, a PLA₂ inhibitor, showed a marked reduction in arachidonate mobilization. The role of Ca²⁺ in PLA₂ activation was also investigated. Cells incubated with VEGF in the presence of EGTA showed a marked decrease in arachidonate mobilization, whereas incubation with the calcium ionophore A23187 alone produced an increase in arachidonate, although to a lesser extent compared with the VEGF stimulation. Incubation with A23187 in association with PMA produced the same increase in arachidonate as the VEGF treatment. Mitogen-activated protein kinase (MAPK) activity was also found to increase as a consequence of VEGF stimulation. Taken together, these results suggest that the VEGF-mediated activation of PLA₂ in HUVEC is dependent on both MAPK-mediated phosphorylation and Ca²⁺ increase. Furthermore, the increase in stearate and palmitate likely is brought about by the activation of a pathway involving phospholipase D, phosphatidate phosphohydrolase (PAP), and DAG lipase. In fact, the increase in those FFA was prevented when HUVEC were stimulated with VEGF in the presence of ethanol (which inhibits the formation of phosphatidate), propranolol (a specific inhibitor of PAP), or RHC-80267 (a specific inhibitor of DAG lipase).     

Determination of the phospholipase activity of patatin by a continuous spectrophotometric assay

Patatin is a family of glycoproteins that accounts for 30–40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported to serve as a storage protein and also to exhibit lipid phospholipase activity. This paper describes a simple continuous spectrophotometric method for assaying patatin phospholipase activity. The procedure is based on a coupled enzymatic assay using [1,2-dilinoleoyl]PC as the phospholipase substrate and lipoxygenase as the coupling enzyme. In the procedure developed in this work, lipoxygenase oxidizes the linoleic acid released by the phospholipase activity of patatin. This activity can then be followed spectrophotometrically by recording the increase in absorbance at 234 nm that results from the formation of the corresponding hydroperoxide from linoleic acid by the action of lipoxygenase. The optimal assay concentrations of patatin and lipoxygenase were established. Phospholipase activity varied with pH, reaching its optimal value at pH 9.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3 mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V _m=9.8×10⁻³ μmol/min·μg protein, K _m=7.8 μM, V _m/K _m=1.3 min⁻¹·μg protein). This method proved to be specific since there was no activity in the absence of patatin. It also had the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase.     

Effect of temperature on the stereoselectivity of phospholipase D toward glycerol in the transphosphatidylation of phosphatidylcholine to phosphatidylglycerol

In this study, the effect of temperature on the stereoselectivity of phospholipase D (PLD) toward the two primary hydroxyl groups of glycerol in the transphosphatidylation reaction of phosphatidylcholine to phosphatidylglycerol (PtdGro) was investigated. For this purpose, PLD from bacteria (Streptomyces septatus TH-2, S. halstedii subsp. scabies K6, and Actinomadura sp.) and cabbage were tested. At the reaction temperatures employed (0–60°C), the proportions of the two PtdGro diastereomers, namely, 1,2-dioleoyl-sn-glycero-3-phospho-3′-sn-glycerol (R,R configuration) and 1,2-dioleoyl-sn-glycero-3-phosphol-1′-sn-glycerol (R,S configuration), which were produced with PLD from Streptomyces TH-2 and Actinomadura sp., changed gradually from 50% R,R and 50% R,S at 50–60°C to 70% R,R and 30% R,S at O°C. These alterations suggested that the stereoselectivity of the bacterial PLD toward the two primary hydroxyl groups of prochiral glycerol was significantly influenced by reaction temperature. PLD from Streptomyces K6 showed relatively little effect of temperature on stereoselectivity, giving 65–69% R,R in the temperature range of 60–10°C examined. The plots of In ([R,R]/[R,S]) vs. 1/T gave good linear fits for these three bacterial PLD. No temperature effect was observed for cabbage PLD, which gave an almost equimolar mixture of the R,R and R,S diastereomers in the range from 0 to 40°C. The temperature-dependent change in enantiomeric selectivity of the bacterial PLD promises potentially profitable commercial exploitation.     

Parathyroid hormone stimulates phosphatidylethanolamine hydrolysis by phospholipase D in osteoblastic cells

Parathyroid hormone (PTH) and phorbol-12,13-dibutyrate (PDBu) stimulate phospholipase D (PLD) activity and PC hydrolysis in UMR-106 osteoblastic cells {Singh, A.T., Kunnel, J.G., Strieleman, P.J., and Stern, P.H. (1999) Parathyroid Hormone (PTH)-(1–34), [Nle^{8, 18}, Tyr³⁴]PTH-(3–34) Amide, PTH-(1–31) Amide, and PTH-Related Peptide-(1–34) Stimulate Phosphatidylcholine Hydrolysis in UMR-106 Osteoblastic Cells: Comparison with Effects of Phorbol 12,13-Dibutyrate, Endocrinology 140, 131–137}. The current studies were designed to determine whether ethanolamine-containing phospholipids, and specifically PE, could also be substrates. In cells labeled with ¹⁴C-ethanolamine, PTH and PDBu treatment decreased ¹⁴C-PE. In cells co-labeled with ³H-choline and ¹⁴C-ethanolamine, PTH and PDBu treatment increased both ³H-choline and ¹⁴C-ethanolamine release from the cells. Choline and ethanolamine phospholipid hydrolysis was increased within 5 min, and responses were sustained for at least 60 min. Maximal effects were obtained with 10 nM PTH and 50 nM PDBu. Dominant negative PLD1 and PLD2 constructs inhibited the effects of PTH on the phospholipid hydrolysis. The results suggest that both PC and PE are substrates for phospholipase D in UMR-106 osteoblastic cells and could therefore be sources of phospholipid hydrolysis products for downstream signaling in osteoblasts.     

Membrane lipid metabolism and phospholipase activity in insect Spodoptera frugiperda 9 ovarian cells

Although there is increasing use of insect ovarian Sf9 cells for the production of recombinant proteins, namely, via the baculovirus vector expression system, little is known about the lipids in the cell membrane and whether endogenous phospholipases are present for regulation of the cell membrane lipids. In this study, analysis of membrane lipids of Sf9 cells indicated the presence of phosphatidylethanolamine (PE) (diacyltype) and phosphatidylcholine as major phospholipids, followed by phosphatidylserine and phosphatidylinositol (PI), and only trace amounts of ethanolamine plasmalogen. These phospholipids contain high proportions of monoenoic fatty acids, e.g., 16∶1 and 18∶1, which comprise more than 70% of the total fatty acids although small amounts of polyunsaturated fatty acids such as 18∶2 and 20∶4 are also present. When Sf9 cells were incubated in a culture medium containing [¹⁴C]oleic acid and [¹⁴C]arachidonic acid, a large portion of the labels were incorporated into membrane phospholipids. Using [¹⁴C]arachidonoyl-phospholipids as substrates for incubation with cell homogenate and subcellular fractions, results indicate the presence of a Ca2⁺-independent phospholipase A (PLA₂) in the Sf9 cell cytosol fraction. This PLA₂ shows a high preference for hydrolysis of PE and is active at a pH range of 7–9. Unlike the brain cells which contain active phospholipase C (PLC) specific for phosphatidylinositol, only limited amount of diacylglycerol (DAG) was released from [¹⁴C]arachidonoyl-PE in the Sf9 cells. Taken together, this study demonstrates active metabolism of membrane phospholipids in Sf9 cells, most likely mediated by acyltransferases and PLA₂. Furthermore, despite the absence of PLC for PI, limited amount of DAG could be generated through hydrolysis of PE.     

Enzymatic method of increasing phosphatidylcholine content of lecithin

An enzymatic method was established to increase the phosphatidylcholine (PC) contents of soybean and egg lecithins. Other phospholipids of lecithin were phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidic acid (PA). Seven preparations of phospholipase D (PLD), PLD-1 to PLD-6 ofStreptomyces origin and PLD-7 of cabbage origin, were tested for their ability to increase PC by transphosphatidylation in the presence of choline chloride (CC). The reactions were carried out at 30 C in a biphasic system that consisted of an aqueous phase containing PLD along with a buffer (optimum pH) having desired concentration of CC and Ca²⁺ and an ethyl acetate phase containing lecithin phospholipids. Intermitttent samples were extracted and analyzed by HPLC. Four of six PLD’s ofStreptomyces origin showed good transphosphatidylation (increase of PC contents of soybean lecithin from approximately 35% to 60–70% on a phospholipid basis) at 2.5 M CC, but the other two microbial PLD’s completely hydrolyzed the phospholipids to PA. Cabbage PLD-7 showed poor transphosphatidylation. PLD-3 gave the highest PC contents (70%) at 1.75 M CC. One hundred percent transphosphatidylation of pure PE to PC was achieved with PLD-3. PI was inert to the attack of most PLD preparations examined with the exception that PLD-3 hydrolyzed PI significantly. Purified PI could not be transphosphatidylized to PC; 100% PA was formed. Soybean lecithin containing about 80% PC and purified egg yolk lecithin with 75% PC could be converted to products having 95% PC and almost 100% PC, respectively, by PLD-3 at 1.75M CC. Studies on Enzymatic Conversion of Phospholipids (v)     

Metal ion stimulation of phospholipase D-like activity of isolated rat intestinal mitochondria

Presence of phospholipase D-like (PLD) activity in the intestinal mitochondria was identified using endogenous phospholipids as substrate. The enzyme had a pH optimum of 6.5, did not show trans-phosphatidylation activity in the presence of ethanol or butanol, and the product formed was phosphatidic acid (PA). This was confirmed by separation of reaction products by high-performance liquid chromatography and analysis of composition of the PA formed which gave phosphate/fatty acid ratio of 1∶2. PLD-like activity was further confirmed by the formation of ethanolamine and choline as products of enzyme action. This activity was stimulated by various metal ions; when stimulated by Mg²⁺ and Ba²⁺, it hydrolyzed both phosphatidylcholine and phosphatidylethanolamine, and when stimulated by Ca²⁺, it preferentially hydrolyzed phosphatidylethanolamine. There was no requirement for sodium oleate for the PLD-like activity in mitochondria. These results suggest that intestinal mitochondria have an active PLD-like enzyme which differs in certain properties from phospholipase D from other tissues.     

Simple synthesis of diastereomerically pure phosphatidylglycerols by phospholipase D-catalyzed transphosphatidylation

A simple method for synthesizing diastereomerically pure phosphatidylglycerols (PtdGro), namely, 1,2-diacyl-sn-glycero-3-phospho-3′-sn-glycerol (R,R configuration) and 1,2-diacyl-sn-glycero-3-phospho-1′-sn-glycerol (R,S configuration) was established. For this purpose, diastereomeric 1,2-O-isopropylidene PtdGro were prepared from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho) and enantiomeric 1,2-O-isopropylideneglycerols by transphosphatidylation with phospholipase D (PLD) from Actinomadura sp. This species was selected because of its higher transphosphatidylation activity and lower phosphatidic acid (PtdOH) formation than PLD from some Streptomyces species tested. The reaction proceeded well, giving almost no hydrolysis of PtdCho to PtdOH in a biphasic system consisting of diethyl ether and acetate buffer at 30°C. The isopropylidene protective group was removed by heating the diastereomeric isopropylidene PtdGro at 100°C in trimethyl borate in the presence of boric acid to obtain the desired PtdGro diastereomers. The purities of the products, which were determined by chiral-phase HPLC, were exclusively dependent on the optical purities of the original isopropylideneglycerols used. The present method is simple and can be utilized for the synthesis of pure PtdGro diastereomers having saturated and unsaturated acyl chains.     

An aqueous suspension system for phospholipase D-mediated synthesis of PS without toxic organic solvent

Enzymatic synthesis of PS by phospholipase D (PLD)-mediated transphosphatidylation in an aqueous media was investigated. The purpose of this study was to establish a novel synthetic method where no toxic organic solvents were used. An attempt to react soybean lecithin (simply dispersed in an aqueous buffer) with an aqueous solution of l-serine and PLD was unsuccessful, giving only 20% of PS. By contrast, a suspension of lecithin adsorbed on fine powders such as silica was effectively converted into PS in an aqueous solution of l-serine and PLD. After screening various powders for use as the lecithin adsorbent, calcium sulfate was found to be the best with respect to lecithin conversion. In addition, calcium sulfate did not require prior adsorption of lecithin (i.e., the reaction proceeded effectively simply by adding the powder to an aqueous mixture of lecithin, l-serine, and PLD). With this “aqueous suspension system” of calcium sulfate, up to 180 mg/mL lecithin was completely converted, resulting in more than 80% PS in 24 h. The synthesized PS could easily be recovered from the powder by extracting with a mixture of n-hexane, ethanol, and diluted HCl.     

On the specificity of a phospholipase A2 purified from the 106,000×g pellet of bovine brain

Assessment has been made of the specificity of a purified phospholipase A₂ from the 106,000×g pellet (microsomal fraction) of bovine grey matter which shows strong activity toward phosphatidylinositol (PI). In the first series of experiments involving the utilization as substrates of PI with different¹⁴C- or³H-labeled fatty acids in the 2-position, the purified phospholipase A₂ most readily removed 16∶0 palmitic acid, followed by 18∶0 stearic acid, 18∶1 oleic acid and 20∶4 arachidonic acid. In the second series of experiments, the purified phospholipase A₂ showed preferential action toward PI (100%) compared to phosphatidylcholine (PC, 62.5%), phosphatidic acid (PA, 32.6%), phosphatidylethanolamine (PE, 25.1%) and phosphatidylserine (PS, 21.5%), where each phosphoglyceride was labeled in the 2-position with [1-¹⁴C] oleic acid. In the third series of experiments, fatty acids were shown to cause inhibition of action of the purified phospholipase A₂ on 1-acyl, 2-[1-¹⁴C] oleoyl PI in the order 20∶4>18∶1>18∶0>16∶0 which is the reverse order to that just noted. In the final series of experiments, the addition of the phosphoglycerides PC, PE, PS and PA in amounts of 5 or 10 μM caused either no inhibition (PE, 2%), slight inhibition (PC, 15%) or reasonably significant inhibition (PA, 20% and PS, 40%) of action of the purified phospholipase A₂ on 1-acyl, 2-[1-¹⁴C]-oleoyl PI. The pattern of specificity observed for the purified phospholipase A₂ combined with its microsomal location are the expected properties of a phospholipase A₂ that might function in a deacylation-reacylation cycle for modifying the fatty acid distribution in PI.     

Thermophilic phospholipase A2 in the cytosolic fraction from the archaeon Pyrococcus horikoshii

Hyperthermophilic archaeon Pyrococcus horikoshii produced phospholipase A² in a cytosolic fraction. The enzyme displayed optimal activity at 90°C and pH 7 and preferentially hydrolyzed sn-2 fatty acids in the following order: linoleoyl> oleoyl>arachidoyl phosphatidylcholine. Phospholipase A₂ had similar activities toward l-α-1-palmitoyl-2-arachidoyl derivatives of phosphatidylcholine and phosphatidylethanolamine. Phospholipase A₂ activity was unaffected by the addition of 0.0001–1 mM calcium, but was inhibited slightly by the addition of 2–10 mM calcium. The activity was enhanced more than 5–18-fold in the presence of 3–20% (vol/vol) glycerol. The activity was unaffected by the addition of 1–5 mM EDTA or 0.01–20 mM dithiothreitol. A caldarchaetidic acid derivative having a molecular weight of 1544 disappeared upon incubation of the cytosolic fraction with total lipid. The phospholipase A₂ was difficult to purify by general chromatography because it existed as an aggregate. Electrophoresis was carried out using 10–15% polyacrylamide gels containing sodium dodecyl sulfate (SDS-PAGE). No activity of phospholipase A₂ activity was observed in the absence of proteins less than 19 kD in size, as fractionated by SDS-PAGE. Portions of this article were presented at the Biocatalysis Symposium at the 91st Annual Meeting and Expo of the American Oil Chemists’ Society in San Diego, CA, April, 2000.     

Asymmetric <Emphasis Type="Italic">in vitro</Emphasis> synthesis of diastereomeric phosphatidylglycerols from phosphatidylcholine and glycerol by bacterial phospholipase D

Using chiral-phase HPLC, we determined the stereochemical configuration of the phosphatidylglycerols (PtdGro) synthesized in vitro from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho, R configuration) or 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PtdEtn, R configuration) and glycerol by transphosphatidylation with bacterial phospholipase D (PLD). The results obtained with PLD preparations from three Streptomyces strains (S. septatus TH-2, S. halstedii K5, and S. halstedii subsp. scabies K6) and one Actinomadura species were compared with those obtained using cabbage and peanut PLD. The reaction was carried out at 30°C in a biphasic system consisting of diethyl ether and acetate buffer. The resulting PtdGro were then converted into bis(3,5-dinitrophenylurethane) derivatives, which were separated on an (R)-1-(1-naphthyl)ethylamine polymer. In contrast to the cabbage and peanut PLD, which gave equimolar mixtures of the R,S and R,R diastereomers, as previously established, the bacterial PLD yielded diastereomixtures of 30–40% 1,2-diacyl-sn-glycero-3-phospho-1′-sn-glycerol (R,S configuration) and 60–70% 1,2-diacyl-sn-glycero-3-phospho-3′-sn-glycerol (R,R configuration). The highest disproportionation was found for the Streptomyces K6 species. The present study demonstrates that bacterial PLD-catalyzed transphosphatidylation proceeds to a considerable extent stereoselectively to produce PtdGro from PtdCho or PtdEtn and prochiral glycerol, indicating a preference for the sn-3′ position of the glycerol molecule.     

Activation and solubilization by triton X-100 of membrane-bound phospholipase D of rat brain

In the present study, we examined the ability of detergents to stimulate and solubilize phospholipase D (PLD) of a particulate fraction of rat brain. PLD activity was assayed by measuring the [³H]choline produced from the exogenous substrate dipalmitoyl phosphatidyl[³H]choline (dipalmitoyl [³H]PC). In the absence of detergents, PLD activity was not detectable. Of the detergents examined, Triton X-100 was found to markedly enhance PLD activity, whereas other detergents including sodium doexycholate, sodium cholate, CHAPS and Lubrol-PX caused only a small, if any increase in activity. Enhancement by Triton X-100 was maximal at 0.1–0.2% (w/v) and decreased at higher concentrations. The optimal pH was 7.1–7.3. Both Ca²⁺ and Mg²⁺ inhibited enzyme activity stimulated by Triton X-100 in a concentration-dependent manner. Triton X-100 effectively solubilized PLD from the particulate fraction of rat brain; more than 70% of the activity of the particulate fraction was extracted by 0.5–1.0% (w/v) Triton X-100. Furthermore, when the PLD activities in brains of three different species (rat, rabbit and bovine) were measured under optimal conditions, the activities were found to differ greatly. PLD activity was highest in rabbit brain, followed by rat and bovine brains; the activity in bovine brain was extremely low compared to the activities in rat and rabbit brains. We conclude that Triton X-100 is potentially useful for the purification of PLD and that rabbit and rat brains are the preferred sources.     

Association of the intestinal brush-border membrane phospholipase A2 and lysophospholipase activities (phospholipase B) with a stalked membrane protein

We have attempted to determine the size and membrane orientation of a recently described rat jejunal brushborder protein possessing phospholipase A₂ and lysophospholipase activities (phospholipase B) (Pind, S. and Kuksis, A. [1988]Biochim. Biophys Acta 938, 211–221). The phospholipase A₂ and lysophospholipase activities were renatured following nonreducing sodium dodecyl sulphate polyacrylamide gel electrophoresis of the total membrane proteins and were shown to migrate as a component of a protein band having a relative molecular mass of 170 kDa. This band accounted for approximately 1% of the total Coomassie Blue staining proteins. Phospholipase B was also shown to be solubilized from the membranes, in an active form, by a proteolytic digestion with papain. Papain solubilization resulted in a loss of the hydrophobic properties observed for the intact phospholipase. These results suggest that the active site of the phospholipase projects from the luminal surface of the membrane vesicles. In support of this, phospholipase activity towards exogenous, detergent-solubilized phosphatidylcholine was demonstrated under conditions in which the membranes remained intact. We conclude that the phospholipase B has the characteristics of a stalked, brush-border membrane protein and may be considered as another digestive enzyme anchored in this membrane.     

Preparation of n‐3 Polyunsaturated Phosphatidylglycerol from Salmon Roe Lipids by Phospholipase D and In Vitro Digestion

Phosphatidylglycerol (PG) is a highly functional phospholipid (PL), which has many physiological functions. However, naturally occurring PG binding n‐3 polyunsaturated fatty acid (n‐3 PUFA) is low in content, resulting in a scarcity of industrial bio‐resources of n‐3 PUFA enriched PG. The current study investigates the preparation of salmon roe PG (SRPG) from three types of salmon roe lipids and glycerol via phospholipase D (PLD)‐mediated transphosphatidylation. The yields of SRPG obtained from salmon roe total lipid (SRTL) and salmon roe PL (SRPL) are higher than those obtained from purified salmon roe phosphatidylcholine (SRPC) in aqueous system. Following a 24 h reaction with 0.75 U PLD, SRTL, and SRPL yield up to 96.4 mol% and 96.7 mol% SRPG, respectively. In addition, more fatty acids are released from synthesized SRPG via hydrolysis by pancreatic enzymes than from SRPC and soybean PC in in vitro digestion model. Fatty acids at the sn‐2 position of SRPG are completely liberated by 0.04 U of phospholipase A₂ (PLA₂) during a 6 h reaction, whereas fatty acids of SRPC are partially unhydrolyzed even after a 24 h reaction. Our results suggest that SRPG converted from salmon lipids by PLD is a functional PL with high bioavailability of n‐3 PUFAs. Practical Applications: Phosphatidylglycerol rich in n‐3 PUFAs is prepared from salmon roe lipids (SRPG) catalyzed by PLD. The SRPG yields reach 96.4 mol% and 96.7 mol% of phosphatidylcholine contained in SRTL and SRPL, respectively, in aqueous reaction system. Fatty acids rich in n‐3 PUFAs at sn‐2 position of prepared SRPG are rapidly liberated by PLA₂ in an in vitro digestion model.