Transphosphatidylation Activity Research Articles

Phospholipase D Activity in the Intestinal Mitochondria: Activation by Oxygen Free Radicals

A prominent feature of cell damage caused by oxidative stress is morphological and functional changes in the mitochondria. The present study looked at the effect of free radical exposure on intestinal mitochondrial lipids. Free radical exposure did not alter neutral lipids, but among the phospholipids, phosphatidylethanolamine (PE) content was decreased on exposure to superoxide anion, generated by xanthine-xanthine oxidase or menadione with a concomitant increase in the level of phosphatidic acid (PA), suggesting activation of phospholipase D (PLD). This enzyme did not show transphosphatidylation 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 HPLC. This alteration in mitochondrial phospholipid was abolished by the presence of superoxide dismutase. Exposure to H 2O 2 did not have any significant effect. Activation of PLD by free radicals was further confirmed by quantitation of ethanolamine released from PE. Absence of any change in the content of lysophospholipid or diglyceride following exposure of mitochondria to superoxide ruled out the involvement of phospholipase A2 or C in the altered lipid composition. Moreover, inclusion of phospholipase A 2 inhibitors, chlorpromazine, or p-bromophenacyl bromide did not prevent the generation of PA on exposure to free radicals. These findings suggest that superoxide anion stimulates intestinal mitochondrial PLD resulting in PE degradation and PA formation. These alterations in mitochondrial lipids may play a role in causing the functional alteration seen in oxidative stress.

Identification of a novel, Ca 2+-dependent phospholipase D with preference for phosphatidylserine and phosphatidylethanolamine in Saccharomyces cerevisiae

A membrane-bound phospholipase D (PLD) from Saccharomyces cerevisiae was solubilized from mitochondrial and plasma membranes and partially purified. The enzyme has an apparent molecular weight of approximately 60 kDa, is strictly Ca 2+-dependent and preferentially hydrolyses phosphatidylserine and phosphatidylethanolamine. Enzyme activity is significantly increased in membranes from cells grown on a non-fermentable carbon source. The Ca 2+-dependent PLD is distinct from PLD encoded by the SPO14/PLD1 gene. The 195 kDa SPO14/PLD1 gene product is specific for PtdCho, Ca 2+-independent and is activated by PIP 2. Furthermore, Pld1p has transphosphatidylation activity in the presence of ethanol and thus resembles the prototypic PLD activity found in mammalian cells and plants. In contrast, the Ca 2+-dependent PLD described here is not affected by PIP 2 and does not catalyze transphosphatidylation. Thus, the Ca 2+-dependent PLD characterized in this study appears to be a member of a novel family of phospholipases D.

Purification and characterization of multiple forms of phosphatidylinositol-specific phospholipases D from suspension cultured Catharanthus roseus cells

Two soluble and two membrane bound phosphatidylinositol (PI) specific phospholipases D (EC 3.1.4.4) were identified, partially purified and characterized from Catharanthus roseus suspension cultured cells. In order to compare the chromatographic properties of the four enzymes, column chromatography was performed under the same conditions for all four enzymes. The soluble enzymes have apparent molecular weights of 132 kD and 48 kD, the plasma membrane bound enzymes of 132 kD and 58 kD. Based on their origin and their molecular weight the types were designated S132, S48, PM132 and PM58, respectively. Isoelectric points of the native type S132, S48, PM132, PM58 phospholipases D were 4.89, 4.89, 4.6 and 4.8. After ammonium sulphate precipitation in the presence of Triton X-100, the cytosolic enzymes precipitated, whereas the plasma membrane localized enzymes floated up. In the presence of Triton X-100 all enzymes do not depend on divalent cations. Maximum activity is obtained at a Triton X-100 PI ratio of 8:1 and half maximum activity at a ratio of 14:1 to 20:1. Using Triton X-100 PI mixed micelles, the 48 and 58 kD enzymes depended on the surface concentration of PI as well as on the bulk concentration of PI, whereas the 132 kD proteins depended nearly exclusively on the surface concentration of PI. A detailed analysis of the 48 kD and 58 kD phospholipases show that they very closely follow the surface dilution kinetic model as proposed by Deems et al. (R.A. Deems, B.R. Eaton, E.A. Dennis. J. Biol. Chem., 250 (1975) 9013–9020). The interfacial Michaelis constants ( K m B) for phosphatidylinositol were 0.017 mol fraction for the 48 kD cytosolic protein and 0.062 for the 58 kD membrane-bound phospholipase, respectively. As has been reported for microsomal PI-specific phospholipases D (A. Becher, J.B. Wissing, K.G. Wagner. Plant Sci., 97 (1994) 143–151) all these enzymes do not display any transphosphatidylation activity. The formation of diphosphatidylglycerol was also not observed. Further Mg 2+ or Ca 2+ are not needed for activity, EDTA, EGTA and DTPA show no effect. The pH optimum is 5.25 for the low molecular weight and 6.5 for the high molecular weight isoforms. Half maximum activity is observed at pH 4.2 and 6.2 for the smaller enzymes and at pH 4.5 and 7.5 for the larger enzymes.

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.

Primary alcohols and phosphatidylcholine metabolism in rat brain synaptosomal membranes via phospholipase D.

Phospholipase D of rat brain synaptosomal membranes was tested with phosphatidylcholine as the substrate for its specificity in the use of primary alcohols as transphosphatidylation co-substrates. The efficiency of the reaction was related to the hydrophobicity and the membrane penetrating capacity of the alcohol molecule. Phosphatidylalcohol formation could be detected up to 1-octanol but not for alcohols with longer hydrocarbon chains (C(9), C(10)). With increasing alcohol concentration the transphosphatidylation activity of the phospholipase D reached an optimum and then declined abruptly. Alcohol concentrations required for maximal transphosphatidylation reaction generally decreased with increasing hydrophobicities of the alcohols. Nevertheless 1-butanol and 4-chloro-1-butanol were the most efficient cosubstrates, sharing identical optimal conditions. Transphosphatidylation works at the cost of phosphatidic acid formation. Phosphatidic acid itself was transformed to diacylglycerol, probably by a contaminating phosphatidic acid phosphohydrolase.

Acidic phospholipids inhibit the phospholipase D activity of rat brain neuronal nuclei

An oleate dependent form of phospholipase D is present in rat brain neuronal nuclei and both the hydrolytic and transphosphatidylation activities measured. Several acidic phospholipids were found to inhibit this activity in a dose dependent manner. The IC 50 values varied from 3.5 μM for PIP 2 to 200 μM for phosphatidic acid. The hydrolysis of PIP 2 by phospholipase C would be expected to result in the disinhibition of the oleate dependent phospholipase D activity.

Kinetics of myocardial phospholipase D.

Myocardial phospholipase D (PLD) is located in different subcellular membranes, including sarcolemma (SL) and sarcoplasmic reticulum (SR). In this study, the kinetics of PLD-dependent hydrolytic and transphosphatidylation activities were examined in SL and SR fractions isolated from rat heart by measuring the formation of phosphatidic acid and phosphatidylethanol, respectively. The results showed that, compared to SR PLD, SL PLD had a higher Vmax, i.e. 373 vs. 70 nmol/mg protein/h for the hydrolytic activity and 415 vs. 60 nmol/mg protein/h for the transphosphatidylation activity. In comparison with the SR enzyme, SL PLD had a lower Km value for the hydrolytic activity (0.46 vs. 0.65 mM), buy a higher Km for the transphosphatidylation activity (225 vs. 179 mM). These distinctive kinetic parameters suggest that SL PLD and SR PLD may be isoforms of the enzyme and/or have different membrane domain. Therefore, SL- and SR-localized PLD activities may be under independent control mechanism(s) and play distinct roles in normal conditions and pathological processes.

Differential Phospholipid-Labeling Suggests Two Subtypes of Phospholipase D in Rat Leydig Cells

The aim of the present study was to compare the transphosphatidylation activity of phospholipase D (PLD) under different substrate labeling conditions, in order to investigate whether PLD in rat Leydig cells exhibited any substrate preferences for the alkyl- or acyl-form of phosphatidylcholine (PtdCho). The [3H]phosphatidylethanol formation in response to 4β-phorbol 12-myristate 13-acetate (PMA), sphingosine, or Ca++-ionophore A23187, was lower when Leydig cells were labeled with 1-O-[3H]alkyl lysoPtdCho compared with the responses when [3H]myristic acid was employed. In contrast, the results for the receptor agonists (vasopressin, bradykinin, and lysophosphatidic acid), using the two labels, showed more consistency. Thus, the PLD-activity induced by PMA, sphingosine, or A23187 has a more selective substrate range (i.e. mainly acyl-linked PtdCho) than the PLD-activity stimulated via a receptor. Our data suggests the existence of PLD isozymes that differ with respect to substrate specificity and activation mechanisms.

Immunoprecipitation of a phospholipase D activity with antiphosphotyrosine antibodies.

When granulocyte-macrophage colony-stimulating factor (GM-CSF)-treated human neutrophils were challenged with the chemotactic factor fMet-Leu-Phe, it was possible to detect a time-dependent increase in the hydrolytic (as measured by the production of phosphatidic acid, PA) and the transphosphatidylation (as measured by the production of phosphatidylethanol, PEt) activities of phospholipase D in intact cells prelabeled with a radioactive fatty acid. Both activities were inhibited by preincubation of cells with genistein. Appropriate conditions were developed to test the PLD transphosphatidylation activity against exogenous phosphatidylcholine (PCho) in an in vitro system. As in intact cells, increased PLD activity could be detected in cell lysates obtained from fMet-Leu-Phe-treated cells compared with controls. When lysates were immunoprecipitated with antiphosphotyrosine antibodies, a PLD activity was found only in immune complexes that were prepared from fMet-Leu-Phe-treated cells. Conversely, no activity was found in lysates immunoprecipitated with an irrelevant antibody (GTPase-activating protein, GAP) that nevertheless was able to recognize a tyrosylphosphorylated form of GAP, as demonstrated by western blotting. These data suggest that a PCho-PLD, or a tightly bound protein, is tyrosine phosphorylated during cell activation.

Purification and properties of two phospholipases D from Streptomyces sp.

Two enzymes with phospholipase D activity were purified from Streptomyces strains (PMF and PM43) by column chromatography on Fractogel TSK CM-650(S), Sephadex G-100 and Fractogel EMD DEAE-650(M). The purified preparations were found to be homogeneous by SDS-PAGE, capillary electrophoresis and analytical gel filtration. The molecular masses, assessed by MALDI-MS spectrometry, were 53.864 kDa for PMF and 54.147 kDa for PM43. The isoelectric point was 9.1 for both enzymes. The enzymes were most active at around 60 degrees C and stable between pH 4 and 9 and below 50 degrees C. The pH optima were between 4 and 6 for PMF and between 6 and 7 for PM43. Both phospholipases displayed high transphosphatidylation activity but PMF was more selective than PM43.

Enzymatic Conversion of Phospholipids

Various natural phospholipids which are difficult to be obtained from natural sources can be synthesized from lecithin by phopholipase D-catalyzed transphosphatidylation. It is also able to synthesize novel unnatural phopholipids which contain biologically active compounds such as vitamins at the polar heads of the phospholipids. Initinally phospholipase D from cabbage leaves were mostly used, but recently bacterial enzymes having high transphosphatidylation activities have been mainly employed.Lipase-catalyzed ester exchange and phospholipase A2-catalyzed ester synthesis can be used to introduce arbitrary fatty acid to phospholipids under some mild reaction conditions. Howev er, the efficiency of these enzymatic acyl exchange reactions are still low, and further research will be needed.

Transphosphatidylation of sugar alcohols and its implications for the pathogenesis of diabetic complications

Glucose-induced sorbitol accumulation and attendant alterations in cellular myo-inositol and phosphoinositide metabolism have been invoked in the pathogenesis of diabetic complications; however, direct effects of sorbitol on membrane phospholipid composition or metabolism have never been evaluated. Phospholipase D catalyses the transphosphatidylation of ethanol into phosphatidylcholine to yield phosphatidylethanol, an "abnormal" phospholipid whose content in rat brain is increased by chronic ethanol ingestion. Analogous transphosphatidylation of sorbitol or other polyols whose concentration is elevated in diabetes was explored in vitro and in glucose-exposed cultured human retinal pigment epithelial cells. Phosphatidylcholine and varying concentrations of sorbitol, galactitol, mannitol and glucose were incubated with peanut phospholipase D in sodium acetate buffer for varying time periods. Thin layer chromatography revealed new phospholipid bands whose hydrolysis by phospholipase D liberated a water-soluble compound that cochromatographed with sorbitol on gas-liquid chromatography, and whose concentration increased in a time- and concentration-dependent fashion. Identical transphosphatidylation activity was demonstrated in a rat brain synaptosomal fraction. Phospholipase D hydrolysis of lipids from human retinal pigment epithelial cells constitutively overexpressing the aldose reductase gene yielded a sorbitol-like compound whose appearance was increased by glucose exposure and was decreased by an aldose reductase inhibitor. Thus, glucose-induced aldose reductase inhibitor sensitive sorbitol accumulation might induce the formation of "phosphatidylsorbitol" through a transphosphatidyl mechanism that may contribute to altered membrane phospholipid metabolism in diabetes.

Phospholipase D activity of isolated rat brain plasma membranes

With [ 14C]oleate-labeled phosphatidylcholine as a substrate for phospholipase D the hydrolytic activity was measured by phosphatidic acid formation and the transphosphatidylation activity was measured by the phosphatidylethanol formed in the presence of ethanol. The pH optimum was 6.5 with dimethylglutarate as the buffer. EGTA inhibited the transphosphatidylation activity to a greater extent than the hydrolytic activity. In contrast CaCl 2, BaCl 2, MgCl 2 and SrCl 2 stimulated the hydrolytic activity without effecting the transphosphatidylation activity. BeCl 2 another member of the group IIa transition metals was a very potent inhibitor of both the hydrolytic and transphosphatidylation activity. GTPγS, an activator of G protein-mediated events, was an inhibitor of both activities.

Phosphatidylinositol specific isoenzymes of phospholipase D from Catharanthus roseus. Purification and characterization

Microsomal membranes from suspension cultured Catharanthus roseus cells were found to contain phospholipase D activity (EC 3.1.4.4) towards phosphatidylinositol (PI). After extraction with buffer containing Triton X-100, two isoenzymes with apparent molecular weights of 50 000 and 125 000 were partially purified. The enzymes accepted only PI as substrate and transphosphatidylation activity could not be detected. Whereas several phospholipids were slightly inhibitory, phosphatidyl glycerol had stimulatory properties. The enzymes required divalent cations for activity, Ca 2+ and Mg 2+ were equally effective. In the presence of deoxycholate and Ca 2+, K m values for PI were found to be in the range 20–40 μM. Although the smaller protein had a slightly higher temperature stability and a slightly lower p I value, both isoenzymes revealed equal enzymic properties. This is the first report on a plant PI-specific phospholipase D.

Interaction between Phosphoinositide-Phospholipase C and Phosphatidylcholine- Phospholipase D in Plasma Membranes of Retinal Capillary Pericytes

The existence of phosphatidylcholine-specific phospholipase D (PC-PLD) was demonstrated in the isolated plasma membranes of retinal capillary pericytes. We determined PC-PLD activity, taking advantage of the transphosphatidylation activity of PC-PLD in the presence of ethanol, with phosphatidylethanol (PEt) formation. In the membrane environment, both phosphoinositide-specific phospholipase C (PI-PLC) and PC-PLD were activated by GTP gamma S. Neomycin, an inhibitor of PI-PLC, at a concentration (500 microM) capable of inhibiting 90% of the PI-PLC activity, only slightly inhibited the basal level of PC-PLD, but significantly decreased the GTP gamma S-activated PC-PLD by 66%. These findings indicate that the inhibitory effects of neomycin on PC hydrolysis may exert through the regulatory mechanisms of G proteins. Exogenous phosphatidic acid, a product of PC-PLD, stimulated, whereas PC, the substrate of PC-PLD, inhibited PI-PLC. These data implicate one mechanism by which PC-PLD may modulate PI-PLC activity through changing the phospholipid composition in the membrane, specifically the ratio of PA/PC. These findings are useful for the further studies on the communication between membrane-associated PI-PLC- and PC-PLD-mediated signal transduction pathways.

Rapid attenuation of receptor-induced diacylglycerol and phosphatidic acid by phospholipase D-mediated transphosphatidylation: formation of bisphosphatidic acid.

Generation and attenuation of lipid second messengers are key processes in cellular signalling. Receptor-mediated increase in 1,2-diacylglycerol (DG) levels is attenuated by DG kinase and DG lipase. We here report a novel mechanism of DG attenuation by phospholipase D (PLD), which also precludes the production of another (putative) second messenger, phosphatidic acid (PA). In the presence of an alcohol, PLD converts phosphatidylcholine (PC) into a phosphatidylalcohol (by transphosphatidylation) rather than into PA. We found in bradykinin-stimulated human fibroblasts that PLD mediates transphosphatidylation from PC (donor) to the endogenous 'alcohol' DG (acceptor), yielding bis(1,2-diacylglycero)-3-sn-phosphate (bisphosphatidic acid; bisPA). This uncommon phospholipid is thus a condensation product of the phospholipase C (PLC) and PLD signalling pathways, where PLC produces DG and PLD couples this DG to a phosphatidyl moiety. Long-term phorbol ester treatment blocks bradykinin-induced activation of PLD and consequent bisPA formation, thereby unveiling rapid formation of DG. BisPA formation is rapid (15 s) and transient (peaks at 2-10 min) and is also induced by other stimuli capable of raising DG and activating PLD simultaneously, e.g. endothelin, lysophosphatidic acid, fetal calf serum, phorbol ester, dioctanoylglycerol or bacterial PLC. This novel metabolic route counteracts rapid accumulation of receptor-induced DG and PA, and assigns for the first time a physiological role to the transphosphatidylation activity of PLD, that is signal attenuation.

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.

V-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine.

Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with arachidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG production and enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.

V-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine.

Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with arachidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG production and enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.

Two Streptomyces strains that produce phospholipase D with high transphosphatidylation activity.

Two Streptomyces strains that produce phospholipase D with high transphosphatidylation activity.