Biochemical characterization of phospholipase D activity from human neutrophils

We have found a phospholipase D activity in the postnuclear fraction of human neutrophils, employing phosphatidylinositol as exogenous substrate. This phospholipase D activity was assessed by both phosphatidate formation and by free inositol release in the presence of 15 mM LiCl in the reaction mixture and in the absence of Mg2+ ions to prevent inositol-1-phosphate phosphatase activity. To assess further the phospholipase D activity, we studied its capacity to catalyze a transphosphatidylation reaction, as a unique feature of the enzyme. It was detected as [14C]phosphatidylethanol formation when the postnuclear fraction was incubated with [14C]phosphatidylinositol in the presence of ethanol. The phospholipase D showed a major optimum pH at 7.5 and a minor one at pH 5.0. Neutral and acid phospholipase D activities were differentially located in subcellular fractionation studies of resting neutrophils, namely in the cytosol and in the azurophilic granules, respectively. Neutral phospholipase D required Ca2+ ions to the active, whereas the acid enzyme activity was Ca2(+)-independent. The neutral phospholipase D activity showed a certain specificity for phosphatidylinositol, as it was able to hydrolyze phosphatidylinositol at a much higher rate than phosphatidylcholine, in the absence and in the presence of different detergents. This neutral phospholipase D activity behaved as a protein of high molecular mass (350-400 kDa) by gel filtration chromatography. Moreover, neutral phospholipase D activity was detected in the postnuclear fraction of human monocytes, by measuring free inositol release from phosphatidylinositol as exogenous substrate, under the same experimental conditions as those used with neutrophils. The enzyme displayed similar specific activities in both cell types as well as the same degree of activation after cell stimulation with the calcium ionophore A23187. These results demonstrate the existence of two phospholipase D activities with different pH optima and intracellular location in human neutrophils. Furthermore, these results suggest that this phospholipase D can play a role in signal-transducing processes during cell stimulation in human phagocytes.     

Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A2 and activation by a 70 kDa protein.

Plasma membranes were isolated from carrot (Daucus carota L.) cells grown in suspension culture and treated with phospholipase A2 from snake or bee venom for 10 min. As a result of this treatment, phosphatidylinositol kinase activity was recovered in the soluble fraction. There was no detectable diacylglycerol kinase or phosphatidylinositol monophosphate kinase activity released from the membranes after the phospholipase A2 treatment. Treating the plasma membranes with phospholipase C or D did not release PI kinase activity. The phospholipase A2-released PI kinase was activated over 2-fold by a heat stable, soluble 70 kDa protein. The partially purified 70 kDa activator increases the Vmax but does not affect the Km of the phospholipase A2-released PI kinase.     

Activation of phospholipase D by sphingoid bases in NG108-15 neural-derived cells

Recent studies suggest that signal-dependent formation of phosphatidic acid by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is a novel trans-membrane signaling pathway in mammalian cells. We here demonstrate that sphingosine, as well as some other long chain bases, activates phospholipase D in neural-derived NG108-15 cells. Sphingosine potently stimulated phosphatidic acid and, in the presence of ethanol, phosphatidylethanol formation. (Phosphatidylethanol is a nonphysiological phospholipid which is characteristically produced by phospholipase D in the presence of ethanol.) Elevated phosphatidic acid levels were accompanied by increased phosphatidylinositol and phosphatidylglycerol production and a decrease in diacylglycerol levels. Sphingosine stimulated phospholipase D activity in a time- and concentration-dependent manner. A long aliphatic chain and a free 2-amino group were important structural requirements for the activation of phospholipase D by sphingosine-related molecules. We propose that phospholipase D may constitute an important cellular target for sphingosine action under both physiological and pathological circumstances.     

Enzymatic degradation and partial biosynthetic reconstitution of microsomal and mitochondrial membranes.

Guinea pig liver microsomal and mitochondrial membranes were degraded with phospholipase C and D followed by partial biosynthetic reconstitution. Activities of phosphatidylinositol synthetase in microsomal membranes and NADPH-cytochrome c reductase were almost completely lost after phospholipase C and D treatment; almost complete restoration of the original activity was achieved after biosynthesis of phosphatidylcholine in degraded microsomes, but was not reparable after biosynthesis of cytidinediphosphodiglycerides (CDP-diglycerides). The mitochondrial biosynthesis of polyglycerophosphatides was completely retained after degradation of these membranes with phospholipase C, but after similar treatment with phospholipase D, only about one-quarter of the original activity remained, the relative composition of polyglycerophosphatides being significantly different. The activity of NADPH-cytochrome c reductase of microsomes represented about 76% of the original activity after phospholipase C treatment, but only approximately 1% after treatment with phospholipase D. Although this activity could not be restored with CDP-diglyceride synthesis, it was restored to about 75% of the original activity after the biosynthesis of phosphatidylcholine in these fragments. These and additional experimental findings are discussed in terms of the relation between structural organization of lipids and proteins and enzymatic activities of membrane-bound phospholipid-synthesizing enzymes in microsomal and mitochondrial membranes isolated from guinea pig liver.     

Coupled inositide phosphorylation and phospholipase D activation initiates clathrin-coat assembly on lysosomes.

Adaptors appear to control clathrin-coat assembly by determining the site of lattice polymerization but the nucleating events that target soluble adaptors to an appropriate membrane are poorly understood. Using an in vitro model system that allows AP-2-containing clathrin coats to assemble on lysosomes, we show that adaptor recruitment and coat initiation requires phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis. PtdIns(4,5)P2 is generated on lysosomes by the sequential action of a lysosome-associated type II phosphatidylinositol 4-kinase and a soluble type I phosphatidylinositol 4-phosphate 5-kinase. Phosphatidic acid, which potently stimulates type I phosphatidylinositol 4-phosphate 5-kinase activity, is generated on the bilayer by a phospholipase D1-like enzyme located on the lysosomal surface. Quenching phosphatidic acid function with primary alcohols prevents the synthesis of PtdIns(4, 5)P2 and blocks coat assembly. Generating phosphatidic acid directly on lysosomes with exogenous bacterial phospholipase D in the absence of ATP still drives adaptor recruitment and limited coat assembly, indicating that PtdIns(4,5)P2 functions, at least in part, to activate the PtdIns(4,5)P2-dependent phospholipase D1. These results provide the first direct evidence for the involvement of anionic phospholipids in clathrin-coat assembly on membranes and define the enzymes responsible for the production of these important lipid mediators.     

Inositol is the water-soluble product of acetylcholine-stimulated breakdown of phosphatidylinositol in mouse pancreas.

The water-soluble products of acetylcholine-stimulated breakdown of phosphatidylinositol in mouse pancreas were analyzed by two different and independent procedures. There was an increased formation of free inositol throughout the period of phosphatidylinositol breakdown; no evidence was obtained for acetylcholine-stimulated formation of either inositol 1,2-cyclic phosphate or inositol 1-phosphate under any of the conditions used. The observations suggest that the acetylcholine-stimulated reaction is phosphatidylinositol → phosphatidic acid + inositol. This might occur by either phospholipase D activity, or through complete or partial reversal of the cytidine nucleotide pathway of phosphatidylinositol biosynthesis.     

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.     

Evidence for multiple metabolic pools of phosphatidylinositol in stimulated platelets

Stimulation of platelets with ionophore A23187 or thrombin indicates the existence of three distinct metabolic fractions of phosphatidylinositol. Two of those pools of phosphatidylinositol are degraded by phosphatidylinositol-specific phospholipase C and the third one by a phospholipase A2 activity. Low concentrations of ionophore A23187 (100 nM) or thrombin (0.25 units/ml) induce the degradation by phospholipase C of a minor fraction of phosphatidylinositol which is involved in the phosphatidylinositol cycle. In addition, thrombin, but not ionophore A23187, leads to the degradation by phospholipase C of a larger fraction of phosphatidylinositol and the subsequent accumulation of phosphatidic acid. A third fraction of phosphatidylinositol, sensitive to thrombin (0.5-2 units/ml) or ionophore A23187 (0.5-2 microM), can be degraded by phospholipase A2 to lysophosphatidylinositol with the concomitant liberation of arachidonic acid. Degradation of phosphatidylinositol by the phospholipase C pathway precedes that of the phospholipase A2 pathway. The results also suggest that the phosphatidylinositol cycle is sensitive to a small rise in cytosolic Ca2+ concentration. A further mobilization of cytosolic Ca2+ interrupts the phosphatidylinositol cycle by inhibiting conversion of phosphatidic acid to phosphatidylinositol and also activates phospholipases of the A2 type.     

Phospholipase D function in Saccharomyces cerevisiae

Phosphatidylinositol 4,5-bisphosphate-regulated phosphatidylcholine-specific phospholipase D is conserved from yeast to man. The essential role of this enzyme in yeast is to mediate the fusion of Golgi and endosome-derived vesicles to generate the prospore membrane during the developmental program of sporulation, through the production of the fusogenic lipid phosphatidic acid. In addition to recruiting proteins required for fusion, phosphatidic acid is believed to lower the energy barrier to stimulate membrane curvature. During mitotic growth, phospholipase D activity is dispensable unless the major phosphatidylinositol/phosphatidylcholine transfer protein is absent; it also appears to play a nonessential role in the mating signal transduction pathway. The regulation of phospholipase D activity during both sporulation and mitotic growth is still not fully understood and awaits further characterization.     

Membrane electrical activity elicits inositol 1,4,5-trisphosphate-dependent slow Ca2+ signals through a Gbetagamma/phosphatidylinositol 3-kinase gamma pathway in skeletal myotubes

Tetanic electrical stimulation of myotubes evokes a ryanodine receptor-related fast calcium signal, during the stimulation, followed by a phospholipase C/inositol 1,4,5-trisphosphate-dependent slow calcium signal few seconds after stimulus end. L-type calcium channels (Cav 1.1, dihydropyridine receptors) acting as voltage sensors activate an unknown signaling pathway involved in phospholipase C activation. We demonstrated that both G protein and phosphatidylinositol 3-kinase were activated by electrical stimulation, and both the inositol 1,4,5-trisphosphate rise and slow calcium signal induced by electrical stimulation were blocked by pertussis toxin, by a Gbetagamma scavenger peptide, and by phosphatidylinositol 3-kinase inhibitors. Immunofluorescence using anti-phosphatidylinositol 3-kinase gamma antibodies showed a clear location in striations within the cytoplasm, consistent with a position near the I band region of the sarcomere. The time course of phosphatidylinositol 3-kinase activation, monitored in single living cells using a pleckstrin homology domain fused to green fluorescent protein, was compatible with sequential phospholipase Cgamma1 activation as confirmed by phosphorylation assays for the enzyme. Co-transfection of a dominant negative form of phosphatidylinositol 3-kinase gamma inhibited the phosphatidylinositol 3-kinase activity as well as the slow calcium signal. We conclude that Gbetagamma/phosphatidylinositol 3-kinase gamma signaling pathway is involved in phospholipase C activation and the generation of the slow calcium signal induced by tetanic stimulation. We postulate that membrane potential fluctuations in skeletal muscle cells can activate a pertussis toxin-sensitive G protein, phosphatidylinositol 3-kinase, phospholipase C pathway toward modulation of long term, activity-dependent plastic changes.     

Cross-talk between receptor-regulated phospholipase D and phospholipase C in brain

Because receptors, G proteins, and phospholipases all exist within a membrane lipid environment, it is not unreasonable to assume that an enzyme capable of changing the lipid environment can affect the coupling relationship among these signal transducing components. Our previous study showed that a muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D via a G protein in brain. We demonstrate here that phosphatidylinositol phospholipase C and phosphatidylcholine phospholipase D are simultaneously activated within 15 s by muscarine in the presence of 1 microM GTP gamma S. More important, inhibition of phospholipase D by zinc attenuated carbamylcholine-induced activation of phospholipase C by 30%. Our additional evidence strongly indicates that the receptor-regulated phospholipase D plays an important modulatory role in agonist-stimulated phosphatidylinositol breakdown. This modulatory effect may be achieved by changing the membrane microenvironment in which phospholipase C and phosphoinositol lipids reside, consequently amplifying the inositol phospholipid signaling process. Our results lead us to postulate that the potential interaction between two different signaling pathways may provide a cell with intracellular coordination and enable the cell to achieve functional responses.     

Inhibition of phosphatidylinositol-specific phospholipase C by phosphonate substrate analogues

Non-hydrolysable analogues of phosphatidylinositol were synthesized and tested as inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus. In these molecules, the phosphodiester bond of phosphatidylinositol hydrolyzed by the phospholipase was replaced by a phosphonate linkage and a simpler hydrophobic group replaced the diacylglycerol moiety. One of the phosphonates also contained a carboxylate functional group suitable for matrix attachment. All three synthetic phosphonates inhibited the phospholipase C activity in a concentration-dependent manner, with the analogue most closely resembling the structure of the natural substrate, phosphatidylinositol, being the most potent inhibitor. The data indicate that phosphonate analogues of phosphatidylinositol may be useful for study of phospholipase C and other proteins interacting with myo-inositol phospholipids.     

Phosphatidic acid regulation of phosphatidylinositol 4-phosphate 5-kinases

Phosphatidic acid (PA) production by receptor-stimulated phospholipase D is believed to play an important role in the regulation of cell function. The second messenger function of PA remains to be elucidated. PA can bind and affect the activities of different enzymes and here we summarise the current status of activation of Type I phosphatidylinositol 4-phosphate 5-kinase by PA. Type 1 phosphatidylinositol 4-phosphate 5-kinase is also regulated by ARF proteins as is phospholipase D and we discuss the contributions of ARF and PA towards phosphatidylinositol(4,5)bisphosphate synthesis at the plasma membrane.     

Phospholipase C from human melanoma: purification and characterization of a phosphatidylinositol-selective enzyme.

Phospholipase C was purified from human melanoma grown as solid tumors in nude mice. The specific activity of the pure enzyme was approx. 100 mumol/min per mg; its apparent molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 150 kDa. The enzyme required calcium for activity and was activated by deoxycholate in the presence of the substrate phosphatidylinositol. The melanoma phospholipase C has a distinctly different substrate preference than those identified from normal tissues; it prefers phosphatidylinositol to phosphatidylinositol bisphosphate. The tumor enzyme was approx. 4-5-fold more active using phosphatidylinositol than phosphatidylinositol bisphosphate as the substrate.     

The action of phosphatidylinositol-specific phospholipases C on membranes.

A phospholipase C prepared from lymphocytes readily hydrolysed pure phosphatidyl-inositol but was relatively ineffective against phosphatidylinositol in erythrocyte "ghosts" and rat liver microsomal fraction and also against sonicated lipid extracts from these membranes. In contrast, a phospholipase C prepared from Staphylcoccus aureus readily hydrolysed phosphatidylinositol in sonicated lipid extracts but had only low activity against purified phosphatidylinositol. Unlike the enzyme from lymphocytes, the S. aureus phospholipase C did not require Ca2+ for its activity and was inhibited by cations. The previously reported specificity of this enzyme was confirmed by our observation of hydrolysis of approx. 75% of the phosphatidylinositol in ox, sheep and cat erythrocyte "ghosts" together with no detectable effect on the major erythrocyte membrane phospholipids. The phosphatidylinositol of rat liver microsomal fraction was hydrolysed only to a maximum of 15%. Some preliminary experiments showed that approx. 60% of the phosphatidylinositol of ox or sheep erythrocytes could be hydrolysed without causing substantial haemolysis.     

Lipocortin I and lipocortin II inhibit phosphoinositide- and polyphosphoinositide-specific phospholipase C. The effect results from interaction with the substrates

Lipocortins I and II, known to inhibit phospholipase A2, have been purified from bovine lung and tested with respect to their ability to affect the enzymatic activities of phosphoinositide- and polyphosphoinositide-specific phospholipase C from human platelets, rat liver cytosol or rat brain membranes. At 0.67 microM, both lipocortins led to complete inhibition of phospholipase C activity with either phosphatidylinositol or phosphatidylinositol 4,5-bisphosphate as substrate. The inhibition could be overcome by increasing the substrate concentration. Ultracentrifugation studies with lipocortin II showed a direct interaction between phosphatidylinositol and the lipocortin, indicating that the lipocortins inhibit phospholipase C not directly but by interacting with the substrate. In experiments with plasma membranes from [3H]inositol-labeled HL-60 cells, lipocortin II did not affect PI-specific phospholipase C activity in the absence or presence of calcium plus or minus GTP-gamma-S.     

Solubilization of trehalase from rabbit renal and intestinal brush-border membranes by a phosphatidylinositol-specific phospholipase C

Trehalase (EC 3.2.1.28) associated with renal and intestinal brush-border membranes was solubilized by highly purified phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) from Bacillus thuringiensis, but not by phosphatidylcholine-hydrolyzing phospholipase C (EC 3.1.4.3) from Clostridium welchii or phospholipase D (EC 3.1.4.4) from cabbage. The solubilized trehalase was not adsorbed on phenyl-Sepharose, indicating that it was hydrophilic. Phosphatidylinositol-specific phospholipase C also converted Triton X-100-solubilized amphipathic trehalase into a hydrophilic form. These results suggest that trehalase is bound to the membrane through a direct and specific interaction with phosphatidylinositol.     

Characterization of the major phosphoinositide-specific phospholipase C of human amnion

Most of the phosphoinositide-specific phospholipase C activity in human amnion at term was found to be attributable to a single isoform (Mr 85,000). Phospholipase C purified from amnion catalyzed the calcium-dependent hydrolysis of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate. The high phospholipase C activity of amnion cells isolated at 38-41 weeks of gestation declined greater than 80% during the initial 2-5 days of culture to values characteristic of amnion tissue in early gestation. Activities of phospholipase A2 and phosphatidylinositol synthase remained essentially unaltered during this period of culture. Loss of phospholipase C activity was apparently due neither to the appearance of an inhibitor nor to the loss of an activator and most likely reflected a decrease in the amount of enzyme in amnion cells. Basal production of prostaglandin E2 (PGE2) by amnion cells also declined greatly during the period of loss of phospholipase C activity. Involvement of phospholipase C in the regulation of amnion prostaglandin production was also supported by the finding that the phospholipase C inhibitor, U-73122, potently inhibited amnion cell PGE2 production. In contrast, vasopressin, which appears to stimulate prostaglandin production in amnion cells by a phospholipase C-dependent mechanism, was equipotent in stimulating PGE2 production by amnion cells on Day 2 and Day 5 of culture, even though phospholipase C activity had declined by more than 75%. Furthermore, epidermal growth factor stimulation of PGE2 production by amnion cells appeared to be largely attributable to an increase in prostaglandin H synthase activity and did not involve an increase in phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of myoinositol from solutions of enzymatic biotransformation

The transferase reaction between phospholipids and inositol catalyzed by phospholipase D on phase interface in water-organic solvent systems was studied. Optimal conditions for phosphatidylinositol synthesis in water-organic solvent heterogeneous system were determined. The rapid separation of the hydrophobic components, phospholipids, from water-soluble products, alcohols, was observed in the systems with organic solvents. Displacement of myo-inositol from phosphatidylinositol by methanol, alcohol substrate, added to the reaction medium was shown in hexane-water system. Myo-inositol was isolated from the mixture of its isomers by two-stage transferase reaction catalyzed by phospholipase D.