Isolation of myo-Inositol from Solutions by Enzymatic Biotransformation

The transferase reaction between phospholipids and inositol catalyzed by phospholipase D was studied at interfaces in water–organic solvent systems. Optimum conditions were determined for phosphatidylinositol synthesis in heterogeneous water–organic solvent systems. Hydrophobic components (phospholipids) were readily separated from water-soluble products (alcohols) in systems with organic solvents. In the hexane–water system, addition of methanol (an alcohol substrate) to the reaction medium displaced myo-inositol from the molecule of phosphatidylinositol. myo-Inositol was isolated from the mixture of its isomers using a two-step transferase reaction catalyzed by phospholipase D.     

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.     

A phospholipid requirement for dolichol pyrophosphate N-acetylglucosamine synthesis in phospholipase A2-treated rat lung microsomes

The role of phospholipids in the activity of UDP-Glc-NAc:dolichol phosphate GlcNAc-1-phosphate transferase of rat lung microsomes has been investigated. Treatment of microsomes with phospholipase A2 in the presence of delipidated bovine serum albumin resulted in a time-dependent loss of 65 to 75% of the enzyme activity and approximately 30% of the phospholipids. Addition of phosphatidylglycerol to the enzyme assay system containing phospholipase A2-treated microsomes restored activity to that obtained with native microsomes and phosphatidylglycerol. Addition of phosphatidylinositol, phosphatidylcholine, or cardiolipin resulted in only partial restoration of activity, whereas phosphatidylserine and phosphatidylethanolamine were without effect. Triton X-100 was not by itself capable of restoring activity, but was required for the phospholipid effect. Measurements of the phospholipase A2 hydrolysis products released from the microsomes during digestion, and other control experiments of adding fatty acids and lysophospholipids to the enzyme assay system, indicated that the loss of UDP-GlcNAc:dolichol phosphate GlcNAc-1-phosphate transferase activity was not due to product inhibition.     

The dependence of the effects of calcium ions on enzymatic hydrolysis of phospholipids on the physical state of the substrate]

The role of calcium ions in the phospholipid hydrolysis by phospholipase D was studied. It was shown that the enzyme does not split egg lecithine in the absence of Ca2+. In the presence of Ca2+ the reaction occurs via different routes, depending on the type of initiation of the reaction. The optimal concentrations of Ca2+ necessary for activation of phospholipase D are different in the systems activated by various treatments (organic solvents, detergents and solid adsorbents). Optimal concentrations of Ca2+ for the hydrolysis and methanolysis catalyzed by phospholipase D are also different. It was found that the need for Ca2+ and their optimal concentrations are determined by the state of phospholipids at the substrate phase. The data suggest that the enzymatic hydrolysis may occur in the absence of Ca2+. Thus, Ca2+-induced activation is merely an alternative pathway of catalytically active conformation of lypolytic enzymes.     

Freezing Injury and Phospholipid Degradation in Vivo in Woody Plant Cells: III. Effects of Freezing on Activity of Membrane-bound Phospholipase D in Microsome-enriched Membranes

Freeze-thawing of microsome-enriched membranes from living bark tissues of black locust trees, especially those from less hardy tissues, caused a drastic increase in sensitivity to Ca²⁺ and a complete loss of the regulatory action of Mg²⁺ in membrane-bound phospholipase D activity with endogenous (membrane-bound) substrates. Also, the freeze-thaw cycle made phospholipase D in these membranes more resistant to digestion by proteases. Thus, the regulatory properties of the membrane-bound phospholipase D seem to be dependent on the nature of the membranes and on the interaction between the enzyme and membranes as well. The alteration of regulatory properties by freezing was protected by sucrose, at lower concentrations, and more effectively for membranes from hardy tissues than for membranes from less hardy tissue. Addition of partially purified soluble phospholipase D to the reaction system containing membranes caused only a slight stimulation of the degradation of endogenous phospholipids. Phospholipid degradation in vivo during freezing of less hardy tissue may be catalyzed mainly by the bound enzyme. Disintegration of the tonoplast, however, besides releasing soluble phospholipase D into the cytosol, would release organic acids (lowering the pH) and free Ca²⁺. Both factors would stimulate drastically the membrane-bound phospholipase D, causing degradation of membrane phospholipids.     

The formation of phosphatidylglycerol and other phospholipids by the transferase activity of phospholipase D

1. Purified phospholipase D can catalyse the transfer of a `phosphatidyl' unit from lecithin to various aliphatic alcohols such as glycerol, ethanolamine, methanol and ethylene glycol with the formation of the equivalent phospholipid. 2. The transferase reaction occurs simultaneously with hydrolase activity but at high alcohol concentrations the former predominates. 3. The acceptor molecule must contain a primary alcoholic grouping. 4. The chromatographic and ionophoretic mobilities of the deacylation products of many enzymically synthesized phospholipids are reported. 5. Enzymically prepared phosphatidylglycerol has been isolated in good yield. Chemical degradation showed that the `phosphatidyl unit' of lecithin had been transferred predominantly to the α-hydroxyl groups of glycerol. 6. Water-soluble alcohols can markedly stimulate the liberation of choline from ultrasonically treated lecithin by phospholipase D. The stimulation is usually due to an increase in hydrolase activity although often the associated transferase activity contributes.     

Substrate specificity of neutral phospholipase D from rat brain studied by selective labeling of endogenous synaptic membrane phospholipids in vitro.

We have designed a novel approach for studying the specificity of neutral phospholipase D from rat brain synaptic plasma membranes for endogenous phospholipid substrates in native membranes. A procedure was established that provides synaptic membranes labeled in selected phospholipids. This labeling procedure exploits the presence of endogenous acyl-coenzyme A synthetase and acyl-coenzyme A:lysophospholipid acyltransferase in synaptosomes for acylating various lysophospholipid acceptors with radioactive fatty acid. With [3H]arachidonate for acylation and optimal concentrations of the respective lysophospholipids, membranes were labeled in either of the following phospholipids: phosphatidylcholine (93% of total label in phospholipids), 1-O-alkyl-phosphatidylcholine (87%), phosphatidylinositol (90%), phosphatidylethanolamine (85%), phosphatidylethanolamine-plasmalogen (81%) or phosphatidylserine (59%). These membranes were employed to study the substrate specificity of the neutral, oleate-activated rat brain phospholipase D. This phospholipase exhibited almost absolute specificity for the choline-phospholipids phosphatidylcholine and 1-O-alkyl-phosphatidylcholine: 0.34% of the former labeled substrate were transphosphatidylated to phosphatidylpropanol during the assay and 0.28% of the latter. Activity toward other phospholipids was barely detectable and could largely be accounted for by utilization of residual labeled phosphatidylcholine present in those preparations. The phospholipase D exhibited some preference for fatty acids in the C-2 position of phosphatidylcholine in the following order: 2-oleoyl-phosphatidylcholine (0.67% of this labeled phosphatidylcholine were converted to phosphatidylpropanol), 2-myristoyl-phosphatidylcholine (0.60%), 2-palmitoyl-phosphatidylcholine (0.46%) and 2-arachidonoyl-phosphatidylcholine (0.34%). The present approach of labeling membrane phospholipids in vitro could be useful in studies of phospholipase specificity as an alternative to the use of sonicated vesicles or mixed detergent-phospholipid micellar systems.     

Phospho-N-acetylmuramoyl-pentapeptide-transferase of Escherichia coli K12. Properties of the membrane-bound and the extracted and partially purified enzyme.

Phospho-N-acetylmuramoyl-pentapeptide-transferase (UDP-N-acetyl-muramoyl-L-alanyl-D-gamma-glutamyl-L-lysyl-D-alanyl-D-alanine:undecaprenoid-alcohol-phosphate-phospho-N-acetylmuramoyl-pentapeptide-transferase, EC 2.7.8.13) was solubilized by repeated freezing and thawing of crude envelopes of Escherichia coli K12. The solubilized enzyme was partially purified by gel filtration and ion-exchange chromatography. This preparation contained small amounts of phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol but no endogenous lipid substrate, C55-isoprenyl phosphate, could be detected. Some catalytic properties (exchange reaction) of the solubilized enzyme were compared to those of membrane-bound transferase. The transfer activity of the partially purified transferase was restored by the addition of an aqueous lipid dispersion. All the transferase activity was found to become incorporated into the liposomes. Preincubation of the transferase preparation with phospholipase A2 or D strongly reduce both exchange and transfer activity. This suggests that phospholipids sensitive to phospholipases are necessary for the enzymatic reaction. Different effects of some neutral detergents on the exchange activity were reported.     

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.     

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.     

Cytosolic and particulate phosphatidylinositol phospholipase C activities in pollen tubes of Lilium longiflorum

Pollen tubes of Lilium longiflorum Thunb. cv. White Europe contain three distinguishable phosphatidylinositol phospholipase C activities (EC 3.1.4.10). Two of these are particulate and have optima at pH 5.2 and 7.0, respectively. The third one, a cytosolic activity, has an optimum at pH 6.0. The distribution of radioactivity in reaction products from phosphatidylinositol, labeled in either the inositol, glycerol or phosphate moiety, indicates that the three phospholipase activities cleave only the bond between glycerol and phosphate. The dependence on divalent cations slightly differs, though Ca²⁺ is the most stimulatory ion species for all the three enzyme activities. Activity is not observed in the presence of EDTA. When anionic phospholipids are mixed with phosphatidylinositol substrate an increase in phosphatidylinositol phospholipase C activities is observed, except for the particulate activity with an optimum at pH 5.2. Phosphatidylcholine and phosphatidylethanolamine are inhibitory.     

Phosphatidylethanolamine and phosphatidylcholine are sources of diacylglycerol in ras-transformed NIH 3T3 fibroblasts

Ras-transformation of cells is accompanied by an increase of the level of diacylglycerol (DAG), which participates in the signal transduction pathways. DAG could be generated from phospholipids either by activation of phospholipase C or by a more complex pathway involving phospholipase D and phosphatidate phosphohydrolase. To clarify which phospholipids produce DAG and which pathways are involved, we examined the DAG generating enzyme activities, using phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) as substrates. The study showed that the breakdown of PC and more markedly of PE by phospholipases C and D was stimulated in membranes from ras-transformed cells. Phosphatidate phosphohydrolase activity was also elevated in oncogene-expressing cells. The increase in glycerol uptake was most pronounced in cells given PE, followed by PC. The fatty acid analysis revealed apparent similarities between the acyl chains of PE and DAG only in the transformed cells. These findings suggest that PE is a source of DAG in ras-fibroblasts but does not rule out the role of PC in DAG production, due to the activation of the PC-specific phospholipases C and D.     

Rapid degradation and limited synthesis of phospholipids in the cotyledons of mung bean seedlings

Seedling growth of mung bean is accompanied by the rapid catabolism of the three major phospholipids in the cotyledons (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). The decline starts 24 hours after the beginning of imbibition and by the 4th day of growth more than 50% of the phospholipids have been catabolized. Extracts of cotyledons of 24-hour-imbibed beans contain enzymes capable of degrading membrane-associated phospholipids in vitro. This degradation involves phospholipase D and phosphatase activity.     

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.     

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.     

Glycosylphosphatidylinositol-specific phospholipase D of human serum--activity modulation by naturally occurring amphiphiles

The enzymatic properties of glycosylphosphatidylinositol-specific phospholipase D (EC 3.1.4.50) were characterized using a 6,000-fold purified enzyme. This was obtained in 100 microg amounts from human serum with a recovery of 35%. Pure alkaline phosphatase containing one anchor moiety per molecule was used as substrate. The enzyme is stimulated by n-butanol, but in contrast to other phospholipases this activation is not produced by a transphosphatidylation reaction. The previously reported non-linearity of the specific activity with respect to phospholipase concentration in the test was no longer observed upon purification, indicating inhibitor removal. The serum inhibitor(s) co-chromatograph with serum proteins and lipoproteins. The main part of the inhibitory activity was found in the lipid fraction after protein denaturation and can be subfractionated into acid phospholipids, cholesteryl esters and triacylglycerides. Added phosphatidyl-serine, phosphatidylinositol, phosphatidylglycerol, gangliosides, cholesteryl esters, and sphingomyelins turned out to be strong inhibitors, as well as phosphatidic acid. Phosphatidylethanolamine and various monoacylglycerols were found to be activators. The low glycosylphosphatidylinositol-specific phospholipase activity found in native serum did not increase significantly upon 90% removal of phospholipids by n-butanol. High serum concentrations of strongly inhibiting compounds, complex kinetic interactions among aggregates of these substances, and compartmentalization effects are discussed as possible reasons for the observed inactivity.     

Pulmonary surfactant protein D specifically binds to phosphatidylinositol.

Alveolar type II cells produce and secrete a complex mixture of lipids and proteins called pulmonary surfactant of which phospholipids are the major components. Surfactant proteins (SP) A, B, and C interact with phospholipids and are believed to play important roles in alveolar spaces. However, whether surfactant protein D (SP-D) interacts with phospholipids is unknown. In the present study, we examined whether SP-D binds to phospholipids and investigated phospholipid specificities of SP-D binding and the structural requirements of phospholipids for that binding using 125I-SP-D as a probe. 125I-SP-D bound exclusively to phosphatidylinositol (PI) in various phospholipids or a fraction containing phospholipids extracted from surfactant, which were developed on thin layer chromatography. 125I-SP-D also bound to PI coated on microtiter wells in a manner dependent upon the SP-D concentration. Unlabeled SP-D competed well with 125I-SP-D for PI binding and the antibody against SP-D abolished 125I-SP-D binding to PI. PI liposome also attenuated 125I-SP-D binding to the solid phase PI. Ca2+ is absolutely required for the binding of SP-D to PI. SP-D failed to bind to lyso-PI, fatty acids derived from PI digested with phospholipase A2, or diacylglycerol obtained after phospholipase C treatment of PI. SP-D bound to neither phosphatidylinositol 4-monophosphate nor phosphatidylinositol 4,5-diphosphate. We conclude that SP-D specifically binds to PI. This is the first report that demonstrates that SP-D interacts with surfactant phospholipids.     

Hydrolysis of N-acylated glycerophospholipids by phospholipases A2 and D: a method of identification and analysis

We have previously identified N-acylethanolamine phospholipids in infarcted dog heart and in normal fish brain by chemical and enzymatic degradation. We now report that hydrolysis with phospholipase D from Streptomyces chromofuscus removes N-acylethanolamine from N-acylethanolamine phospholipids and lyso N-acylethanolamine phospholipids, or N-acylserine from lyso N-acylserine phospholipids. At acidic pH, a phosphatase present in the phospholipase D preparation further hydrolyzes the resulting phosphatidic acid (PA) or lyso-PA to diacyl- or monoacylglycerol. Because N-acylserine phospholipids are a poor substrate for the phospholipase D, pretreatment with phospholipase A2 (Trimeresurus flavoviridis venom) is used to remove the 2-O-acyl group. Thus, both types of N-acylated phospholipids can be analyzed by consecutive phospholipase A2 and phospholipase D treatment. Reaction products, i.e., free fatty acids, monoacylglycerols and N-acylethanolamine or N-acylserine, are separable by thin-layer chromatography. Both N-acyl components can be further characterized by conversion to the t-butyldimethylsilyl derivatives. The method was used to identify and analyze the N-acylserine phospholipids of bovine brain.     

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.     

Regulation of membrane phospholipid catabolism in senescing carnation flowers

Evidence has been obtained for the involvement of μ M levels of Ca²⁺ in phospholipid catabolism during petal senescence by following the breakdown of [U-¹⁴C]-phosphatidylcholine by microsomal membranes from cut carnation ( Dianthus caryophyllus L. cv. White-sim) flowers. Phospholipid degradation was mediated by three membrane-associated lipases, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4) and lipolytic acyl hydrolase. The activities of phospholipase D and phosphatidic acid phosphatase were stimulated by 30 and 100%, respectively, in the presence of 40 μ M free Ca²⁺, and the Ca²⁺-stimulation of phosphatidic acid phosphatase was calmodulin-dependent. When L-3-phosphatidyl-[2-³H]-inositol and L-3-phosphatidyl-[N-methyl-³H]-choline were used as substrates, inositol and choline accounted for 95 and 99%, respectively, of the water-soluble radiolabelled products. This suggests a predominance of phospholipase D activity over phospholipase C activity in these membranes.
Breakdown of membrane phospholipids in senescing carnations is known to be accelerated by treatment of young flowers with ethylene. To determine whether this involves a specific turnover of phosphatidylinositol as observed in animal systems in response to certain agonists, young flowers pre-labelled with ³²PO^3-₄ were treated with 10 ppm ethylene. All phospholipids incorporated the label, but no enhanced turnover of phosphatidylinositol was observed. Inositol 1,4,5-triphosphate did not release Ca²⁺ from preloaded microsomal vesicles at concentrations known to be effective in animal systems (i.e. < 5 μ M ) although release of Ca²⁺ was observed when a higher (20 μ M ) concentration was used.