Phosphatidylcholine Substrate Research Articles

Pasteurella haemolytica leukotoxin-induced increase in phospholipase A2 activity in bovine neutrophils.

Exposure of bovine neutrophils to Pasteurella haemolytica leukotoxin (LKT) stimulates the production of leukotriene B4 (LTB4), which is believed to be an important chemotactic agent in the development of acute fibrinopurulent pneumonic infection in cattle. The involvement of phospholipase A2 (PLA2) in LKT-induced synthesis of LTB4 was studied by using bovine neutrophils labeled with 3H-arachidonate ([3H]AA). Incubation of isolated neutrophils with [3H]AA resulted in incorporation of radioactivity in the PLA2 substrates phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Exposure of radiolabeled neutrophils to LKT caused concentration- and time-dependent release of radioactivity and redistribution of radioactivity in neutrophil membranes consistent with utilization of phosphoglyceride substrate and release of free fatty acid and eicosanoid products. These LKT-induced effects could be inhibited by pretreatment with arachidonyl trifluoromethyl ketone, an inhibitor of type IV cytoplasmic PLA2, and were dependent on extracellular calcium. These results support the conclusion that LKT-induced synthesis of LTB4 involves a calcium-mediated increase in PLA2 activity.

Identification of lipid inhibitor of mammalian phospholipase D.

Phospholipase D (PLD) is implicated in important cellular processes, such as hormone action, inflammation, secretion, mitogenesis, and neural activity. Recent studies using cell-free systems have shown that the enzyme activity is modulated by both positive and negative regulators. During an attempt to purify PLD from pig colon mucosa, we noted the presence of a PLD inhibitor in the tissue extract. The inhibitor was purified and identified as comprising lysophosphatidylserine, phosphatidylinositol, and lysophosphatidylinositol, of which lysophosphatidylserine was the most potent. These lipids affected all of the PLD isoforms examined, oleate-dependent PLD, ARF-dependent PLD (PLD1a, PLD1b), and phosphatidylinositol 4,5-bisphosphate-dependent PLD (PLD2), in the concentration range of the 1 or 10 microM order. In contrast to lysophosphatidylserine, the diacyl counterpart phosphatidylserine was without effect in the same concentration range. PLD inhibition by lysophosphatidylserine could not be reversed by an increase in the concentration of the substrate phosphatidylcholine or activator phosphatidylinositol 4,5-bisphosphate.

Utilization of phosphatidylcholine and production of diradylglycerol as a consequence of sphingomyelin synthesis.

1. After the degradation of cell-surface sphingomyelin (SM) by exogenous sphingomyelinase (SMase), the resynthesis of SM by baby-hamster kidney (BHK) and human leukaemia-60 (HL-60) cells was examined in relation to utilization of substrate phosphatidylcholine (PtdCho) and generation of the expected product, diradylglycerol (DRG). Using [3H]choline-labelled BHK cells incubated in non-radioactive medium, SMase caused a release of phosphocholine, which was derived approximately equally from SM and PtdCho, consistent with the anticipated resynthesis of SM at the expense of PtdCho. However, with choline-labelled cells incubated in radioactive medium or [14C]acetate-labelled cells treated with SMase, no loss of radioactivity from PtdCho or accumulation of labelled DRG was observed, suggesting that any DRG produced as a consequence of SM synthesis must have been rapidly converted back into PtdCho. In contrast, SMase treatment of HL-60 cells caused more than a doubling of DRG levels at the expense of PtdCho, and this appears to be the first demonstration of a rise in DRG related to the synthesis of SM. The DRG produced consisted of about 80% 1,2-diacylglycerol and 18% 1-O-alkyl-2-acylglycerol species, a similar composition to that of the DRG backbone of total cell PtdCho. 2. The requirement for cell-surface PtdCho in the biosynthesis of SM by BHK cells was also investigated. Treatment of [3H]choline-labelled BHK cells with Bacillus cereus PtdCho-specific phospholipase C (PLC) rapidly degraded about 6% of the total PtdCho, which was assumed to represent the cell-surface pool. This did not appear to be the pool of PtdCho required for SM synthesis, since (a) the released phosphocholine was additional to that derived from PtdCho in cells treated with SMase and (b) treatment with PLC did not affect SM synthesis, either de novo or in response to degradation of cell-surface SM by SMase. These findings suggest either that there is no SM synthase in the plasma membrane or, if it is present, then it does not utilize cell-surface PtdCho as a substrate.

General base catalysis by the phosphatidylcholine-preferring phospholipase C from Bacillus cereus: the role of Glu4 and Asp55.

To assess what roles the active site residues Glu4 and Asp55 of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus (PLCBc) might play in binding and catalysis, selected mutants were prepared through site-directed mutagenesis of the plc gene. The mutants were then expressed in Escherichia coli and purified as fusion proteins with the maltose binding protein (MBP). Kinetic analysis showed that mutations at Glu4 had only modest effects on the catalytic activity, whereas those at Asp55 led to proteins whose values for kcat/KM were 10(4)-10(6) times less than that of the wild-type enzyme. The modest decrease in catalytic activity and the pH-dependent profile of the E4L mutant strongly suggest that glutamic acid at position 4 is not the general base in the PLCBc-catalyzed reaction. Rather, the results support the hypothesis that Glu4 is primarily involved in substrate binding, perhaps by electrostatic stabilization of the positive charge of the choline moiety of the phosphatidylcholine substrate. Examination of X-ray crystallographic data of PLCBc and its various complexes reveals that the carboxylate side chain of Asp55 is positioned such that it could activate a water for nucleophilic attack on the substrate or serve as a ligand for Zn1. However, the involvement of the side chain of Asp55 as an important Zn1 ligand is not consistent with the atomic absorption and thermostability data obtained for the D55L mutant, which are virtually identical with that of the wild-type enzyme. The large reduction in the measured kcat/KM of the D55E, D55N, and D55L mutants of PLCBc indicates that Asp55 plays a critical role in catalysis and likely serves as the general base in the hydrolysis of phosphatidylcholine by PLCBc.

Hexadecylphosphocholine and 2-modified 1,3-diacylglycerols as effectors of phospholipase D

The kinetic behaviour of phospholipase D (PLD) from cabbage has been studied in the presence of several substrate-like compounds such as hexadecylphosphocholine (HPC) and 1,3-didodecanoylglycero-2-phosphatides. 1,3-Didodecanoylglycero-2-phosphocholine (1,3-DiC 12PC) was found being not cleft by PLD, whereas HPC is hydrolyzed by PLD with small rate. The plot of initial velocity vs. substrate concentration for HPC is more sigmoidal than those for the common substrate phosphatidylcholine (PC)/sodium dodecylsulfate (SDS) (1:0.5) or the short-chain 1,2-dihexanoyl- sn-glycero-3-phosphocholine (DiC 6PC). The anionic amphiphiles 1,3-didodecanoylglycero-2-sulfate and 1,3-didodecanoylglycero-2-phosphate act as activators of PLD towards PC similar to SDS. In contrast, 1,3-DiC 12PC shows inhibitory properties with an increase in the sigmoidicity of the initial velocity as a function of substrate concentration in the PC/SDS assay. Also HPC inhibits the hydrolysis of PC/SDS, whereas it acts as activator or inhibitor in the hydrolysis of DiC 6PC. The results suggest that PLD possesses two substrate-binding sites, where one binds substrate in function of an effector without catalytic activity while the other is the catalytic site. HPC and 1,3-DiC 12PC are assumed to compete with the substrate for both binding sites with effects depending on the ratio of concentrations and affinities of substrates and effectors.

Inhibitory effects of lipid oxidation on the activity of plasma lecithin-cholesterol acyltransferase.

We investigated the effects of free radical generation on the esterification of cholesterol by lecithin-cholesterol acyltransferase (LCAT). A water-soluble free radical initiator, 2,2'-azobis-amidinopropane dihydrochloride (AAPH), inhibited the activity of plasma LCAT as a function of the incubation time after its addition. When a small amount of oxidized HDL was added to plasma, LCAT activity was dose-dependently inhibited. To identify the effects of HDL oxidation on LCAT activity, a purified enzyme and cofactor in a vesicle solution (an artificial substrate) were used. i) LCAT activity was inhibited by the oxidation of substrate vesicles, this inhibition being related to the degree of oxidation. ii) This inhibition was observed even if apolipoprotein A-I was not oxidized. iii) Oxidized phosphatidylcholine, but not oxidized cholesterol, in the vesicles affected LCAT activity. iv) The addition of 0-40% of oxidized vesicles to normal substrate vesicles resulted in the activity of LCAT being inhibited in a dose-dependent manner. These results suggest that the esterification of cholesterol by LCAT may be affected by the oxidation of substrate phosphatidylcholine via free radical generation in the plasma.

Substrate binding and catalytic mechanism in phospholipase C fromBacillus Cereus: A molecular mechanics and molecular dynamics study

For the first time a consistent catalytic mechanism of phospholipase C from Bacillus cereus is reported based on molecular mechanics calculations. We have identified the position of the nucleophilic water molecule, which is directly involved in the hydrolysis of the natural substrate phosphatidylcholine, in phospholipase C. This catalytically essential water molecule, after being activated by an acidic residue (Asp55), performs the nucleophilic attack on the phosphorus atom in the substrate, leading to a trigonal bipyramidal pentacoordinated intermediate (and structurally similar transition state). The subsequent collapse of the intermediate, regeneration of the enzyme, and release of the products has to involve a not yet identified second water molecule. The catalytic mechanism reported here is based on a series of molecular mechanics calculations. First, the x-ray structure of phospholipase C from B cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme-substrate complex was determined by evaluation of nonbonded interaction energies between the complex and a water molecule. The nucleophilic water molecule is positioned at a distance (3.8 A) from the phosphorus atom in the substrate, which is in good agreement with experimentally observed distances. Finally, the stability of the complex between phospholipase C, the substrate, and the nucleophilic water molecule was verified during a 100 ps molecular dynamics simulation. During the simulation the substrate undergoes a conformational change, but retains its localization in the active site. The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations, but remain within reasonable limits, thereby confirming the stability of the enzyme-substrate-water complex. The protocol developed for energy minimization of phospholipase C containing three zinc ions located closely together at the bottom of the active site cleft is reported in detail. In order to handle the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered. Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular mechanics calculations, and two different sets of partial atomic charges (MNDO-Mulliken and AMI-ESP) were applied. After careful assignment of partial atomic charges, a complete energy minimization of the protein was carried out by a stepwise procedure without explicit solvent molecules. Energy minimization with either set of charges yielded structures, which were very similar both to the x-ray structure and to each other, although using AMI-ESP partial atomic charges and a dielectric constant of 4, yielded the best protein structure.

Purification and characterization of phospholipase A2 from the pyloric caeca of red sea bream, Pagrus major

A phospholipase A2 was purified 55,000-fold in a yield of 10% from the lipid-free extract of powder of the pyloric caeca of red sea bream to near homogeneity by sequential column chromatography on S-sepharose fast flow, butyl-cellulofine, Asahipak ES-502C cation-exchange HPLC, TSK gel G3000SW gel-filtration HPLC, and Asahipak ODP-50 reversed-phase HPLC. The final preparation showed a single band with the apparent molecular mass of 14 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and an estimated specific activity was 717 µmol min-1 mg-1 protein. The purified enzyme had a pH optimum in the range of pH 8.0–9.0 and required the presence of both 8 mM of Ca2+ and from 2 to 10 mM of sodium deoxycholate for its maximal activity, using 2 mM of phosphatidylcholine as a substrate. The purified enzyme preferentially hydrolyzed the 2-acyl ester bonds of both phosphatidylglycerol and phosphatidylcholine in the presence of sodium deoxycholate, followed in order by phosphatidylethanolamine and phosphatidyl-serine. In contrast to porcine pancreatic PLA2, pyloric caeca PLA2 hydrolyzed mixed-micellar phosphatidylcholine substrate effectively, regardless of the kinds of bile salts used. These results indicate that Ca2+-dependent low molecular mass PLA2, so called secretory PLA2, occurs in the pyloric caeca of red sea beam.

Vanadate is a potent competitive inhibitor of phospholipase C from Bacillus cereus

Monomeric vanadate is a potent competitive inhibitor of phospholipase C from Bacillus cereus, much better than other oxyanions (e.g., phosphate or iodate). The apparent efficiency of inhibition depends on the substrate aggregate structure. The measured inhibition constant with respect to monomeric phosphatidylcholine substrate is 0.21 mM under conditions where the K m is 0.12 mM; for micellar substrate the apparent K i appears much lower and in fact tracks the apparent K m, which decreases 10-fold. Vanadate inhibition is removed by addition of exogenous diacylglycerol, which by itself is an inhibitor. In contrast to its effect with monomeric or micellar substrate, vanadate does not strongly inhibit the PLC-catalyzed hydrolysis of small unilamellar vesicles of phosphatidylcholine. These results are interpreted in terms of the surface binding of the enzyme. Because of its ability to mimic the transition state of phosphate ester hydrolysis vanadate is also used to investigate the constraints on the occurrence of strained cyclic intermediates in phospholipid hydrolysis by PLC.

Membrane-associated phospholipase D activity in neural cell line PC12

Stimulation of PC12 cells with carbachol (Cch) or phorbol 12-myristate 13-acetate (PMA) induced [ 3H]phosphatidylbutanol (PBut) production. Depletion of extracellular Ca 2+ abolished the Cch-mediated phospholipase D (PLD) activation, indicating the requirement of Ca 2+ influx. Two different types of PLD activity, oleate-dependent and GTPγS-dependent, were examined by using exogenous [ 3H]phosphatidylcholine substrate. PLD activity of the membrane fraction of PC12 cells was highly dependent on oleate and independent of GTPγS. This profile is in sharp contrast to that observed in HL60 cells showing the profound GTPγS-induced activation of PLD. The oleate-dependent PLD activity of PC12 membrane was inhibited by high concentrations of Ca 2+ and Mg 2+. These results indicate that Ca 2+ may not directly activate PLD but through some Ca 2+-dependent mechanism(s) in Cch-stimulated cells.

Intracellular location of oleate desaturase and associated constituents in developing sunflower ( Helianthus annuus) seeds

The location of sunflower seed oleate desaturase (ODS; EC 1.3.1.35) activity and its putative component cytochrome b has been investigated in fractions obtained by isopycnic centrifugation of seed homogenates prepared in the absence and in the presence of added Mg 2+ ions. Electron microscopy showed that the addition of 1 mM Mg 2+ resulted in maximal retention of ribosome association with the separated microsomes, whilst omission of this ion from the homogenisation medium yielded preparations containing denuded vesicles. Both ODS activity and cytochrome b were present in the fractions containing smooth vesicles which co-sedimented with the ER marker enzyme NCR, in fractions of density 1.09 g ml −1, in the latter and with vesicles having a slightly less than maximal population of associated ribosomes, sedimenting at a density of 1.18 g ml −1, in the former preparations. Whilst the enzymes LPCAT and CPT, which may be concerned in the formation of the phosphatidylcholine substrate of ODS, co-sedimented with the desaturase enzyme in the smooth microsomal preparations of the homogenates prepared in the absence of Mg 2+, addition of this ion resulted in preparations showing a polydisperse distribution of both enzymes and the marker NCR in which negligible CPT activity and 38% of the LPCAT activity was present in the fractions containing ODS activity.

Characteristics of phospholipase D in reverse micelles of Triton X-100 and phosphatidylcholine in diethyl ether

Phospholipase D, from cabbage, is active in reverse micelles formed from its substrate phosphatidylcholine and Triton X-100 in diethyl ether. The activity is optimum at w0=12.5. The increase of the molar ratio of Triton X-100/substrate from 1:4 to 2:1 results in an activity decrease by 25 %. At 136 mM Triton X-100 the KM value in reverse micelles is 136 mM, whereas it is 0.40 mM in the aqueous system containing SDS.

Entry of polyunsaturated fatty acids into the brain: evidence that high-density lipoprotein-induced methylation of phosphatidylethanolamine and phospholipase A2 are involved.

The conversion of phosphatidylethanolamine (PE) into phosphatidylcholine (PC) by a sequence of three transmethylation reactions is shown to be stimulated by the apolipoprotein E-free subclass of high-density lipoprotein (HDL3) in isolated bovine brain capillary (BBC) membranes, HDL3-induced stimulation of BBC membranes pulsed with [methyl-14C]methionine causes a transient increase in each methylated phospholipid, i.e. phosphatidyl-N-monomethylethanolamine (PMME), phosphatidyl-NN-dimethylethanolamine (PDME) and PC. PC substrate arising from the activation of PE N-methyltransferase (PEMT) is hydrolysed by a phospholipase A2 (PLA2), as demonstrated by the accumulation of lysophosphatidylcholine (lyso-PC). When PE containing [14C]arachidonic acid in the sn-2 position ([14C]PAPE) is incorporated into BBC membranes, HDL3 stimulation induces the formation of PMME, PDME, PC and lyso-PC and the release of [14C]arachidonic acid, which correlates with the previous production of lyso-PC, suggesting that HDL3 stimulates a PLA2 that can release polyunsaturated fatty acids (PUFA). Both PEMT and PLA2 activities depend on a HDL3 concentration in the range 0-50 micrograms/ml and are strictly dependent on HDL3 binding, because HDL3 modified by tetranitromethane is no longer able to bind to specific receptors and to trigger PEMT and PLA2 activation. Moreover, HDL3 prelabelled with [14C]PAPE can stimulate PDME and lyso-PC synthesis in BBC membranes in the presence of S-adenosylmethionine, suggesting that HDL3 can supply BBC membranes in polyunsaturated PE and can activate enzymes involved in PE N-methylation and PUFA release. The results support the hypothesis of a close relationship between HDL3 binding, PE methylation and PUFA release, and suggest that the PC pool arising from PE could be used as a pathway for the supply of PUFA to the brain.

A selenium-containing phospholipid-hydroperoxide glutathione peroxidase in Schistosoma mansoni.

The 100000Xg supernatant parasite platyhelminth Schistosoma mansoni exhibits a glutathione peroxidase activity with the substrate phosphatidylcholine hydroperoxide. Purification yielded a protein of 20 kDa molecular mass both on gel filtration column chromatography and SDS/PAGE, thus suggesting that S. mansoni expresses a protein similar to the mammalian selenoenzynic phospholipid-hydroperoxide glutathione peroxidase. Kinetic analysis and substrate specificity corroborated this assumption, the second-order rate constants for the oxidation of the ground-state enzyme (k+1) being higher with phosphatidylcholine hydroperoxide than with other peroxide substrates, such as cumene liydroperoxide or H2O2, and quantitatively similar to those of mammalian phospholipid-hydroperoxide glutathione peroxidase. Partial sequencing of the protein and selenium measurement by neutron activation analysis established that the purified peroxidase corresponded to the product of the S. mansoni gene previously reported and supposed to encode a selenium-containing glutathione peroxidase [Roche, C., Williams, D. L., Khalife, J., LePresle, T., Capron, A. & Pierce, R. J. (1994) Cloning and characterization of gene encoding Schistosoma mansoni glutathione peroxidase, Gene 138, 149 - 152]. S. mansoni thus contains a scienoperoxidase sharing molecular mass, catalytic efficiency and substrate specificity with phospholipid-hydroperoxide glutathione peroxidase, dismantling the concept that those enzymes are unique to vertebrate organisms.

Comparative studies on the substrate specificity of lecithin:cholesterol acyltransferase towards the molecular species of phosphatidylcholine in the plasma of 14 vertebrates.

Comparative studies indicate that plasma cholesteryl ester (CE) composition is correlated with susceptibility to atherosclerosis. We previously showed that the lecithin:cholesterol acyltransferases (LCATs) of susceptible species such as rabbit, pig, and chicken (group I) differ in their substrate and positional specificities from the LCATs of resistant species such as rat and mouse (group II). However, the relative importance of enzyme specificity and substrate phosphatidylcholine (PC) composition in determining the CE composition is not known. To address this, we analyzed the molecular species composition of plasma PC in the same 14 vertebrates in which we previously studied the CE composition and LCAT specificity. The utilization of native PC species by LCAT was studied by determining the loss of each PC after incubation of plasma at 37 degrees C. The major contributor for LCAT reaction was either 16:0-18:2 PC or 18:0-18:2 PC in all species except dog, in which it was 18:0-20:4 PC. The formation of 20:4 CE correlated more with the consumption of 18:0-20:4 PC in group I, and with the consumption of 16:0-20:4 PC in group II. The group II enzymes exhibited higher selectivity for sn-2-20:4 PCs, whereas the group I enzymes showed preference for sn-2-18:2 PCs. The synthesis of high percentage of 20:4 CE in dog plasma was found to be due to the presence of unusually high concentration of 18:0-20:4 PC, rather than due to enzyme selectivity. These results show that the PC molecular species composition, especially the concentrations of sn-2-20:4 phosphatidylcholines has profound influence on plasma CE composition, and possibly on atherogenic risk.

Glucose-induced phosphorylation and activation of a high molecular weight cytosolic phospholipase A2 in neonatal rat pancreatic islets

Previous studies have shown that stimulus-secretion coupling for the release of insulin from the pancreatic islet is potentiated by phospholipase A2 activity. Several biochemically distinct phospholipase A2 activities have been described in the islet. A recently identified cytosolic high molecular weight phospholipase A2, which requires Ca2+ for association with cellular membranes but not for catalytic activity can be activated in a protein kinase C-dependent manner in other cell-types. We determined its phosphorylation and activation in response to phorbol ester and glucose in cultured islet cells from neonatal rats. Islet cell monolayers were labelled to equilibrium with [32P]orthophosphate. Following stimulation cytosolic phospholipase A2 was immunoprecipitated and, after electrophoretic separation and transfer to nitrocellulose membrane, 32P-labelled protein was detected by autoradiography. Phospholipase A2 activity of islet cell cytosol was determined by hydrolysis of exogenous 1-stearyl-2[14C]arachidonyl phospha-tidylcholine substrate. It could be shown that phosphorylation of immunoprecipitated phospholipase A2 was augmented by prolonged glucose exposure (> 1 hr) in a protein kinase C-dependent manner. Phosphorylation occurred concomitant with a glucose-induced increase in total cellular phospholipase A2 activity (177 ± 3 nmol substrate hydrolysed/mg protein at glucose 5.6 mM vs 267 ± 32 (SEM, n = 4) at glucose 25 mM, P < 0.05). Both acute protein kinase C (459 ± 71) and glucose-activated phospholipase A2 activities were reduced in the presence of a specific arachidonic acid analogue inhibitor of cytosolic phospholipase A2 (to 231 ± 10 and 161 ± 17, respectively). The finding of phosphorylation and activation of cytosolic Ca2+-requiring phospholipase A2 by a nutrient agonist indicates a role in amplification of glucose action in the islet.

Licorice-derived compounds inhibit linoleic acid (C:18:2ω6) desaturation in soybean chloroplasts

Although glycyrrhizic acid, a major constituent of licorice root, has important pharmacological effects in humans, the biological activity of glycyrrhizic acid and its aglycone glycyrrhetinic acid in plants is unknown. Here we report that these licorice-derived compounds and the analog carbenoxolone inhibit desaturation of linoleic acid (C18:2ω6) in soybean chloroplasts using monogalactosyldiacylglycerol and phosphatidylcholine substrates in an in vitro assay for desaturase activity. At 10 nM glycyrrhetinic acid, there is significant inhibition of desaturation of linoleic acid suggesting that licorice-derived compounds could prove useful in investigating biochemical pathways of linoleic acid desaturation in plant chloroplasts and plant desaturase regulation, which has application in modification of plant response to environmental stress, as well as optimization of oil seed composition.

Activation of rat brain phospholipase D by ADP-ribosylation factors 1,5, and 6: separation of ADP-ribosylation factor-dependent and oleate-dependent enzymes.

Two major forms of phospholipase D (PLD) activity, solubilized from rat brain membranes with Triton X-100, were separated by HPLC on a heparin-5PW column with buffer containing octyl glucoside. One form was completely dependent on sodium oleate for activity. The other, which was dramatically activated by the addition of ADP-ribosylation factor (ARF) 1 and guanine 5' [gamma-thio]triphosphate, required the presence of phosphatidylinositol 4,5-bisphosphate in the phosphatidylcholine substrate for demonstration of activity, as described by others. Oleate-dependent activity was unaffected by guanine 5' [gamma-thio]triphosphate, or phosphatidylinositol 4,5-bisphosphate. Both sodium oleate-and ARF-dependent activities catalyzed transphosphatidylation, thus identifying them as PLDs. ARF-dependent PLD was activated by recombinant ARF5 (class II) and ARF6 (class III), as well as ARF1 (class I). Myristoylated recombinant ARFs were more effective than their nonmyristoylated counterparts. ARFs were originally identified as activators of cholera toxin ADP-ribosyltransferase activity. The effects of recombinant ARF proteins from the three classes on cholera toxin activity (assayed under conditions identical to those used to assay PLD activity) did not, however, correlate with those on PLD, consistent with the notion that different aspects of ARF structure are involved in the two functions.

Diacylglycerol and alkylacylglycerol stimulate ram sperm phospholipase A 2

1. 1. We have investigated the susceptibility of ram sperm phospholipase A 2 (PLA 2) to stimulation by diacyi- and alkylacylglycerols and by monoacyi- and monoalkylglycerols. 2. 2. PLA 2 activity in sonicates from ram spermatozoa was enhanced when l-stearoyl-2-arachidonoyl- sn-glycerol, the diacylglycerol usually generated by polyphosphoinositide breakdown, was added to a radioactive phosphatidylcholine substrate; the effect was time- and Ca 2+-dependent. 3. 3. Both diacyi- and alkylacylglycerol considerably enhanced PLA 2 activity; 1-O-hexadecyl-2-O-methyl- rac-glycerol, however, only showed slight stimulatory ability. 4. 4. The monoradylglycerols 1-monohexadecanoyl- rac-glycerol, 2-monohexadecanoylglycerol, and 1-O-hexadecyl-. sn-glycerol had very little effect on the enzyme's activity. 5. 5. Exposure of spermatozoa to 1-oleoyl-2-acetyl-. sn-glycerol (OAG) or 1-O-hexadecyl-2-acetyl- rac-glycerol (1-O-C 16/2-C 2), when cells were stimulated with the ionophore A23187 and Ca 2+, resulted in higher PLA 2 activity in sperm sonicates. Furthermore, parallel experiments showed that exocytosis was enhanced if spermatozoa were treated with A23187/Ca 2+ and either OAG or 1-O-C 16/2-C 2. Since both diacyi- and alkylacylglycerols increased PLAi activity and exocytosis, stimulation of PLA 2 activity by these diglycerides may take place independently from protein kinase C activation.

Conductimetric Assays for the Hydrolase and Transferase Activities of Phospholipase D Enzymes

Measurement of solution electrical conductance (conductimetry) is a simple direct assay method for the protogenic, hydrolytic reactions catalyzed by all phospholipase enzymes. The technique is especially suitable for assay of phospholipase D (PLD) enzymes where cleavage of zwitterionic substrates reinforces the pH dependent conductance change and allows the method to be used over a much wider pH range than the equivalent titrimetric assay. The ability to detect zwitterion cleavage enables the method to assay reactions in which phospholipase D transfers neutral, or anionic, alcohol species to the zwitterionic substrates phosphatidyl choline and phosphatidyl ethanolamine. The method can follow the sequential attack by different phospholipases and provides a simple technique for investigating the effect of substrate structure on susceptibility to various phospholipase enzymes. The results confirm that PLD from Streptomyces chromofuscus can attack lysophospholipids, but cannot transfer primary alcohols to the phosphatidyl residue, while the PLD from savoy cabbage is an efficient transferase, but cannot attack lysophospholipids. The data suggest that the bacterial PLD fails to act as a transferase because it hydrolyzes the transphosphatidylation products. Some phosphatidyl alcohols are more highly susceptible to PLA2 attack than the parent phosphatidyl choline derivatives.