Purification,Biochemical Characterization,and Cloning of Phospholipase D from <Emphasis Type="Italic">Streptomyces racemochromogenes</Emphasis> Strain 10-3

We previously isolated Streptomyces racemochromogenes strain 10-3, which produces a phospholipase D (PLD) with high transphosphatidylation activity. Here, we purified and cloned the PLD (PLD103) from the strain. PLD103 exerted the highest hydrolytic activity at a slightly alkaline pH, which is in contrast to the majority of known Streptomyces PLDs that have a slightly acidic optimum pH. PLD103 shares only 71–76% amino acid sequence identity with other Streptomyces PLDs that have a slightly acidic optimum pH; thus, the diversity in the primary structure might explain the discrepancy observed in the optimum pH. The purified PLD displayed high transphosphatidylation activity in the presence of glycerol, l-serine, and 2-aminoethanol hydrochloride with a conversion rate of 82–97% in a simple one-phase system, which was comparable to the rate of other Streptomyces PLDs in a complicated biphasic system.     

Regulation of Phospholipase D Activity by Light and Phytohormones in Oat Seedlings

The effect of synthetic analogs of phytohormones and red light absorbed by phytochrome on the phospholipase D activity (PLD) was studied in oat (Avena sativa L.) seedlings. ABA manifested a short-term stimulating effect on PLD activity in the green seedlings and inhibited phospholipase activity in the etiolated plants. Kinetin inhibited enzyme activity in the etiolated seedlings and did not affect its activity in light. GA did not markedly affect PLD activity in the etiolated plants and activated this enzyme in the green seedlings. Finally, IAA did not affect the enzyme activity. The relationship of the regulatory effects of phytohormones and light on PLD activity is discussed.     

Phospholipase Dδ negatively regulates plant thermotolerance by destabilizing cortical microtubules in Arabidopsis

The pattern of cortical microtubule arrays plays an important role in plant growth and adaptation in response to hormonal and environmental changes. Cortical microtubules are connected with the plasma membrane (PM); however, how the membrane affects cortical microtubule organization is not well understood. Here, we showed that phospholipase Dδ (PLDδ) was associated with the PM and co‐localized with microtubules in cells. In vitro analysis revealed that PLDδ bound to microtubules, resulting in microtubule disorganization. Site‐specific mutations that decreased PLDδ enzymatic activity impaired its effects on destabilizing microtubule organization. Heat shock transiently activated PLDδ, without any change of its PM localization, triggering microtubule dissociation from PM and depolymerization and seedling death in Arabidopsis, but these effects were alleviated in pldδ knockout mutants. Complementation of pldδ with wild‐type PLDδ, but not mutated PLDδ, restored the phenotypes of microtubules and seedling survival to those of wild‐type Arabidopsis. Thus, we conclude that the PM‐associated PLDδ negatively regulates plant thermotolerance via destabilizing cortical microtubules, in an activity‐dependent manner, rather than its subcellular translocation.     

Mutual regulation of plant phospholipase D and the actin cytoskeleton

Membrane lipids and cytoskeleton dynamics are intimately inter‐connected in the eukaryotic cell; however, only recently have the molecular mechanisms operating at this interface in plant cells been addressed experimentally. Phospholipase D (PLD) and its product phosphatidic acid (PA) were discovered to be important regulators in the membrane–cytoskeleton interface in eukaryotes. Here we report the mechanistic details of plant PLD–actin interactions. Inhibition of PLD by n‐butanol compromises pollen tube actin, and PA rescues the detrimental effect of n‐butanol on F‐actin, showing clearly the importance of the PLD–PA interaction for pollen tube F‐actin dynamics. From various candidate tobacco PLDs isoforms, we identified NtPLDβ1 as a regulatory partner of actin, by both activity and in vitro interaction assays. Similarly to published data, the activity of tobacco PIP₂‐dependent PLD (PLDβ) is specifically enhanced by F‐actin and inhibited by G‐actin. We then identified the NtPLDβ1 domain responsible for actin interactions. Using sequence‐ and structure‐based analysis, together with site‐directed mutagenesis, we identified Asn323 and Thr382 of NtPLDβ1 as the crucial amino acids in the actin‐interacting fold. The effect of antisense‐mediated suppression of NtPLDβ1 or NtPLDδ on pollen tube F‐actin dynamics shows that NtPLDβ1 is the active partner in PLD–actin interplay. The positive feedback loop created by activation of PLDβ by F‐actin and of F‐actin by PA provides an important mechanism to locally increase membrane–F‐actin dynamics in the cortex of plant cells.     

Identification of Actinomycetes Producing Phospholipase D with High Transphosphatidylation Activity

Previously we isolated six actinomycetes strains, 9-4, 10-1, 10-2, 10-3, 10-6, and 21-4, that produce phospholipase D (PLD) with high transphosphatidylation activity. In this study, we identified these strains, and the PLD activities were compared with those of reference strains. 16S rDNA sequences and DNA–DNA hybridization tests indicated taxonomic affiliations of strain 9-6 with Streptomyces senoensis, strains 10-1 and 10-6 with S. vinaceus, and strains 10-2 and 10-3 with S. racemochromogenes. Strain 21-4, though identified as a Streptomyces sp., could not be identified with any known species. Meanwhile, most of the culture supernatants of reference strains demonstrated no or very weak PLD activity, while those of our strains exhibited significantly higher activity. All of the strains in this study were identified as Streptomyces species. The PLD activity of our strains exceeded most of the reference Streptomyces strains. The findings in this study imply that the Streptomyces strains, although they are members of the same species, can produce different quantities of PLD enzyme.     

Light-Dependence of Phospholipase D Activity in Oat Seedlings

The effect of light on the activity of phospholipase D (PLD) in oat (Avena sativa L.) seedlings and the dependence of this enzyme activity on the regime of their illumination were studied. The PLD activity in etiolated seedlings was 1.5–2.0-fold higher than in green plants. The illumination of etiolated seedlings with white light resulted in a decrease in PLD activity to its level in the seedlings grown under light. In contrast, the transfer of green seedlings to darkness enhanced the activity of the enzyme up to its level in etiolated seedlings. The illumination of etiolated seedlings with red light inhibited the PLD as well. It was shown that this photoeffect decreased with seedling aging and correlated with a phytochrome content in plants. Far-red light reversed the effect of red light. The involvement of phytochrome in the control of the PLD activity is discussed.     

Phospholipase D Activity in the Tetrahymena pyriformis GL

Phospholipase D (PLD) is an enzyme which participates in the signalling mechanism cleaving phosphatidylcholine (PC) to choline and phosphatidic acid (PA). In Tetrahymena pyriformis GL this enzyme activity is enhanced by different kinds of agonists (sodium orthovanadate, sodium fluoride and phorbol 12-myristate 13-acetate), and its activity can be inhibited by inhibitors such as pertussis toxin, calphostin C, genistein, trifluoperazine. These results suggest that the PLD signalling pathway is connected with the tyrosine kinase, phospholipase C, phosphatidylinositol and G-protein coupled signalling pathways. By demonstrating the PLD activity in Tetrahymena our knowledge on the signalling mechanisms at a unicellular level has been extended. The results support our view that most transducing mechanisms that are characteristic of mammalian cells are also in the protozoan Tetrahymena. © 1997 John Wiley & Sons, Ltd.     

Molecular cloning of the phospholipase D gene from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. YU100 and its expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and hetero-logously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100.     

Phospholipase D: A new mediator during high phosphate-induced vascular calcification associated with chronic kidney disease

Vascular calcification (VC) is the pathological accumulation of calcium phosphate crystals in one of the layers of blood vessels, leading to loss of elasticity and causing severe calcification in vessels. Medial calcification is mostly seen in patients with chronic kidney disease (CKD) and diabetes. Identification of key enzymes and their actions during calcification will contribute to understand the onset of pathological calcification. Phospholipase D (PLD1, PLD2) is active at the earlier steps of mineralization in osteoblasts and chondrocytes. In this study, we aimed to determine their effects during high-phosphate treatment in mouse vascular smooth muscle cell line MOVAS, in the ex vivo model of the rat aorta, and in the in vivo model of adenine-induced CKD. We observed an early increase in PLD1 gene and protein expression along with the increase in the PLD activity in vascular muscle cell line, during calcification induced by ascorbic acid and β-glycerophosphate. Inhibition of PLD1 by the selective inhibitor VU0155069, or the pan-PLD inhibitor, halopemide, prevented calcification. The mechanism of PLD activation is likely to be protein kinase C (PKC)-independent since bisindolylmaleimide X hydrochloride, a pan-PKC inhibitor, did not affect the PLD activity. In agreement, we found an increase in Pld1 gene expression and PLD activity in aortic explant cultures treated with high phosphate, whereas PLD inhibition by halopemide decreased calcification. Finally, an increase in both Pld1 and Pld2 expression occurred simultaneously with the appearance of VC in a rat model of CKD. Thus, PLD, especially PLD1, promotes VC in the context of CKD and could be an important target for preventing onset or progression of VC.     

Rapid activation of specific phospholipase(s) D by cytokinin in Amaranthus assay system

The suggested link between intracellular cytokinin signaling and phospholipase D (PLD, EC 3.1.4.4.) activity (Romanov et al. 2000, 2002) was investigated. The activity of PLD in the early period of cytokinin action was studied in vivo in derooted Amaranthus caudatus seedlings, using the level of phosphatidylbutanol production as a measure of PLD activity. Rapid activation of phosphatidylbutanol synthesis was demonstrated as early as within 5 min of cytokinin administration. Neomycin, a known phosphatidylinositol‐4,5‐bisphosphate (PIP₂) antagonist, strongly repressed both physiological cytokinin effect and cytokinin‐dependent PLD activation. N‐acylethanolamine (NAE 12), an inhibitor of α‐class PLD, did not influence significantly cytokinin effect on Amaranthus seedlings. Together, results suggest the involvement of PIP₂‐dependent non‐class α‐PLD in the molecular mechanism of cytokinin action.     

Phospholipase D fromStreptomyces antibioticus: Cloning,sequencing, expression,and relationship to other phospholipases

The extracellular phospholipase D (PLD) gene fromStreptomyces antibioticus was cloned, sequenced, and expressed inEscherichia coli. Analysis of DNA sequence data revealed a putative ribosome-binding site and an open reading frame encoding a 556-amino-acid protein that included amino acid sequences obtained from the purified enzyme. The protein was expressed in an insoluble form inE. coli, but reacted with antibody against PLD. After solubilization of the protein with guanidine-HCI and 2-mercaptoethanol, subsequent dialysis restored the PLD activity. Comparison of the nucleotide sequence data with the N-terminal protein sequence indicates that this secreted protein is synthesized as a larger precursor with a 47-amino-acid N-terminal extension to the mature enzyme of 509 amino acids. The amino acid sequence of the S.antibioticus PLD was extensively compared with other PLDs and phospholipase C (PLC). The deduced amino acid sequence of the cloned PLD was highly homologous to PLDs from S. acidomyceticus andStreptomyces sp., and contained a conserved region with S.chromofuscus PLD. From comparisons of the structural similarity and properties of the various PLDs, a classification of PLDs into two subgroups has been proposed and the highly conserved region designated tentatively region X_PLD, which may be important in the catalytic function, has been identified. The homology comparison between our PLD and phosphatidylinositol-specific phospholipase C (PI-PLC) is also discussed.     

Production of octadecylphospho-l-serine by phospholipase d

Octadecylphospho-l-serine (OPS), belonging to the family of alkylphosphate esters with anticancer activity, was synthesized by transesterification of octadecylphosphocholine using phospholipase d (PLD). With respect to the yield of product, PLD from cabbage proved to be superior to PLD from Streptomyces spp. Although the addition of n-hexane/2-octanol is advantageous to suppress the hydrolytic byproduct, a purely aqueous reaction medium was preferred because of better recovery of the product.     

Phospholipid and triacylglycerol profiles modified by PLD suppression in soybean seed

Phospholipase D (PLD) is capable of hydrolyzing membrane phospholipids, producing phosphatidic acid. To alter phospholipid profiles in soybean seed, we attenuated PLD enzyme activity by an RNA interference construct using the partial sequence from a soybean PLDα gene. Two transgenic soybean lines were established by particle inflow gun (PIG) bombardment by co‐bombarding with pSPLDi and pHG1 vectors. The lines were evaluated for the presence and expression of transgenes thoroughly through the T₄ generation. PLD‐suppressed soybean lines were characterized by decreased PLDα enzyme activity and decreased PLDα protein both during seed development and in mature seeds. There was no change in total phospholipid amount; however, the PLD‐attenuated transgenic soybean seed had higher levels of di18 : 2 (dilinoleoyl)‐phosphatidylcholine (PC) and ‐phosphatidylethanolamine (PE) in seeds than the non‐transgenic lines. The increased polyunsaturation was at the expense of PC and PE species containing monounsaturated or saturated fatty acids. In addition to increased unsaturation in the phospholipids, there was a decrease in unsaturation of the triacylglycerol (TAG) fraction of the soybean seeds. Considering recent evidence for the notion that desaturation of fatty acids occurs in the PC fraction and that the PC → DAG (diacylglycerol) → TAG pathway is the major route of TAG biosynthesis in developing soybean seed, the current data suggest that PLDα suppression slows the conversion of PC to TAG. This would be consistent with PLD playing a positive role in that conversion. The data indicate that soybean PLD attenuation is a potentially useful approach to altering properties of edible and industrial soybean lecithin.     

Phospholipase D and retromer promote recycling of TRPL ion channel via the endoplasmic reticulum

Plasma membrane protein trafficking is of fundamental importance for cell function and cell integrity of neurons and includes regulated protein recycling. In this work, we report a novel role of the endoplasmic reticulum (ER) for protein recycling as discovered in trafficking studies of the ion channel TRPL in photoreceptor cells of Drosophila. TRPL is located within the rhabdomeric membrane from where it is endocytosed upon light stimulation and stored in the cell body. Conventional immunohistochemistry as well as stimulated emission depletion super-resolution microscopy revealed TRPL storage at the ER after illumination, suggesting an unusual recycling route of TRPL. Our results also imply that both phospholipase D (PLD) and retromer complex are required for correct recycling of TRPL to the rhabdomeric membrane. Loss of PLD activity in PLD^3.1 mutants results in enhanced degradation of TRPL. In the retromer mutant vps35^MH20, TRPL is trapped in a Rab5-positive compartment. Evidenced by epistatic analysis in the double mutant PLD^3.1 vps35^MH20, PLD activity precedes retromer function. We propose a model in which PLD and retromer function play key roles in the transport of TRPL to an ER enriched compartment.     

Activation of muscarinic receptors in porcine airway smooth muscle elicits a transient increase in phospholipase D activity

Phospholipase D (PLD) is a phosphodiesterase that catalyses hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. In the presence of ethanol, PLD also catalyses the formation of phosphatidylethanol, which is a unique characteristic of this enzyme. Muscarinic receptor-induced changes in the activity of PLD were investigated in porcine tracheal smooth muscle by measuring the formation of [³H]phosphatidic acid ([³H]PA) and [³H]phosphatidylethanol ([³H]PEth) after labeling the muscle strips with [³H]palmitic acid. The cholinergic receptor agonist acetylcholine (Ach) significantly but transiently increased formation of both [³H]PA and [³H]PEth in a concentration-dependent manner (>105–400% vs. controls in the presence of 10^–6 to 10^–4 M Ach) when pretreated with 100 mM ethanol. The Ach receptor-mediated increase in PLD activity was inhibited by atropine (10^–6 M), indicating that activation of PLD occurred via muscarinic receptors. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) increased PLD activity that was effectively blocked by the PKC inhibitors calphostin C (10^–8 to 10^–6 M) and GFX (10^–8 to 10^–6 M). Ach-induced increases in PLD activity were also significantly, but incompletely, inhibited by both GFX and calphostin C. From the present data, we conclude that in tracheal smooth muscle, muscarinic acetylcholine receptor-induced PLD activation is transient in nature and coupled to these receptors via PKC. However, PKC activation is not solely responsible for Ach-induced activation of PLD in porcine tracheal smooth muscle.     

Partially Purified RhoA-Stimulated Phospholipase D Activity Specifically Binds to Phosphatidylinositol 4,5-Bisphosphate

Abstract: Phosphatidylinositol 4,5-bisphosphate (PIP₂) is absolutely required for the ADP-ribosylation factor-stimulated phospholipase D (PLD) activity. In the present study, partially purified rat brain PLD was found to be activated by another PLD activator, RhoA, when PIP₂, but not other acidic phospholipids, was included in vesicles comprising phosphatidylethanolamine (PE) and the PLD substrate phosphatidylcholine (PC) (PE/PC vesicles), demonstrating the absolute requirement of PIP₂ for the RhoA-stimulated PLD activation, too. It is interesting that the RhoA-dependent PLD activity in the partially purified preparation was drastically decreased after the preparation was incubated with and separated from PE/PC vesicles containing PIP₂. The PLD activity was extracted by higher concentrations of NaCl from the vesicles containing PIP₂ that were incubated with and then separated from the partially purified PLD preparation. These results demonstrate that RhoA-dependent PLD binds to PE/PC vesicles with PIP₂. The degree of binding of the RhoA-dependent PLD activity to the vesicles was totally dependent on the amount of PIP₂ in the vesicles and correlated well with the extent of the enzyme activation. Furthermore, it was found that a recombinant peptide of the pleckstrin homology domain of β-adrenergic receptor kinase fused to glutathione S-transferase, which specifically binds to PIP₂, inhibited the PIP₂-stimulated, RhoA-dependent PLD activity in a concentration-dependent manner. From these results, it is concluded that in vitro rat brain PLD translocates to the vesicles containing PIP₂, owing to its specific interaction with PIP₂, to access its substrate PC, thereby catalyzing the hydrolysis of PC. PLD appears to localize exclusively on plasma membranes of cells and tissues. An aminoglycoside, neomycin, that has high affinity for PIP₂ effectively extracted the RhoA-dependent PLD activity from rat brain membranes. This indicates that PIP₂ serves as an anchor to localize PLD on plasma membranes in vivo.     

Kinetic Analysis in Mixed Micelles of Partially Purified Rat Brain Phospholipase D Activity and its Activation by Phosphatidylinositol 4,5-Bisphosphate

A partially purified rat brain membrane phospholipase D (PLD) activity was characterized in a mixed micellar system consisting of l-palmitoyl-2-[6-N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)-amino]caproyl-phosphatidylcholine (NBD-PC) and Triton X-100, under conditions where Triton X-100 has a surface dilution effect on PLD activity and the catalytic rate is dependent on the surface concentration (expressed in terms of molar ratio) of NBD-PC. PLD activity was specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP₂), and the curve of activation versus PIP₂ molar ratio fitted a Michaelis-Menten equation with a K_act value between molar ratios of 0.001–0.002. Maximal activation was observed at a PIP₂ molar ratio of 0.01. Similar values were obtained when activities of partially purified PLD as well as membrane-bound PLD were determined towards pure NBD-PC micelles. In the mixed micellar system PIP₂ was shown to elevate by 6–22 fold the specificity constant of PLD towards NBD-PC (K_A, which is proportional to V_max/K_m). Kinetic analysis of PLD trans-phosphatidylation activity towards ethanol, 1-propanol and 1-butanol revealed a Michaelis-Menten type dependence on alcohol concentration up to 1000, 200 and 80 mM, respectively. While V_max values were similar towards all three alcohols, enzyme affinity increased as the alcohol was longer, and K_m values for ethanol, 1-propanol and 1-butanol were 291, 75 and 16 mM (respectively). PLD specificity constants (K_A) towards ethanol, 1-propanol and 1-butanol were shown to be respectively 260, 940 and 5,920 times higher than to water, the competing substrate. 1-Propanol and 1-butanol inhibited PLD activity above 400 and 100 mM, respectively. The present results indicate that partially purified PLD obeys surface dilution kinetics with regard to its phospholipid substrate PC and its cofactor PIP₂, and that in the presence of alcohols, its transphosphatidylation activity may be analyzed as a competitive reaction to the hydrolysis reaction.