Phospholipase D Product Research Articles

Arcanobacterium haemolyticum phospholipase D is genetically and functionally similar to Corynebacterium pseudotuberculosis phospholipase D

Arcanobacterium haemolyticum, a pathogen of the human upper respiratory tract and other systems, has been reported to produce soluble toxins, including a phospholipase D (PLD). We confirmed production of PLD by this organism and cloned and sequenced pld. Arcanobacterial PLD (PLD-A) was found to be a protein of approximately 31.5 kDa with a pI of approximately 9.4. Cosmid cloning, followed by subcloning into phagemid pBluescriptIISK+, yielded Escherichia coli(pAh140), a recombinant with a gene product corresponding to PLD-A. Evidence of PLD activity was found by three assays in supernatant fluid of cultures of E. coli(pAh140) and A. haemolyticum, but not in E. coli(pBluescriptIISK+). Experiments to determine if this protein was secreted were not conducted, but previous work with PLD from Corynebacterium pseudotuberculosis suggested that the presence of the enzyme in culture supernatant fluids was due to lysis of E. coli rather than to active transport. Antibodies in polyclonal sera from goats immunized with native or recombinant PLD-A neutralized native and recombinant PLD-A activity, and antibodies against native or recombinant PLD from C. pseudotuberculosis (PLD-P) partially neutralized native and recombinant PLD-A. Antibodies prepared against recombinant PLD-A labelled both recombinant and native PLD-A in Western blots (immunoblots) and dot blots, but antibodies against PLD-P did not. Sequencing of the insert in pAh140 revealed an open reading frame of 930 bp coding for 309 amino acids, including a putative signal sequence of 26 amino acids (3.2 kDa, determined on the basis of homology with the 24-amino-acid signal sequence of pld from C. pseudotuberculosis bv. ovis) and the mature PLD protein (31.5 kDa). Sequence comparisons of coding regions revealed 65% DNA homology with pld genes from C. pseudotuberculosis and Corynebacterium ulcerans. Comparison of amino acid sequences revealed 64% homology of PLD-A both with PLD-P and with PLD produced by C. ulcerans.

Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.

Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.

A neutral phospholipase D activity from rat brain synaptic plasma membranes. Identification and partial characterization.

Rapid activation of phospholipase D (PLD) in response to cell stimulation was recently demonstrated in many systems, raising the hypothesis that PLD participates in transduction of extracellular signals across the plasma membrane. In the present study, we describe the identification of a neutral PLD activity in purified rat brain synaptic plasma membranes, and the in vitro conditions required to assay its catalytic activity with exogenous [3H]phosphatidylcholine as substrate. Production of [3H]phosphatidic acid, the natural lipid product of PLD and of [3H]phosphatidylethanol, catalyzed by PLD in the presence of ethanol via transphosphatidylation, were measured. The synaptic membrane PLD exhibited its highest activity at pH 7.2 and was thus defined as a neutral PLD. Enzyme activity was absolutely dependent on the presence of sodium oleate and was strongly activated by Mg2+ ions (at 1 mM). Ca2+ at concentrations up to 0.25 mM was as stimulatory as Mg2+, but at 2 mM it completely inhibited enzyme activity. Mg2+ extended the linear phase of PLD activity from 2 to 15 min, suggesting that it may stabilize the enzyme under our assay conditions. The production of [3H]phosphatidylethanol was a saturable function of ethanol concentration. Production of [3H] phosphatidic acid was inversely related to the concentration of ethanol and to the accumulation of phosphatidylethanol, indicating that the two phospholipids are indeed produced by the competing hydrolase and transferase activities of the same enzyme. beta,beta-Dimethylglutaric acid, utilized previously as a buffer in studies of rat brain PLD, inhibited enzyme activity at neutral pH but not at acidic pH. The properties of the neutral synaptic membrane PLD and its relationships with other in vitro, acid, and neutral PLD activities, as well as with the signal-dependent PLD detected in intact cells, are discussed.