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At least 19 recordsLinked to original sources
PubMed1979

Pulmonary phosphatidic acid phosphatase. A comparative study of the aqueously dispersed phosphatidate-dependent and membrane-bound phosphatidate-dependent phosphatidic acid phosphatase activities of rat lung.

1. The properties of the aqueously dispersed phosphatidate-dependent phosphatidic acid phosphatase (EC 3.1.3.4) activities of rat lung have been studied in microsomal and cytosol preparations and compared with the properties of the membrane-bound phosphatidate-dependent activities. 2. The microsomal phosphatidic acid phosphatase displayed a prominent pH optimum at 6.5 with a minor peak which varied between 7.5--8 in different experiments. With the cytosol, the major activity was at the higher pH (7.5--8.0) but a distinct optimum was also observed at pH 6.0--6.5. With the membrane-bound substrate, a single broad optimum was observed between pH 7.4 and 8.0 with the cytosol and 6.5--7.5 with the microsomal fraction. 3. Subcellular fractionation studies revealed that the microsomal fraction possessed the greatest proportion of the total phosphatidic acid phosphatase activity and the highest relative specific activity. However, studies with marker enzymes indicated that the aqueously dispersed phosphatidate-dependent activity could be present in plasma membrane, lysosomes and osmiophilic lamellar bodies as well as in the endoplasmic reticulum. 4. The aqueously dispersed phosphatidic acid-dependent activities present in the microsomal and supernatant fractions were inhibited by Ca2+, Mn2+, F- and by high concentrations of Mg2+. In contrast to the membrane-bound phosphatidate-dependent activities, there was little Mg2+ stimulation and only a very slight inhibitory effect was noted with EDTA. A small EDTA-dependent Mg2+ stimulation could be observed with the microsomal fraction but only at the lower pH optimum (6.5). 5. The presence of a number of phosphate esters tended to stimulate rather than inhibit the microsomal activity, indicating that the hydrolase is relatively specific for lipid substrates. Marked inhibitions were noted with lysophosphatidic acid and phosphatidylglycerol phosphate. Phosphatidylcholine produced a slight inhibition. 6. The results indicate that the bulk of the aqueously dispersed phosphatidate-dependent phosphatidic acid phosphatase activities of rat lung microsomes and cytosol is not related to the activities observed with membrane-bound phosphatidate. The Mg2+-dependent hydrolase activities may be synonymous. However, unequivocal conclusions will only be possible when the polypeptide or polypeptides responsible for these activities can be purified.

Animals↗
PubMed1978

The influence of exogenous and of membrane-bound phosphatidate concentration on the activity of CTP: phosphatidate cytidylyltransferase and phosphatidate phosphohydrolase.

Rat liver microsomes were treated with phospholipase D to obtain microsomal membranes with varying amounts of membrane-bound phosphatidate. This treatment did not impair the activity of two microsomal-bound enzymes acting with phosphatidate as substrate, i.e. CTP: phosphatidate cytidylyltransferase and phosphatidate phosphohydrolase. The dependency of the activity of these enzymes on the concentration of membrane-bound phosphatidate was determined. Both enzymes showed a linear increase in activity with membrane-bound phosphatidate concentrations up to at least 100 nmol phosphatidate/mg microsomal protein. These results indicate that both enzymes have a large reserve capacity and suggest that the enzymes are operating intracellularly, i.e. at phosphatidate concentrations of 5-10 nmol/mg endoplasmic reticulum protein, far below their maximal capacity. The ratio of phosphatidate conversion into CDP-diglyceride and 1,2-diglyceride seems to be constant for a large range of membrane-bound phosphatidate concentrations. The membrane-bound enzymes cannot utilize phosphatidate substrate present in heat-denatured membranes, but are active on phosphatidate incorporated into membranes of phospholipid vesicles.

Animals↗
PubMed1979

Pulmonary phosphatidic acid phosphatase. Properties of membrane-bound phosphatidate-dependent phosphatidic acid phosphatase in rat lung.

1. The membrane-bound phosphatidate-dependent phosphatidic acid phosphatase activity of rat lung has been investigated in cytosol and microsomal fractions using as a substrate [32P]phosphatidate bound to heat inactivated rat liver microsomes. Both activities demonstrated broad pH optima with a maximum of 7.4--8 for the cytosol and a maximum of 6.5--7.5 with microsomal preparations. 2. At low concentrations (0--5 mM) Mg2+ produced a slight stimulation of the cytosol activity but at higher concentrations an inhibition was observed. Low concentrations (1.0--2.0 mM) of EDTA abolished the cytosol activity and reduced the microsomal activity to half. In both cases, the addition of Mg2+ in the presence of EDTA resulted in an activity which was more than 2-fold greater than that observed in the absence of chelator or divalent cation. 3. The cytosol activity was relatively resistant to the addition of ionic and nonionic detergents. In general, the addition of a number of phosphate esters increased rather than decreased the release of 32Pi, indicating a relative specificity for phosphate groups associated with a hydrophobic environment. The addition of aqueous dispersions of phosphatidate, lysophosphatidic acid or phosphatidylglycerophosphate markedly reduced the hydrolysis of membrane-bound [32P]phosphatidate. The cytosol activity was slightly inhibited by the addition of phosphatidylcholine. 4. In an attempt to estimate the relative contributions of the cytosol and microsomal activities in vivo, these activities were assayed using [32P]phosphatidate endogenously generated on rat lung microsomes. With the 32P-labelled microsomes, the hydrolysis remained linear over the 45 min of the experiment. Addition of high speed supernatant produced a rapid release of 32Pi during the first 10 min followed by a more gradual release similar to that oberved with the microsomes alone. The cytosol activity remained greater than the microsomal activity at all times studied. 5. When [14C]phosphatidate-labelled microsomes were incubated in the presence of nonradioactive CDPcholine, the addition of cytosol markedly stimulated the incorporation of radioactivity into phosphatidylcholine. This observation suggests that the phosphatidic acid phosphatase activity associated with the cytosol has a role in phosphatidylcholine (and presumably surfactant) biosynthesis in rat lung.

Animals↗
PubMed1985

Phosphatidate phosphatase in rat liver: the relationship between the activities with membrane-bound phosphatidate and aqueous dispersion of phosphatidate.

Gel filtration of rat liver cytosol on Bio-Gel A-5m resolved the phosphatase activities into four peaks, all of which showed activity with either phosphatidase bound to microsomal membrane (PAmb) or phosphatidate dispersed in sonicated microsomal lipid (PAaq) as the substrate. A major part of the PAmb phosphatase activity (52%) was eluted in a peak with an apparent molecular weight (Mr) of 500,000 where the PAaq phosphatase activity was very low. A major PAaq phosphatase activity peak (48%) was obtained in the void volume (Vo), where the PAaq phosphatase activity was higher than the PAmb phosphatase activity. The addition of 0.075% Tween 20 to the elution buffer gave only the 500 kilodalton (kDa) peak. When the activity in the Vo peak obtained in the absence of Tween 20 was rechromatographed in the presence of the detergent, a part of the activity was dissociated into 500 kDa molecules having a preference for PAmb. These results suggest that the enzymes obtained in the Vo peak are formed by the association of the 500 kDa molecules with macromolecules and that the substrate preference of phosphatidate phosphatase is modified by the change in the physical state of the enzyme. The microsomal phosphatidate phosphatase activities were also separated on Bio-Gel A-5m after solubilizing by sonication. Most of both the PAmb and PAaq phosphatase activities were coeluted in the Vo peak, in which the latter activity was higher than the former. When the gel filtration was performed in the presence of Tween 20, a major activity peak with a preference for PAmb was obtained at the elution volume corresponding to apparent Mr 500,000, indicating a potential relationship between the cytosolic and microsomal activities.

Animals↗
PubMed1960

Studies on the carrier function of phosphatidic acid in sodium transport. I. The turnover of phosphatidic acid and phosphoinositide in the avian salt gland on stimulation of secretion.

Incubation of slices of the salt gland of the albatross with acetylcholine, which is the physiological secretogogue for this tissue, led to a 13-fold increase in the rate of incorporation of P(32) into phosphatidic acid and a 3-fold increase in the incorporation of P(32) and inositol-2-H(3) into phosphoinositide. The incorporation of P(32) into phosphatidyl choline and phosphatidyl ethanolamine was increased relatively slightly or not at all. Respiration was doubled. The "phospholipid effect" occurred in the microsome fraction, which is known to contain fragments of the endoplasmic reticulum. The enzymes, diglyceride kinase and phosphatidic acid phosphatase, which catalyze the stimulated turnover of phosphatidic acid in brain cortex, were also found in highest concentration in the microsome fraction. The phosphatides which respond to acetylcholine are bound to protein in the membrane. On the basis of these findings it appears that phosphatidic acid and possibly phosphoinositide participate in sodium transport. A scheme, termed the phosphatidic acid cycle, is presented as a working hypothesis, in which the turnover of phosphatidic acid in the membrane, catalyzed by diglyceride kinase and phosphatidic acid phosphatase, functions as a sodium pump.

Acetylcholine↗
PubMed1992

Stimulation of phosphatidate synthesis in endothelial cells in response to P2-receptor activation. Evidence for phospholipase C and phospholipase D involvement, phosphatidate and diacylglycerol interconversion and the role of protein kinase C.

To investigate the stimulation of phosphatidic acid formation in bovine aortic endothelial cells by P2-purinergic agonists, we labelled AG4762 cells with [32P]P1 and stimulated in the presence of butanol. Under these conditions phospholipase D generated [32P]phosphatidylbutanol, whereas the [32P]phosphatidic acid from phospholipase C and diacylglycerol kinase was unchanged. The action of various purinergic agonists on both [32P]phosphatidic acid and [32P]phosphatidylbutanol was consistent with the presence of a P2Y receptor. The stimulation of phospholipase D was dependent on extracellular Ca2+ and was mostly transient (completed within 3 min), whereas the initial stimulation of phospholipase C was independent of extracellular Ca2+, followed by a Ca(2+)-dependent phase. The agonist stimulation of phospholipase D was dependent on protein kinase C, as judged by its sensitivity to the relatively selective protein kinase C inhibitor Ro 31-8220. These results show that purinergic-receptor-mediated stimulation of phosphatidic acid has three phases: an initial Ca(2+)-independent stimulation of phospholipase C, an early but transient Ca(2+)- and protein kinase C-dependent stimulation of phospholipase D, and a sustained Ca(2+)-dependent stimulation of phospholipase C. Using propranolol to inhibit phosphatidate phosphohydrolase, we provide evidence that phosphatidic acid derived from purinergic-receptor-mediated stimulation of the phospholipase C/diacylglycerol kinase route can itself be converted back into diacylglycerol.

Adenosine Triphosphate↗
PubMed1977

Factors controlling the activities of phosphatidate phosphohydrolase and phosphatidate cytidylyltransferase. The effects of chlorpromazine, demethylimipramine, cinchocaine, norfenfluramine, mepyramine and magnesium ions.

1. Microsomal membranes from rat liver were incubated with ATP, CoA, Mg2+, [14C]palmitate, F- and sn-glycerol 3-phosphate in order to label them with [14C]phosphatidate. These membranes were isolated and used in a second incubation in which [3H]CTP was present, and the simultaneous synthesis of [14C]diacylglycerol and [3H]CDP-diacylglycerol was measured. 2. The addition of phosphatidate phosphohydrolase, which had been partially purified from the particle-free supernatant, supplemented the activity of the endogenous phosphohydrolase, but it did not alter the rate of CDP-diacylglycerol formation. 3. Adding EDTA inhibited phosphatidate cytidylyl-transferase activity and stimulated the activity of the phosphohydrolases by removing excess of Mg2+. 4. Increasing the concentration of Mg2+, norfenfluramine or chlorpromazine in the assay system stimulated cytidylyltransferase activity, but decreased the activities of both phosphohydrolases. 5. The mechanism for the stimulation of cytidylyl=transferase activity by the cationic drugs and Mg2+ was investigated with emulsions of phosphatidate and the microsomal fraction of rat liver. 6. There was a threshold concentration of about 5mM-MgCl2 below which no cytidylyltransferase activity was detected in the presence or absence of norfenfluramine. Just above this threshold concentration norfenfluramine stimulated cytidylyltransferase activity, but this stimulation disappeared as the Mg2+ concentration was raised to its optimum of 20mM. Norfenfluramine therefore partially replaced the bivalent-cation requirement. 7. At 30 mM-MgCl2 amphiphilic cationic drugs inhibited cytidylyltransferase activity at relatively high concentrations in a non-competitive manner with respect to phosphatidate. 8. The implications of these results are discussed with respect to the regulation of the synthesis of the acidic phospholipids compared with the synthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol.

Animals↗
PubMed2003

Functional analysis of a phosphatidic acid binding domain in human Raf-1 kinase: mutations in the phosphatidate binding domain lead to tail and trunk abnormalities in developing zebrafish embryos.

Previously, we and others identified a 35-amino acid segment within human Raf-1 kinase that preferentially binds phosphatidic acid. The presence of phosphatidic acid was found to be necessary for the translocation of Raf-1 to the plasma membrane. We have now employed a combination of alanine-scanning and deletion mutagenesis to identify the critical amino acid residues in Raf-1 necessary for interaction with phosphatidic acid. Progressive mutations within a tetrapeptide motif (residues 398-401 of human Raf-1) reduced and finally eliminated binding of Raf-1 to phosphatidic acid. We then injected zebrafish embryos with RNA encoding wild-type Raf-1 kinase or a mutant version with triple alanine mutations in the tetrapeptide motif and followed the morphological fate of embryonic development. Embryos with mutant but not wild-type Raf-1 exhibited defects in posterior axis formation exemplified by bent trunk and tail structures. Molecular evidence for lack of signaling through mutated Raf-1 was obtained by aberrant in situ hybridization of the ntl (no tail) gene, which functions downstream of Raf-1. Our results demonstrate that a functional phosphatidate binding site is necessary for Raf-1 function in embryonic development.

Alanine↗
PubMed1999

Activation of human neutrophil NADPH oxidase by phosphatidic acid or diacylglycerol in a cell-free system. Activity of diacylglycerol is dependent on its conversion to phosphatidic acid.

The superoxide-generating neutrophil NADPH oxidase can be activated in cell-free reconstitution systems by several agonists, most notably arachidonic acid and the detergent sodium dodecyl sulfate. In this study, we show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system. Stimulation of superoxide generation by these lipids was dependent upon both Mg(2+) and agonist concentration. Activation of NADPH oxidase by phosphatidic acids did not appear to require their conversion to corresponding diacylglycerols by phosphatidate phosphohydrolase, since diacylglycerols were much slower than phosphatidic acids to activate the system and required the presence of ATP. Stimulation of the oxidase by dioctanoylglycerol proved to be by a means other than the activation of protein kinase C. Instead, dioctanoylglycerol was converted to dioctanoylphosphatidic acid by an endogenous diacylglycerol kinase present in the cell-free reaction system. This conversion was sensitive to the diacylglycerol kinase inhibitor R59949 and explains the markedly slower kinetics of activation and the novel ATP requirement seen with dioctanoylglycerol. The level of dioctanoylphosphatidic acid formed was suboptimal for NADPH oxidase activation but could synergize with the unmetabolized dioctanoylglycerol to activate superoxide generation.

Cell-Free System↗
PubMed1975

[Fat-stabilizing properties of phosphatides. 1. Response of selected phosphatides to oxygen].

The authors investigated the formation of hydroperoxides and carbonyl compounds in raw phosphatides from soya-beans and rape-seeds during storage at 50 degrees C. According to the results obtained, the chemical deterioration of the raw phosphatides depends upon their intrinsic contents of prooxidative and antioxidative compounds and upon the consituent fatty acids. Purified preparations are considerably more stable as comparable fatty acid methyl esters; and phosphatides containing nitrogenous bases are less sensitive to atmospheric oxygen than their nitrogen-free analogues.

Antioxidants↗
PubMed1981

Pulmonary phosphatidic acid phosphohydrolase: further studies on the activities in rat lung responsible for the hydrolysis of membrane-bound and aqueously dispersed phosphatidate.

Rat lung cytosol and microsomal fractions both contain phosphohydrolase activity towards membrane-bound phosphatidic acid (PAmb) and aqueously dispersed phosphatidic acid (PAaq) which cannot be explained through contamination with the other fraction. The phosphohydrolase activities with PAaq demonstrated Km and Vmax values which were more than an order of magnitude greater than those observed with PAmb and with vesicles prepared from the lipids extracted from [32P]PA-labelled microsomes. The PAaq-dependent activities in both fractions were stimulated by preparing mixed liposomes with phosphatidylcholine. The PAmb-dependent activities in rat lung microsomes and cytosol were markedly stimulated by high concentrations of Triton X-100 and Nonidet P-40. The PAmb- and PAaq-dependent activities in the microsomes were stimulated by deoxycholate. Although no difference was observed in the inhibition profiles of the PAmb- and PAaq-dependent activities of the cytosol in the presence of various mercurials, the PAmb-dependent activity in the microsomes was somewhat more susceptible than the PAaq-dependent activity. The PAmb-dependent activities in both fractions were more susceptible to inhibition by iodoacetamide. These results support the view that separate rat lung enzymes were involved in the hydrolysis of PAmb and PAaq. The relative abilities of rat lung cytosol and microsomes to hydrolyse PA endogenously generated on the microsomes were compared using relative concentrations of cytosol corresponding to the levels in intact rat lung. During the initial period (5-10 min) the cytosol phosphohydrolase activity was more effective than the microsomal activity. At later stages (10-20 min), the rates were comparable.

Animals↗
PubMed1996

Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells.

Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., and Bell, R. M. (1994) J. Biol. Chem. 269, 10000-10007). Further analysis showed that full-length Raf-1 bound to both phosphatidylserine and phosphatidic acid (PA). Specifically, a carboxyl-terminal domain of Raf-1 kinase (RafC; residues 295 648 of human Raf-1) interacted strongly with phosphatidic acid. The binding of RafC to PA displayed positive cooperativity with Hill numbers between 3.3 and 6.2; the apparent Kd ranged from 4.9 +/- 0.6 to 7.8 +/- 0.9 mol % PA. The interaction of RafC with PA displayed a pH dependence distinct from the interaction between the cysteine-rich domain of Raf-1 and PA. Also, the RafC-PA interaction was unaffected at high ionic strength. Of all the lipids tested, only PA and cardiolipin exhibited high affinity binding; other acidic lipids were either ineffective or weakly effective. By deletion mutagenesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphatidyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the translocation of Raf-1 from the cytosol to the membrane following stimulation with 12-O-tetradecanoylphorbol-13-acetate. These results suggest a potential role of the lipid second messenger, PA, in the regulation of translocation and subsequent activation of Raf-1 in vivo.

Amino Acid Sequence↗
PubMed1976

[Fat stabilizing action of phosphatides. Part 3. Synergistic action of various phosphatides].

Phosphatides do not regnerate tocopherol worn with supplying oxygen; their synergistic action seems to be a reaction competing with that of hydroperoxide with tocopherol and, consequently, it must be regarded as "tocopherol-saving". Phosphatide mixtures act to some extent as metal capturers. Their effects are functions of the heavy metal contents of the substrates and of their contents of specific metallic pro-oxidants and must be attributed to more complex carbohydrate-containing compounds; phosphatidyl choline and ethanolamine are not able to form metal complexes.

Copper↗
PubMed1989

Interactions of thiophosphatidic acid with enzymes which metabolize phosphatidic acid. Inhibition of phosphatidic acid phosphatase and utilization by CDP-diacylglycerol synthase.

Thiophosphatidic acid (1,2-diacyl-sn-glycero-3-phosphorothioate; thioPA) was chemically synthesized from egg phosphatidylcholine-derived 1,2-diacylglycerol and PSCl3 and tested for its effects on enzymes which utilize phosphatidic acid (PA) in phospholipid biosynthesis. The compound was not a substrate for rat liver cytosolic PA phosphatase and strongly inhibited this enzyme activity. ThioPA was also a potent inhibitor of purified membrane-associated PA phosphatase from Saccharomyces cerevisiae in a competitive manner and exhibited an apparent Ki = 60 microM. In contrast, purified CDPdiacylglycerol synthase (PA:CTP cytidylyltransferase) from this organism was able to convert thioPA to CDP-diacylglycerol. The apparent Vmax for thioPA was 7-fold lower than that for PA, whereas the apparent Km for thioPA (70 microM) was 4-fold lower than that for PA. Calculation of the specificity constant (Vmax/Km) demonstrated that PA was the preferred substrate. These properties of thioPA indicate that this substance may prove useful in studies of phospholipid metabolism and function.

Animals↗
PubMed1989

Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide-stimulated human neutrophils. Involvement of phosphatidate phosphohydrolase in signal transduction.

Human neutrophils have been labeled in 1-O-alkyl-phosphatidylcholine (alkyl-PC) with 32P by incubation with alkyl-[32P]lysoPC. Upon stimulation with the chemotactic peptide, formylMet-Leu-Phe (fMLP), these 32P-labeled cells produce 1-O-alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) and, in the presence of ethanol, 1-O-alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Because the cellular ATP contains no 32P, alkyl-[32P]PA and alkyl-[32P]PEt must be formed from alkyl-[32P]PC by phospholipase D (PLD)-catalyzed hydrolysis and transphosphatidylation, respectively. Analyses of the sn-1 bonds by selective hydrolysis and mass measurements reveal that the PA and PEt formed during stimulation contain both ester and ether bonds with distributions similar to that in the endogenous PC. Furthermore, in neutrophils labeled in alkyl-[32P]PC, the specific activities of the diradyl-PA and diradyl-PEt formed during stimulation are similar to that of diradyl-PC. These results demonstrate that the fMLP-induced PLD utilizes diradyl-PC as the major substrate. It is further concluded that, at early times (30 s), PA and PEt are both formed almost exclusively by PLD. Following stimulation with fMLP, neutrophils double-labeled in alkyl-PC by incubation with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC generate [3H]alkyl-DG and [32P]orthophosphate [( 32P]PO4) with superimposable kinetics, indicating degradation of PA by a phosphohydrolase. Generation of [3H]alkyl-DG and [32P]PO4 lags behind PA formation and parallels the decline in PA accumulation. In addition, generation of both [3H]alkyl-PA and [3H]alkyl-DG requires extracellular Ca2+ and cytochalasin B. Furthermore, the phosphohydrolase inhibitor, propranolol, decreases both [3H]alkyl-DG and [32P]PO4 while increasing [3H]alkyl-PA and not altering [3H]alkyl-PEt. Moreover, the decreases in DG are accounted for by increases in PA. These results demonstrate that PLD-derived alkyl-PA is degraded by a phosphohydrolase to produce alkyl-DG. DG formed during stimulation contains both ester and ether-linked species and this DG formation is inhibited completely by propranolol. Upon stimulation, alkyl-[32P]PC-labeled neutrophils do not produce [32P]phosphocholine, suggesting that PC is not hydrolyzed by phospholipase C. In addition, PA is formed in amounts sufficient to account for all of the DG formed during stimulation. It is concluded that the DG formed during fMLP stimulation is derived almost exclusively from PC via the PLD/PA phosphohydrolase pathway.

Diglycerides↗
PubMed1993

A novel ecto-phosphatidic acid phosphohydrolase activity mediates activation of neutrophil superoxide generation by exogenous phosphatidic acid.

Phosphatidic acid (PA) added to intact cells activates a variety of processes including mitogenesis in fibroblasts and superoxide generation in neutrophils. We have investigated the mechanism of activation of superoxide generation in intact human neutrophils by a short-chain (dioctanoyl) PA (diC8PA). After a lag, diC8PA caused a high rate of superoxide production (19.6 nmol of cytochrome c reduced/min/10(6) cells). Activation did not require extracellular Ca2+ and coincided with near quantitative conversion of diC8PA to dioctanoylglycerol (diC8-glycerol). diC8PA also activated cellular phospholipase D with release of long-chain PA and secondary production of long-chain diradylglycerol (sn-1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol). The metabolism of diC8PA to diC8-glycerol was catalyzed by a novel PA phosphohydrolase on the outer leaflet of the plasma membrane as demonstrated by the exclusive release of Pi into the extracellular medium. This enzyme also showed activity toward PA containing long-chain unsaturated fatty acids. The ecto-PA phosphohydrolase differed from the intracellular PA phosphohydrolase based on its relative insensitivity to desipramine and N-ethylmaleimide. The enzyme was also present in Chinese hamster ovary (CHO) cells and its activity did not change in transfected CHO cells expressing the two membrane-associated isoforms of alkaline phosphatase, indicating that the PA phosphohydrolase was not alkaline phosphatase. Non-hydrolyzable phosphonate analogs of diC8PA poorly stimulated superoxide production. Activation of superoxide generation by diC8PA was inhibited by staurosporine, suggesting a protein kinase C-dependent mechanism. We suggest that the action of a novel ecto-PA phosphohydrolase permits exogenously added short-chain PA to serve as "timed-release diacylglycerol" and that its biological effects in neutrophils are secondary to diacylglycerol-mediated protein kinase C activation.

Animals↗
PubMed1996

Identification and cDNA cloning of 35-kDa phosphatidic acid phosphatase (type 2) bound to plasma membranes. Polymerase chain reaction amplification of mouse H2O2-inducible hic53 clone yielded the cDNA encoding phosphatidic acid phosphatase.

We previously described the purification of an 83-kDa phosphatidic acid phosphatase (PAP) from the porcine thymus membranes (Kanoh, H., Imai, S.-i., Yamada, K. and Sakane, F.(1992) J. Biol. Chem. 267, 25309-25314). However, we found that a minor 35-kDa protein could account for the PAP activity when the purified enzyme preparation was further analyzed. We thus determined the N-terminal sequence of the 35-kDa candidate protein and prepared antipeptide antibody against the determined sequence, MFDKTRLPYVALDVL. The antibody almost completely precipitated the purified enzyme activity. Furthermore, the antibody precipitated from the radioiodinated enzyme preparation a single 35-kDa protein, which was converted to a 29-kDa form when treated with N-glycanase. We also found that the immunoprecipitable PAP activity was exclusively associated with the plasma membranes of porcine thymocytes. These results indicated that the 35-kDa glycosylated protein represents the plasma membrane-bound (type 2) PAP. We surprisingly noted that the N-terminal sequence of the porcine PAP was almost completely conserved in the internal sequence encoded by a mouse partial cDNA clone, hic53, reported as a H2O2-inducible gene (Egawa, K., Yoshiwara, M., Shibanuma, M., and Nose, K.(1995) FEBS Lett. 372, 74-77). We thus amplified from the mouse kidney RNA the hic53 clone by polymerase chain reaction, and obtained a cDNA encoding a novel protein of 283 amino acid residues with a calculated Mr of 31,894. Methionine reported as an internal residue was found to serve as an initiator, and the C-terminal 64 residues were lacking in hic53. The protein contains several putative membrane-spanning domains and two N-glycosylation sites. When transfected into 293 cells, the cDNA gave more than 10-fold increase of the membrane-bound PAP activity, which could be precipitated by the antipeptide antibody. In [35S]methionine-labeled cells, the translational product was confirmed to be a 35-kDa protein, which became 30 kDa in cells treated with tunicamycin, an inhibitor of N-glycosylation. We thus succeeded first in identifying the porcine type 2 PAP and subsequently in determining the primary structure of a mouse homolog of the PAP.

Amino Acid Sequence↗