Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PHOSPHATIDES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources
PubMed1985

Mg2-dependent phosphatidate phosphohydrolase of rat lung: development of an assay employing a defined chemical substrate which reflects the phosphohydrolase activity measured using membrane-bound substrate.

An assay of pulmonary phosphatidate phosphohydrolase activity has been developed that employs a chemically defined liposome substrate of equimolar phosphatidate and phosphatidylcholine. Enzyme assays employing this substrate resolved two distinct activities based upon their requirements for Mg2+. Assays were performed in the presence and absence of 2 mM MgCl2 and the Mg2+-dependent phosphatidate phosphohydrolase activity calculated by difference. The Mg2+-independent phosphatase activity resembled that found using aqueous dispersions of phosphatidate (PAaq). Approximately 90% of the Mg2+-dependent phosphatidate phosphohydrolase activity was recovered in the cytosol and the remainder was associated with the microsomal fraction. The Mg2+-dependent phosphatidate phosphohydrolase activity has kinetic parameters of Km = 55 microM, Vmax = 1.6 nmol/min/mg protein for the microsomal fraction, and Km = 215 microM, Vmax = 6.8 nmol/min/mg protein for the cytosolic fraction. These parameters resembled those found using the microsomal membrane-bound (PAmb) substrate. In addition, the pH optima and sensitivity to detergents and thermal inactivation are equal to those for the PAmb-dependent phosphatidate phosphohydrolase activity. In the course of these studies the microsomal and cytosolic activities were qualitatively equal, indicative of a single enzyme in two subcellular locations. In conclusion, the assay of Mg2+-dependent phosphatidate phosphohydrolase activity measured using equimolar phosphatidate and phosphatidylcholine liposomes is equivalent to that activity previously described using microsomal membrane-bound substrate. However, the chemically-defined system provides a more simplified starting point for further studies on this important enzyme.

Animals↗
PubMed1984

Glycerolipid biosynthesis in rat adipose tissue 12. Properties of Mg2+-dependent and -independent phosphatidate phosphohydrolase.

The properties of Mg2+-dependent and Mg2+-independent phosphatidate phosphohydrolase activities were investigated in different subcellular fractions in rat adipose tissue. Phosphatidate phosphohydrolase activity was measured in the presence of aqueous dispersed phosphatidate as substrate, and the release of inorganic phosphate was taken as a measure of phosphatidate phosphohydrolase activity. The Mg2+-dependent phosphatidate phosphohydrolase was inhibited in the presence of N-methyl- or N-ethylmaleimide, whereas the Mg2+-independent activity was unaffected by these agents. The Mg2+-dependent phosphatidate phosphohydrolase was more sensitive to proteolysis and to high temperature (55 degrees C) compared to the Mg2+-independent enzyme. The Mg2+-dependent phosphatidate phosphohydrolase activity was reduced significantly during aging without any appreciable effects on the Mg2+-independent phosphatidate phosphohydrolase activity. These studies demonstrate that, in addition to Mg2+-dependency, these two forms of phosphatidate phosphohydrolases differ in several respects irrespective of their location in the adipose cell.

Adipose Tissue↗
PubMed1984

Developmental changes in molecular species of phosphatidic acid in rat lung and liver during the perinatal stage.

A new and sensitive method for the determination of the molecular species of phosphatidic acid was developed. The developmental profiles of the structural heterogeneity of phosphatidic acid in rat lung and liver are presented. The individual molecular species of phosphatidic acid at different stages of development were analyzed as [14C]dimethylphosphatidates. The content of phosphatidic acid gradually increased from 20 to 40 nmol/g wet tissue during development of both lung and liver. The relative distribution of the disaturated species of lung phosphatidic acid accounted for 10.1 +/- 2.4% at -2 days of gestation and slightly decreased at -1 day and 1 day after birth (8.4 +/- 1.1%), then increased significantly to the adult level of 14.6 +/- 3.2%. The developmental pattern of the disaturated species of phosphatidic acid was not similar to that of diacylglycerol or phosphatidylcholine. On the other hand, the relative distribution of the disaturated species of liver phosphatidic acid did not change (4.1-6.0%) during development. The level of the disaturated species of phosphatidic acid was significantly higher in lung than in liver at any stage of development. The overall results strongly suggest that disaturated phosphatidic acid could be effectively formed via the de novo pathway in fetal as well as in adult lung.

Aging↗
PubMed1990

Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes.

Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 3-times higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C16-18) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.

Alkaline Phosphatase↗
PubMed1987

Role of acetaldehyde in ethanol-induced increase in the activity of phosphatidate phosphatase in rat liver.

The effect of ethanol on the activity of phosphatidate phosphatase was studied in rat liver using membrane-bound phosphatidate and phosphatidate emulsion as substrate. A single large dose of ethanol (5 g/kg body wt) caused an increase in the enzyme activity measured with membrane-bound phosphatidate after an approximate 2-hr lag period in both cytosolic and microsomal fraction and the increase was approximately 2.2- and 1.8-fold that in control rats at 5 hr in cytosol and microsomes, respectively. A similar time-course of the increase was obtained with phosphatidate emulsion as substrate. These ethanol-induced increases in the activity of cytosolic and microsomal phosphatidate phosphatase were blocked by the pretreatment of rats with actinomycin D. The ethanol-induced rise in the activity of cytosolic and microsomal phosphatidate phosphatase measured with membrane-bound phosphatidate was abolished when rats were injected with pyrazole prior to ethanol administration. On the other hand, pretreatment with cyanamide enhanced the increase in cytosolic activity produced by a suboptimal dose of ethanol (1 g/kg), while microsomal activity was not affected by the same treatment, suggesting that acetaldehyde may be selectively involved in the ethanol-induced increase in the activity of cytosolic phosphatidate phosphatase. This hypothesis was supported by a finding that administration of paraldehyde, a cyclic trimer of acetaldehyde, produced an increase (35%) in cytosolic activity, but not in microsomal activity.

Acetaldehyde↗
PubMed1982

Regulation of phosphatidate synthesis by secretagogues in parotid acinar cells.

The metabolism of phosphatidate in rat parotid acinar cells was investigated, particularly with regard to the actions of agonists known to act by mobilizing Ca2+. When cells were incubated in medium containing 10 microM-[32P]Pi, phosphatidate was rapidly labelled, approaching an apparent steady-state with a half-time of approx. 20 min. Methacholine provoked a more than doubling of phosphatidate radioactivity, which was reversed by the muscarinic antagonist atropine. These results suggest that phosphatidate labels to near steady-state rapidly and that in cells prelabelled for 60 min the increase in radioactivity induced by agonists probably reflects net synthesis rather than an increase in specific radioactivity. Phosphatidate synthesis in response to methacholine was rapid and occurred, within the resolution of a few seconds, with no measurable latency. Adrenaline and substance P also stimulated phosphatidate synthesis but both agonists were less efficacious than methacholine. A Ca2+ ionophore, ionomycin, did not provoke phosphatidate synthesis. By using a protocol that eliminates the receptor-regulated Ca2+ pool, it was demonstrated that methacholine-induced phosphatidate formation does not come about as a consequence of Ca2+ influx nor of Ca2+ release. These results indicate that the phosphatidate synthesis response has characteristics compatible with its previously suggested role as a primary mediator of membrane Ca2+-gating.

Animals↗
PubMed1989

The effects of Triton X-100 and chlorpromazine on the Mg2+-dependent and Mg2+-independent phosphatidate phosphohydrolase activities of rat lung.

Lung contains both Mg2+-dependent and Mg2+-independent phosphatidate phosphohydrolase activities. Addition of Triton X-100 (0.5%) or chlorpromazine (1 mM) leads to a marked increase in the total phosphatidate phosphohydrolase activity in rat lung microsomes (microsomal fractions), but a decrease in the Mg2+-dependent activity. These observations suggest that the Mg2+-independent activity is stimulated, whereas the Mg2+-dependent activity is inhibited. However, the possibility exists that Triton X-100 could stimulate the Mg2+-dependent enzymic activity in an Mg2+-independent manner. In addition, the positively charged amphiphilic drug could be replacing the enzyme's requirement for Mg2+. These two possibilities were examined by using subcellular fractions in which the Mg2+-dependent phosphatidate phosphohydrolase had been abolished by heat treatment at 55 degrees C for 15 min. Heat treatment does not affect the microsomal Mg2+-independent phosphohydrolase to any great extent. Since the 6-8-fold stimulations due to Triton X-100 and chlorpromazine are retained after heat treatment of this fraction, the Mg2+-independent activity must be involved. Addition of Triton X-100 and chlorpromazine to cytosol virtually abolishes the Mg2+-dependent phosphatidate phosphohydrolase activity and decreases the Mg2+-independent activity by half. Heat treatment also abolishes the Mg2+-dependent activity and decreases the Mg2+-independent activity by over half. The Mg2+-independent phosphatidate phosphohydrolase activity remaining after heat treatment was not affected by Triton X-100 or chlorpromazine. These studies demonstrate that Triton X-100 and chlorpromazine specifically stimulate the heat-stable Mg2+-independent phosphatidate phosphohydrolase activity in rat lung microsomes. In contrast, the heat-labile Mg2+-independent phosphatidate phosphohydrolase activities in cytosol are inhibited by these reagents. Triton X-100 and chlorpromazine inhibit the Mg2+-dependent phosphatidate phosphohydrolase activities in both rat lung microsomes and cytosol. These results are consistent with the view that a single Mg2+-dependent phosphatidate phosphohydrolase present in both microsomes and cytosol is specifically involved in glycerolipid metabolism.

Animals↗
PubMed1993

Plasma membrane form of phosphatidate phosphohydrolase: a possible role in signal transduction during liver fibrogenesis.

1. Several growth factors important in liver regeneration and fibrosis stimulate phospholipase D in plasma membranes via a receptor/G-protein-coupled mechanism resulting in hydrolysis of phosphatidylcholine to phosphatidate. Phosphatidate can be further hydrolysed to diacylglycerol by phosphatidate phosphohydrolase. Phosphatidate and diacylglycerol can act as 'second-messengers' and regulation of phosphatidate phosphohydrolase activity could control the balance between them. 2. A form of phosphatidate phosphohydrolase, located in the plasma membrane and insensitive to inhibition by N-ethylmaleimide, has recently been identified that is distinct from the 'metabolic' form, which is present in the cytosol and microsomes and is sensitive to N-ethylmaleimide. 3. We have investigated the hypothesis that the balance between regeneration and fibrosis is, in part, determined by the activity of plasma membrane phosphatidate phosphohydrolase through its effect on the phosphatidate/diacylglycerol ratio. N-Ethylmaleimide-insensitive and -sensitive phosphatidate phosphohydrolase activities were measured in three hepatic conditions characterized by regeneration and/or fibrosis: alcoholic liver disease in humans (regeneration and fibrosis) and rat livers after either acute CCl-4-induced injury (regeneration) or common bile duct ligation (fibrosis). 4. In patients with alcoholic liver disease, N-ethylmaleimide-insensitive phosphatidate phosphohydrolase activity was higher in cirrhotic biopsies (5.82 +/- 0.3 nmol of Pi min-1 mg-1 of protein, n = 19) than in non-cirrhotic biopsies (2.17 +/- 0.2, n = 23) or in wedge biopsies from healthy subjects undergoing routine cholecystectomy (2.16 +/- 0.5, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Phosphatidic acid has a potential to promote hair growth in vitro and in vivo, and activates mitogen-activated protein kinase/extracellular signal-regulated kinase kinase in hair epithelial cells.

Phospholipids have recently been discovered to play an important role in cellular regulation. In this study, we focused on phosphatidic acid and lysophosphatidic acid, which are phospholipids known to possess growth-hormonal effects on several types of cells, and examined their growth-promoting effects on murine hair epithelial cells. We discovered that phosphatidic acid possesses intensive growth-promotional effects on hair epithelial cells and epidermal keratinocytes. In contrast, lyso-phosphatidic acid showed lower growth-promoting effects on hair epithelial cells relative to phosphatidic acid and showed minimal or no growth-promoting activity on epidermal keratinocytes. Phosphatidic acid was also shown to have hair-growing activity to induce the anagen phase of the hair cycle in the in vivo murine model. For the purpose of examining the hair-growing mechanisms of phosphatidic acid, we examined its relationship to the mitogen-activated protein kinase cascade linked to cell proliferation and the transforming growth factor beta signal pathway known to be a regulator of catagen induction. We confirmed that phosphatidic acid activates MEK-1/2 and upregulates the expression of MEK-1/2 in cultured murine hair epithelial cells. Addition of transforming growth factor beta1 to hair epithelial cell cultures concentration-dependently decreased cell growth and induced apoptosis; however, addition of phosphatidic acid to the culture neutralized the growth-inhibiting effects of transforming growth factor beta1 and protected the cells from apoptosis. We speculate that the hair-growing activity of phosphatidic acid is at least linked to its growth-promoting effects on hair epithelial cells that follow mitogen-activated protein kinase/extracellular signal-regulated kinase kinase activation and its protective action on transforming-growth-factor-beta1-induced apoptosis that is assumed to trigger catagen induction in the hair cycle.

Animals↗
PubMed2003

Regulators of G-protein signaling (RGS) 4, insertion into model membranes and inhibition of activity by phosphatidic acid.

Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a G alpha(i1)-[gamma-(32)P]GTP single turnover assay with an IC(50) approximately 4 microm and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into m1 acetylcholine receptor-G alpha(q) vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC(50) of RGS4 was increased from 1.5 to 7 nm. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys(20) to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.

DNA Primers↗
PubMed1997

Selective stimulation of a cAMP-specific phosphodiesterase (PDE4A5) isoform by phosphatidic acid molecular species endogenously formed in rat thymocytes.

We have previously reported that concanavalin A (ConA) stimulation of rat thymocytes induces an increase in the cellular phosphatidic acid mass as well as a change in its fatty acid composition. An increase in phosphodiesterase (PDE) activity, mostly due to cAMP-specific (PDE4) isoforms, has also been observed in thymocytes stimulated by ConA. Furthermore, phosphatidic acid was able to stimulate PDE4 activity in vitro. In the present study, cAMP levels have been shown to decrease upon ConA stimulation of thymocytes. Decreasing phosphatidic acid level using diacylglycerol kinase inhibitors induced a parallel decrease of the ConA-stimulated cAMP-specific PDE activity in these cells. Analyses of phosphatidic acid molecular species in cells stimulated for 5 min by ConA revealed a significant increase in 1-stearoyl-2-arachidonoyl-sn-glycerol-3-phosphate and a relative decrease in the other molecular species of phosphatidic acid, mainly species containing palmitate. On the other hand, phosphatidic acid extracted from ConA-stimulated cells activated more efficiently the recombinant PDE4A5 isoform in vitro, as compared to phosphatidic acid extracted from unstimulated cells. In addition, phosphatidic acid species containing unsaturated fatty acids were stimulatory, while those containing two saturated fatty acids had only a marginal effect on the enzyme activity. Taken together, these data suggest that the mitogenic stimulation of thymocytes is accompanied by the synthesis of peculiar phosphatidic acid molecular species able to activate a PDE4 isoform. This activation might be of physiological relevance since cAMP is a major negative effector of the mitogenic response.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Mannosylation of endogenous and exogenous phosphatidic acid by liver microsomal membranes. Formation of phosphatidylmannose.

Hamster liver post-nuclear membranes catalyze the transfer of mannose from GDP-mannose to endogenous dolichyl phosphate and to a second major endogenous acidic lipid. This mannolipid was believed to be synthesized from endogenous retinyl phosphate and was tentatively identified as retinyl phosphate mannose (Ret-P-Man) (De Luca, L. M., Brugh, M. R. Silverman-Jones, C. S. and Shidoji, Y. (1982) Biochem. J. 208, 159-170). To characterize this endogenous mannolipid in more detail, we isolated and purified the mannolipid from incubations containing hamster liver membranes and GDP-[14C]mannose and compared its properties to those of authentic Ret-P-Man. We found that the endogenous mannolipid was separable from authentic Ret-P-Man on a Mono Q anion exchange column, did not exhibit the absorbance spectrum characteristic of a retinol moiety, and was stable to mild acid under conditions which cleave authentic Ret-P-Man. The endogenous mannolipid was sensitive to mild base hydrolysis and mannose was released from the mannolipid by snake venom phosphodiesterase digestion. These properties were consistent with the endogenous acceptor being phosphatidic acid. Addition of exogenous phosphatidic acid, but not phospholipids with a head group blocking the phosphate moiety, to incubations containing hamster liver membranes and GDP-[14C]mannose resulted in the synthesis of a mannolipid with chromatographic and physical properties identical to the endogenous mannolipid. A double-labeled mannolipid was synthesized in incubations containing hamster liver membranes, GDP-[14C]mannose, and [3H]phosphatidic acid. Mannosyl transfer to exogenous phosphatidic acid was saturable with increasing concentrations of phosphatidic acid and GDP-mannose and specific for glycosyl transfer from GDP-mannose. Class E Thy-1-negative mutant mouse lymphoma cell membranes, which are defective in dolichyl phosphate mannose synthesis, also fail to transfer mannose from GDP-mannose to exogenous phosphatidic acid or retinyl phosphate. Amphomycin, an inhibitor of dolichyl phosphate mannose synthesis, blocked mannosyl transfer to the endogenous lipid, and to exogenous retinyl phosphate and phosphatidic acid. We conclude that the same mannosyltransferase responsible for dolichyl phosphate mannose synthesis can also utilize in vitro exogenous retinyl phosphate and phosphatidic acid as well as endogenous phosphatidic acid as mannosyl acceptors.

Animals↗
PubMed1981

Stimulation of phosphatidic acid of calcium influx and cyclic GMP synthesis in neuroblastoma cells.

Phosphatidic acid added to the medium markedly elevated intracellular cyclic GMP content in cultured neuroblastoma N1E 115 cells. There was a significant elevation of cyclic GMP with 1 micrograms/ml and a maximum (70-fold) elevation with 100 micrograms/ml of phosphatidic acid. Other natural phospholipids did not increase, or increased only slightly, the cyclic GMP content in the cells. The elevation of cyclic GMP content by phosphatidic acid was absolutely dependent on extracellular calcium. Phosphatidic acid stimulated the influx of calcium into neuroblastoma cells 2- to 5-fold. The pattern of the calcium influx induced by phosphatidic acid was comparable to that of cyclic GMP elevation. The stimulation of calcium influx by phosphatidic acid was also observed in cultured heart cells, indicating that phosphatidic acid acts as a calcium ionophore or opens a specific calcium-gate in a variety of cell membranes. Treatment of neuroblastoma cells with phospholipase C increased 32Pi labeling of phosphatidic acid, stimulated the influx of calcium, and elevated the cyclic GMP content in the cells. Thus exogenous as well as endogenous phosphatidic acid stimulates the translocation of calcium across cell membranes and, as a consequence, induces the synthesis of cyclic GMP in the neuroblastoma cells.

Animals↗
PubMed1980

Rapid acylation and deacylation of arachidonic acid into phosphatidic acid of horse neutrophils.

Horse neutrophils incorporate exogenous [14C]arachidonate into phosphatidic acid very rapidly. This acylation of phosphatidate with arachidonate is followed quickly and spontaneously by its deacylation. This transient formation of arachidonyl-phosphatidate, which reflects a rapidly turning over pool of arachidonate-associated lipid, is not observed with stearic acid or other phospholipids or triglycerides. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinosinositol, and triglycerides are slowly but increasingly labeled with time. Ionophore A23187 (10 microM) stimulates the extent of labeling of phosphatidate while decreasing the labeling of all the other phospholipids and triglycerides. Phosphatidate is not transiently labeled with [14C]stearate of (32P)orthophosphate, either in the presence or absence of ionophore A23187. When cells are prelabeled for 2 h with very high quantities of (32P)orthophosphate a very substantial fraction (i.e. 20 to 30%) of the phospholipid radioactivity is associated with phosphatidic acid. However, on addition of exogenous arachidonate, there is no increase in [32P]phosphatidate in these prelabeled cells. Thus, the entire phosphatidate molecule does not appear to be turned over during the process described above. Inhibitors of cyclooxygenase and lipoxygenase activities such as BW755C, nordihydroguaiaretic acid, and low concentrations of indomethacin do not affect the labeling of phospholipids. However, eicosatetraynoic acid, an analog of arachidonate, and high concentration (0.1 mM) of indomethacin can block [14C]arachidonate incorporation into lipids. The rapid turnover of the 2-acyl position in phosphatidate might be related to a specific process of fatty acid mobilization within neutrophils.

Acylation↗
PubMed1973

Glycerolipid synthesis in rat adipose tissue. II. Properties and distribution of phosphatidate phosphatase.

The properties and subcellular distribution of phosphatidate phosphatase (EC 3.1.3.4) from adipose tissue have been investigated. The enzyme was assayed using both aqueous phosphatidate and membrane-bound phosphatidate as substrates. When measured with aqueous substrate, activity was detected in the mitochondria, the microsomes, and the soluble fraction. Mg(2+) at low concentration stimulated the phosphatidate phosphatase from soluble and microsomal fractions but had no effect on the mitochondrial phosphatidate phosphatase. At higher concentration Mg(2+) was inhibitory. In the presence of Mg(2+), the phosphatidate phosphatase from soluble and microsomal fractions was active against membrane-bound phosphatidate. No activity was demonstrated with membrane-bound substrate in the absence of Mg(2+). Mitochondria did not contain activity toward the membrane-bound substrate. The rate of utilization of aqueous phosphatidate was always higher than that of membrane-bound substrate. These results indicate that there are at least two different phosphatidate phosphatases in adipose tissue.

Adipose Tissue↗
PubMed1992

Vanadate-sensitive phosphatidate phosphohydrolase activity in a purified rabbit kidney Na,K-ATPase preparation.

Reconstitution of purified rabbit kidney Na,K-ATPase in phosphatidylcholine/phosphatidic acid liposomes resulted in the absence of ATP in a time-, temperature- and protein-dependent formation of inorganic phosphate. This formation of inorganic phosphate could be attributed to a phosphatidate phosphohydrolase activity present in the Na,K-ATPase preparation. A close interaction of the enzyme with the substrate phosphatidic acid was important, since no or little Pi production was observed under any of the following conditions: without reconstitution, after reconstitution in the absence of phosphatidic acid, with low concentrations of detergent or at low lipid/protein ratios. The hydrolysis of phosphatidic acid was not influenced by the Na,K-ATPase inhibitor ouabain but was completely inhibited by the P-type ATPase inhibitor vanadate. Besides Pi diacylglycerol was also formed, confirming that a phosphatidate hydrolase activity was involved. Since the phosphatidate phosphohydrolase activity was rather heat- and N-ethylmaleimide-insensitive, we conclude that the phosphatidic acid hydrolysis was not due to Na,K-ATPase itself but to a membrane-bound phosphatidate phosphohydrolase, present as an impurity in the purified rabbit kidney Na,K-ATPase preparations.

Animals↗
PubMed1994

Cell-permeable ceramides inhibit the stimulation of DNA synthesis and phospholipase D activity by phosphatidate and lysophosphatidate in rat fibroblasts.

The interactions of ceramides with phosphatidate and lysophosphatidate in the regulation of signal transduction in rat fibroblasts were examined. C2- and C6-ceramides (N-acetylsphingosine and N-hexanoylsphingosine, respectively) at 10 microM inhibited the stimulation of DNA synthesis that was produced by 50-100 microM phosphatidate, or lysophosphatidate, or by exogenous phospholipase D. Sphingosine (10 microM) had the opposite effect to the ceramides on DNA synthesis. C2- or C6-ceramides failed to inhibit the stimulation of DNA synthesis by insulin or serum. The ceramides did not modify the actions of phosphatidate, or lysophosphatidate, in decreasing the forskolin-induced increase in cAMP. C2- and C6-ceramides inhibited the stimulation of phospholipase D activity by: (a) phosphatidate, lysophosphatidate, phorbol ester, thrombin, or serum in intact fibroblasts and (b) phorbol ester or guanosine 5'-3-O-(thio)triphosphate in permeabilized fibroblasts. The ceramides can therefore modify cell signaling via phospholipase D, but this effect alone could not explain the decreased DNA synthesis. Incubation of fibroblasts with C2- or C6-ceramides or sphingomyelinase inhibited the interaction of exogenous phosphatidate or lysophosphatidate with the fibroblasts by 42 and 53%, respectively. Furthermore, a greater proportion of the phosphatidate, or lysophosphatidate, that was associated with the fibroblasts was metabolized further when the cells were pretreated with ceramides or sphingomyelinase. This effect was accompanied by an increased activity of N-ethylmaleimide-insensitive phosphatidate phosphohydrolase. Ceramides may therefore produce part of their growth inhibitory effects by blocking some of the signal transducing effects of phosphatidate and lysophosphatidate.

Animals↗
PubMed1976

Biosynthesis of molecular species of CDP-diglyceride from endogenously-labeled phosphatidate in rat liver microsomes.

The biosynthesis of [14C] CDP-diglyceride was studied using rat liver microsomes which were endogenously labeled with [14C] phosphatidic acid by preincubation of unlabeled microsomes with sn-[14C] glycerol-3-phosphate and appropriate cofactors. The formation of CDP-diglyceride from radioactive phosphatidate showed an absolute requirement for CTP and MgCl2. The newly formed [14C] CDP-diglyceride was characterized by thin layer chromatography (TLC), isotopic labeling from radioactive CTP, and its ability to serve as substrate for the microsomal enzyme, CDP-diglyceride: inositol phosphatidyltransferase. The distributions of radioactive glycerol-3-phosphate among the various chemical classes of microsomal [14C] phosphatidate and [14C] CDP-diglyceride were determined following argentation TLC of their 1,2-diglyceride acetate derivatives. Most of the radioactivity among the phosphatidic acids was present in the monoenoic (36%) and dienoic (33%) molecular species, whereas 10, 8, 4, and 8% were associated with the saturates, trienes, tetraenes, and polyenes, respectively. Similar distributions of radioactivity were found among the corresponding classes of newly formed CDP-diglyceride. Only a slight enrichment of radioactivity in the tetraenoic CDP-diglyceride was found relative to the corresponding phosphatidates. Therefore, under the conditions of study, the microsomal CTP: phosphatidate cytidylyltransferase produces mainly monoenoic and dienoic species of CDP-diglyceride and shows little specificity towards different molecular species of phosphatidic acids. The present results suggest also that the arachidonoyl phosphatidate derived from the microsomal acylation of sn-glycerol-3-phosphate is not likely the major source of arachidonic acid in liver phosphatidylinositol.

Animals↗