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PubMed1979

Stimulation of phosphatidic acid production in platelets precedes the formation of arachidonate and parallels the release of serotonin.

Thrombin rapidly induces the formation of labeled phosphatidic acid from platelets prelabeled with [17C]arachidonate or 32PO34- and specifically decreases by 50--75% the content of phosphatidylinositol. Ionophore A23187 also stimulates phosphatidate labeling, but less effectively than thrombin. This effect on phosphatidic acid is blocked by increasing the levels of cyclic AMP by preincubation with dibutyryl cyclic AMP, cyclic AMP-phosphodiesterase inhibitors or prostacyclin. Indomethacin and eicosatetraynoic acid do not alter the production of phosphatidate, indicating independence from cyclooxygenase or lipoxygenase products. Increased turnover of [14C]- or [32P]phosphatidate occurs within 2--5 s after platelet activation by thrombin and is observed before endogenous, 14C-labeled arachidonate can be detected. The rate of phosphatidate formation parallels the induced rate of serotonin release. Release of [3H]serotonin is not affected by eicosatetraynoic acid. Phosphatidate production reflects the generation of diacylglycerol by C-type phospholipase degradation of phosphatidylinositol. Diacylglycerol and phosphatidic acid may participate in the membrane modification related to the early changes in platelet shape, release reactions or aggregation which occur on stimulation.

Animals↗
PubMed1983

Glycerolipid biosynthesis in rat adipose tissue. 11. Effects of polyamines on Mg2+-dependent phosphatidate phosphohydrolase.

The effect of polyamines (spermine, spermidine and putrescine) on the Mg2+-dependent phosphatidate phosphohydrolase was investigated. 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. In the presence of various polyamines there was activation of the Mg2+-dependent phosphatidate phosphohydrolase activity. Under this condition, the Km of enzyme towards phosphatidase decreased from 1.6 x 10(-4) to 9.8 x 10(-5) M and the Mg2+ requirement decreased from 5 to 0.5 mM. These polyvalent cations did not replace Mg2+, but potentiate the phosphohydrolase activity in the presence of Mg2+. The activation of Mg2+-dependent phosphatidate phosphohydrolase activity by polyamines was observed in the presence of 3-sn-phosphatidylcholine, suggesting that these modulators of phosphatidate phosphohydrolase activity may be acting through different mechanisms. These studies demonstrate that polyamines may be important regulators of Mg2+-dependent phosphatidate phosphohydrolase activity in adipose tissue.

Adipose Tissue↗
PubMed1984

The role of Mg2+-dependent phosphatidate phosphohydrolase in pulmonary glycerolipid biosynthesis.

Rat lung microsomes washed with increasing concentrations of NaCl show a displacement of protein from microsomes to the wash supernatant. Among the proteins removed from the microsomal surface was the Mg2+-dependent phosphatidate phosphohydrolase, while the Mg2+-independent activity remained associated with the microsomes. The Mg2+-dependent activity could be quantitatively assayed in the wash supernatant. Microsomes washed with increasing concentrations of NaCl showed a progressive impairment in the synthesis of labelled neutral lipid and phosphatidylcholine from [14C]glycerol 3-phosphate with a concomitant increase in the labelling of phosphatidic acid. The impairment was sigmoidal and correlated highly with the decrease in Mg2+-dependent phosphatidate phosphohydrolase activity. When Mg2+-dependent phosphatidate phosphohydrolase from wash supernatant was incubated with microsomes previously washed with high salt concentrations, the labelling of neutral lipid and phosphatidylcholine was returned to control levels. Labelling of neutral lipids and phosphatidylcholine could be restored upon addition of a cytosolic Mg2+-dependent phosphatidate phosphohydrolase isolated by gel filtration. Mg2+-independent phosphatidate phosphohydrolase isolated from cytosol was incapable of restoring the labelling of neutral lipids and phosphatidylcholine. These findings confirm that the Mg2+-dependent phosphatidate phosphohydrolase of rat lung is involved in pulmonary glycerolipid biosynthesis. The role of the Mg2+-independent phosphatidate phosphohydrolase activity remains unknown.

Animals↗
PubMed1987

A distinction in vitro between rat liver phosphatidate phosphatase and phospholipase C.

Hepatocellular membranes (1000 X g) containing membrane-associated, labeled phosphatidic acid were incubated (1-30 min) with 2 mM oleate or 5 mM bromobenzene in the presence or absence of various metals and NaF. Under the appropriate incubation conditions, membranes displayed rapid and significant oleate- and bromobenzene-dependent increases in the dephosphorylation of labeled phosphatidic acid. However, oleate and bromobenzene activated the dephosphorylation of phosphatidate by phosphatidate phosphatase and phospholipase C, respectively. This conclusion is supported by the observation that the phosphatase stimulated by oleate is: (1) Mg2+ -dependent; (2) inhibited by other metals, such as Ca2+; (3) inhibited by NaF; (4) specific for phosphatidic acid; and (5) associated with a rise in liver cell triacylglycerol production. Bromobenzene, however, activated a phospholipase C that is: (1) stimulated by various metals, such as Mg2+, Ca2+ and Ba2+; (2) insensitive to NaF; (3) associated with the degradation of various membrane phospholipids; (4), associated with liver cell injury; and (5) not associated with a rise in liver cell triacylglycerol formation. These results suggest that under appropriate conditions in vitro the dephosphorylation of phosphatidic acid can be used to assess changes in phosphatidate phosphatase and/or phospholipase C activity. The distinction between these enzymes is important, since phosphatidate phosphatase and phospholipase C regulate key steps in phospholipid biosynthesis and degradation, respectively.

Animals↗
PubMed1988

The formation of phosphatidic acid de novo: a comparison of activities in neuronal nuclei and microsomes isolated from immature rabbit cerebral cortex.

The formation of phosphatidic acid from sn-glycerol 3-phosphate was studied in neuronal nuclear fraction N1 and a microsomal fraction P3, isolated from cerebral cortices of 15-day-old rabbits. Two assays were used, employing dithiothreitol, MgCl2, NaF and (A) sn-glycerol 3-phosphate, [14C]oleate, ATP and CoA or (B) sn-[3H]glycerol 3-phosphate and oleoyl-CoA. In both assays fraction N1 had specific rates of phosphatidic acid labelling (expressed per mumol phospholipid in the fraction) which were 5- to 6-times the corresponding values for P3. In contrast to N1, the formation of phosphatidic acid by fraction P3 was more sensitive to inhibition at high concentrations of oleoyl-CoA and was greatly dependent upon the presence of NaF. In the absence of this salt, P3 showed decreased phosphatidate formation and increased levels of radioactive monoacylglycerols. Using cerebral cortex, rough (R) and smooth (S) microsomal fractions were prepared, as was a microsomal fraction P from isolated nerve cell bodies. P had specific rates of phosphatidic acid labelling which were 2-3 times the values for P3, but were about 50% of the N1 values. This indicates a concentration of phosphatidate synthesis in the nucleus within the nerve cell. Specific rates for fraction R were higher and were similar to those of N1. In S, P3 and R the specific rates of phosphatidic acid synthesis paralleled specific RNA contents and indicated a location for phosphatidic acid synthesis within the rough endoplasmic reticulum.

Animals↗
PubMed2003

Modulation of membrane curvature by phosphatidic acid and lysophosphatidic acid.

The local generation of phosphatidic acid plays a key role in the regulation of intracellular membrane transport through mechanisms which are largely unknown. Phosphatidic acid may recruit and activate downstream effectors, or change the biophysical properties of the membrane and directly induce membrane bending and/or destabilization. To evaluate these possibilities, we determined the phase properties of phosphatidic acid and lysophosphatidic acid at physiological conditions of pH and ion concentrations. In single-lipid systems, unsaturated phosphatidic acid behaved as a cylindrical, bilayer-preferring lipid at cytosolic conditions (37 degrees C, pH 7.2, 0.5 mM free Mg2+), but acquired a type-II shape at typical intra-Golgi conditions, a mildly acidic pH and submillimolar free Ca2+ (pH 6.6-5.9, 0.3 mM Ca2+). Lysophosphatidic acid formed type-I lipid micelles in the absence of divalent cations, but anhydrous cation-lysophosphatidic acid bilayer complexes in their presence. These data suggest a similar molecular shape for phosphatidic acid and lysophosphatidic acid at cytosolic conditions; however, experiments in mixed-lipid systems indicate that their shape is not identical. Lysophosphatidic acid stabilized the bilayer phase of unsaturated phosphatidylethanolamine, while the opposite effect was observed in the presence of phosphatidic acid. These results support the hypothesis that a conversion of lysophosphatidic acid into phosphatidic acid by endophilin or BARS (50 kDa brefeldin A ribosylated substrate) may induce negative spontaneous monolayer curvature and regulate endocytic and Golgi membrane fission. Alternative models for the regulation of membrane fission based on the strong dependence of the molecular shape of (lyso)phosphatidic acid on pH and divalent cations are also discussed.

Calcium Chloride↗
PubMed1983

Phosphatidic acid and phosphatidylinositol labelling in adipose tissue. Relationship to the metabolic effects of insulin and insulin-like agents.

Exposure to phospholipase C increased the incorporation of [32P]Pi into phosphatidate, CMP-phosphatidate and phosphatidylinositol in rat adipose tissue and isolated adipocytes. A similar effect was observed in response to insulin and oxytocin. Theophylline, 3-isobutyl-1-methylxanthine and adenosine deaminase decreased [32P]Pi incorporation, and adenosine and N6-phenylisopropyladenosine reversed these effects. As with insulin, exposure of adipose tissue to phospholipase C stimulated oxidation of glucose, pyruvate and leucine and activated pyruvate dehydrogenase. Oxytocin and adenosine also mimicked the effects of insulin on leucine oxidation and pyruvate dehydrogenase. However, only insulin stimulated glycogen synthase activity, indicating that the regulation of synthase may be achieved by intracellular events distinct from those regulating changes in phospholipid metabolism, sugar transport and mitochondrial enzyme activities. It is postulated that exposure to phospholipase C forms diacylglycerol, which is phosphorylated to yield phosphatidate. The increased labelling of CMP-phosphatidate and phosphatidylinositol results from the conversion of phosphatidate into these lipids. The correlation between the effects of phospholipase C on phosphatidate synthesis and changes in adipose-tissue metabolism suggests the possibility that increased phosphatidate may directly or indirectly produce changes in membrane transport and enzyme activities. The pattern of phospholipid labelling produced by insulin, adenosine and oxytocin suggests that these stimuli may also increase phosphatidate synthesis, and, if so, changes in phospholipid metabolism could account for some of the metabolic actions of these stimuli.

Adenosine↗
PubMed1987

Phosphatidic acid and arachidonic acid each interact synergistically with glucagon to stimulate Ca2+ influx in the perfused rat liver.

The administration of phosphatidic acid to rat livers perfused with media containing either 1.3 mM- or 10 microM-Ca2+ was followed by a stimulation of Ca2+ efflux, O2 uptake and glucose output. The responses elicited by 100 microM-phosphatidic acid were similar to those induced by the alpha-adrenergic agonist phenylephrine. Contrary to suggestions that phosphatidic acid acts like a Ca2+-ionophore, no net influx of Ca2+ was detected until the phosphatidic acid was removed. Sequential infusions of phenylephrine and phosphatidic acid indicate that the two agents release Ca2+ from the same intracellular source. The co-administration of glucagon (or cyclic AMP) and phosphatidic acid, and also of glucagon and arachidonic acid, led to a synergistic stimulation of Ca2+ uptake of the liver, a feature similar to that observed after the co-administration of glucagon and other Ca2+-mobilizing hormones [Altin & Bygrave (1986) Biochem. J. 238, 653-661]. A notable difference, however, is that the synergistic stimulation of Ca2+ uptake induced by the co-administration of glucagon and arachidonic acid was inhibited by indomethacin, whereas that induced by glucagon and phosphatidic acid, or glucagon and other Ca2+-mobilizing agents, was not. The results suggest that the synergistic action of glucagon and arachidonic acid in stimulating Ca2+ influx is mediated by prostanoids, but that of glucagon and phosphatidic acid is evoked by a mechanism similar to that of Ca2+-mobilizing agents.

Animals↗
PubMed1976

Phosphatidic acid metabolism in rat liver microsomes.

Rat liver microsomes contain phosphatidate phosphatases which split phosphatidic acid into inorganic phosphate and diacylglycerol and a system of phospholipases and lipases, which split phosphatidic acid into free fatty acids, glycerol and inorganic phosphate. In the presence of ATP,CoA and [1-14C]palmitate, part of the monoacyl-sn-glycerol 3-phosphate formed by phospholipase action is reesterified, yielding radioactive phosphatidic acid. The sum of di- and triacylglycerols formed from phosphatidic acid in the presence of ATP and CoA exceeded the amount of diacylglycerol formed in their absence. The yield of neutral lipids from sn-glycerol 3-phosphate and monoacyl-sn-glycerol 3-phosphate markedly exceeded that from phosphatidic acid. Comparison of the yields of di- and triacylglcerols from glycerol-labelled and fatty-acid-labelled phosphatidic acid was used to establish the extent of deacylation and reacylation. About 60% of the diacylglycerol was formed by direct dephosphorylation. The triacylglycerols, on the other hand, were formed almost exclusively from recycled phosphatidic acid.

Adenosine Triphosphate↗
PubMed1982

Kinetics of phosphatidylglycerol synthesis in isolated membrane vesicles from Escherichia coli containing different amounts of membrane-bound phosphatidic acid.

Cytoplasmic membrane vesicles of Escherichia coli containing various amounts of phosphatidic acid ranging from 0.2% to more than 50% (mol/mol) of the total lipid has been prepared by de novo synthesis of phosphatidic acid in the isolated membranes from acyl-CoA esters and sn-glycerol 3-phosphate. The kinetics of CTP-initiated phosphatidylglycerol synthesis in the phosphatidic-acid-enriched membranes has been studied. Phosphatidic acid pools with high and low reactivity as substrate for phosphatidylglycerol synthesis were present in the membranes. The two pools were found identical with respect to fatty acid composition and content of molecular species. The rate of phosphatidylglycerol synthesis from the highly reactive phosphatidic acid pool was independent of the phosphatidic acid concentration in membranes containing from 0.2-30% (mol/mol) phosphatidic acid. Ca2+-ions were found to inhibit the synthesis of phosphatidylglycerol. On the basis of the findings presented it is suggested that phosphatidic acid probably plays a minor role as a feed-back modulator of sn-glycerol 3-phosphate acylation in E. coli, and that phosphatidylglycerol synthesis can occur at near maximal rate in growing cells.

Binding Sites↗
PubMed1983

Accumulation of phosphatidic acid in microsomes from propranolol-treated retinas during short-term incubations.

The pool size and synthesis of phosphatidic acid derived from [2-3H]glycerol were studied in bovine whole retinas and subcellular fractions. Microsomal preparations from retinas incubated with [2-3H]glycerol displayed the highest percentage labeling of phosphatidic acid at 5 min of incubation: labeling decreased rapidly thereafter. In drug-treated retinas, 0.5 mM propranolol increased the endogenous content of phosphatidic acid and stimulated [2-3H]glycerol labeling in whole retina and microsomal and postmicrosomal supernatant fractions. This effect was observed during short-term incubations and was reversible. In pulse-chase experiments, 60 min of reincubation greatly reduced the labeling effect, although propranolol still enhanced phosphatidic acid labeling. At the same time, endogenous phosphatidic acid accumulated, and reincubation without propranolol reversed the effect. During accumulation, the amount of palmitate increased and that of oleate decreased, whereas the relatively high level of docosahexaenoate in phosphatidic acid remained unchanged. It was concluded that this propranolol-induced effect is due to cationic amphiphilic drug activity in the endoplasmic reticulum that results in a partial inhibition of phosphatidic acid degradation and a stimulation of its de novo synthesis. Hence, net synthesis of phosphatidic acid can be assessed in the retina during short-term incubation with propranolol.

Animals↗
PubMed1993

Phosphatidic acid stimulates inositol 1,4,5-trisphosphate production in adult cardiac myocytes.

The cellular content of phosphatidic acid can increase in response to several agonists either by phosphorylation of diacylglycerol after phospholipase C-catalyzed hydrolysis of phospholipids or directly through activation of phospholipase D. Although previous findings indicated that the generation of phosphatidic acid was exclusively a means of regulation of the cellular concentration of diacylglycerol, more recent studies have indicated that phosphatidic acid may also directly regulate several cellular functions. Accordingly, the present study was performed to assess whether phosphatidic acid could stimulate cardiac phospholipase C in intact adult rabbit ventricular myocytes. The mass of inositol 1,4,5-trisphosphate [Ins (1,4,5)P3] was determined by a specific and sensitive binding protein assay and by direct mass measurement using anion exchange chromatography for separation of selected inositol phosphates and gas chromatography and mass spectrometry for quantification of inositol monophosphate (IP1), inositol bisphosphate (IP2), inositol trisphosphate (IP3), and inositol tetrakisphosphate (IP4). Phosphatidic acid (10(-9)-10(-6) M) elicited a rapid concentration-dependent increase in Ins (1,4,5)P3 accumulation, with the peak fourfold to fivefold increase at 30 seconds of stimulation; the concentration required for 50% of maximal stimulation was 4.4 x 10(-8) M. The time course of individual inositol phosphates indicated a successive increase in the mass of IP3, IP4, IP2, and IP1 in response to stimulation with phosphatidic acid. The production of Ins (1,4,5)P3 in response to phosphatidic acid was not altered in the absence of extracellular calcium or in the presence of extracellular EGTA (10(-3) M). Thus, these findings indicate that phosphatidic acid is a potent activator of inositol phosphate production in adult ventricular myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Effects of phosphatidic acid on islet cell phosphoinositide hydrolysis, Ca2+, and adenylate cyclase.

Phosphatidic acid may be raised in glucose-stimulated islet cells through hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and de novo synthesis with glucose-derived trioses. The mechanism by which exogenous phosphatidic acid from egg yolk lecithin may augment insulin secretion was investigated in neonatal beta-cells. In whole cells labeled with [2,8-3H]-adenine, a dose-dependent increase in phosphatidic acid-stimulated adenylate cyclase activity was seen, and a small intracellular transient free-Ca2+ rise was seen in Fura 2AM-loaded cells. In [gamma-32P]ATP-labeled membranes from those beta-cells, phosphatidic acid effected PIP2 hydrolysis. These phosphatidic acid-stimulated effects were not sensitive to preincubation with Bordetella pertussis exotoxin. The findings are consistent with a stimulatory effect of exogenous phosphatidic acid on insulin release and indicate an effect at the plasma membrane. It is possible that newly synthesized phosphatidic acid may function similarly to amplify intracellular events in glucose-stimulated islet cells through both local Ca2+ concentration and cyclic AMP-sensitive mechanisms. The participation of newly synthesized phosphatidic acid derived from glucose could provide a link between the metabolism of glucose and insulin release.

Adenylyl Cyclases↗
PubMed1982

Platelet-activating factor stimulates the phosphatidylinositol cycle. Appearance of phosphatidic acid is associated with the release of serotonin in horse platelets.

The addition of platelet-activating factor to horse platelets prelabeled with (32P)orthophosphate induces the rapid formation of [32P]phosphatidic acid. This is subsequently followed by a considerable increase in the labeling of phosphatidylinositol. Activation of the formation of phosphatidic acid by platelet-activating factor is evident at a concentration of 1 nM, is maximal at 0.1 microM, and independent of the presence or absence of plasma in the medium. In horse platelets prelabeled with [14C]arachidonic acid, platelet-activating factor stimulates the rapid formation of [14C]phosphatidic acid, [14C]arachidonic acid, and [14C]arachidonate metabolites. Concomitantly, there is a loss of radioactivity from phosphatidylinostiol., phosphatidylcholine, and phosphatidylethanolamine. Platelet-activating factor at a concentration of 1 nM stimulates formation of phosphatidic acid while the appearance of arachidonate metabolites is seen at a higher concentration (10 nM), without acetate in the 2-position, is unable to induce in platelets the formation of phosphatidic acid, arachidonate metabolites, or the release of [3H]serotonin and, in addition, does not antagonize the action of platelet-activating factor. the release of [3H]serotonin and [14C]arachidonate from platelets stimulated with platelet-activating factor is not affected by indomethacin. Trifluoperazine (50-100 microM) inhibits the platelet-activating factor-stimulated liberation of arachidonic acid from phospholipids, without affecting the formation of phosphatidic acid and the release of serotonin. Prostacyclin, on the other hand, inhibits the platelet-activating factor-induced release of serotonin, arachidonate metabolites, and formation of phosphatidic acid. These data indicate a close relationship between the formation of phosphatidic acid and the release of serotonin in platelets stimulated with the platelet-activating factor.

Animals↗
PubMed1993

The regulation of phospholipase C-gamma 1 by phosphatidic acid. Assessment of kinetic parameters.

A survey of lipids revealed that the anionic phospholipid phosphatidic acid activates both control and tyrosine-phosphorylated PLC-gamma 1. The mechanism by which phosphatidic acid activates both forms of PLC-gamma 1 was investigated using kinetic analysis. In the presence of phosphatidic acid, the substrate concentration response for control PLC-gamma 1 changes from sigmoidal to hyperbolic, while the cooperativity index decreases from 2.5 for control to 1.0 for tyrosine-phosphorylated PLC-gamma 1. The primary influence of phosphatidic acid on the control enzyme is on the cooperativity index and not the association of PLC-gamma 1 with substrate micelles, as phosphatidic acid had little effect on the micellar association constant, Ks. Phosphatidic acid also increases the activity of the tyrosine phosphorylated form of the enzyme. This increase is reflected in a decrease in the Km from 0.3- to 0.03-mol fraction phosphatidylinositol 4,5-bisphosphate. Phosphatidic acid has no effect on the Ks of the tyrosine-phosphorylated enzyme. From this data it is concluded that phosphatidic acid appears to activate PLC-gamma 1 by acting as an allosteric modifier.

Allosteric Regulation↗
PubMed1989

Rapid hydrolysis of diacylglycerol formed during phosphatidate phosphatase assay by lipase activities in rat liver cytosol and microsomes.

Side reactions which may affect the determination of phosphatidate phosphatase activity were investigated in rat liver cytosol and microsomes. Incubation of these subcellular fractions with either 14C-labeled phosphatidate bound to microsomal membranes (PAmb) or that coemulsified with microsomal lipids resulted in rapid formation of water-soluble products, most of which were identified as glycerol, in addition to diacylglycerol. Neither lysophosphatidate nor glycerol 3-phosphate accumulated under any of the conditions used and only a minute amount of activity catalyzing hydrolysis of glycerol 3-phosphate could be detected in cytosol and microsomes, suggesting that glycerol was not formed by the deacylation of phosphatidate to glycerol 3-phosphate and subsequent dephosphorylation. On the other hand, pretreatment of cytosol or microsomes with diisopropylfluorophosphate abolished the formation of water-soluble products, indicating that glycerol was formed from diacylglycerol, the product of the phosphatidate phosphatase reaction, by lipase-type activities. Rapid deacylation of diacylglycerol by these subcellular fractions was also observed with an emulsion of phosphatidate, which has been purified from the total lipid extract of PAmb as substrate. The rate of hydrolysis of diacylglycerol was maximum when the concentration of diacylglycerol was less than 20 microM with either cytosol or microsomes. The present results suggest that it is essential to characterize the reaction products before employing specific assay conditions for phosphatidate phosphatase. At least under the conditions we tested, reliable measurement of the enzyme activity in rat liver cytosol and microsomes can be achieved only by determining the release of Pi or that of water-soluble activity from 32P-labeled phosphatidate.

Animals↗
PubMed1986

Phosphatidate phosphatase activity in isolated rod outer segment from bovine retina.

Phosphatidate phosphohydrolase (EC 3.1.3.4) was detected in isolated bovine rod outer segments and its properties investigated. The enzyme activity was assayed using aqueously dispersed 1,2-diacyl-sn-[2-3H]glycerol 3-phosphate as substrate. The phosphatidic acid concentration was optimal at 1 mM and the estimated Km value was 6.7 X 10(-4) M. The activity was linear for 60 min with a protein concentration of 0.3 mg. A clear pH optimum was observed at 7.5. When the enzyme activity was measured using a substrate phosphatidic acid containing 15% lyso compound, the production of diacylglycerols was inhibited by about 70-75% at all concentrations studied. In rod outer segment preparations containing 0.2 mM Mg2+, further additions of the ion (0.2-2.5 mM) only produced a slight inhibition of the activity at 2.5 mM. F- (50 mM), Ca2+ (1 mM) and EDTA (50 mM) inhibited the dephosphorylation of the substrate by 80, 10 and 70%, respectively. The aqueously dispersed phosphatidic acid-dependent activity present in rod outer segments was stimulated by Triton X-100 and taurocholate. Similar values were observed in the enzyme activity of entire rod outer segments and in that of disks obtained from them, showing the enzyme to be associated with disk membrane. The [3H]diacylglycerol production from [3H]phosphatidic acid was analyzed in synaptosomal-mitochondrial and microsomal fractions and in a crude pigment epithelium preparation. The degree of dephosphorylation of phosphatidic acid in these subcellular fractions was as follows: microsomes greater than synaptosomal-mitochondrial fraction greater than rod outer segment greater than crude pigment epithelium. The present report is the first evidence of phosphatidic acid phosphohydrolase activity associated with rod outer segment membranes.

Animals↗
PubMed1988

Comparison of the HPLC-separated species patterns of phosphatidic acid, CDP-diacylglycerol and diacylglycerol synthesized de novo in rat liver microsomes (a new method).

The species pattern of phosphatidic acid was compared with that of CDP-diacylglycerol and diacylglycerol synthesized de novo by glycerol 3-phosphate acylation in a CoA ester-generating system in liver microsomes. The similarity of the species patterns of phosphatidic acid and CDP-diacylglycerol indicated that the CTP-phosphatidyl cytidylyltransferase showed no selectivity for individual species of its phosphatidic acid substrate. Since the species pattern of diacylglycerol deviated from that of phosphatidic acid, a slight acyl selectivity of the phosphatidic acid phosphohydrolase or a slight inhomogeneity of its substrate pool might be assumed. For the determination of the molecular species of CDP-diacylglycerol, a new method was developed. By incubation of CDP-diacylglycerol with oligonucleate 5'-nucleotidohydrolase (phosphodiesterase), phosphatidic acid was produced. The CDP-diacylglycerol-derived phosphatidic acid was methylated with diazomethane and then separated by reverse-phase HPLC in 15 molecular species.

Animals↗