A rapid sensitive assay for phosphatidate phosphohydrolase.

For a purified preparation of the soluble form of phosphatidate phosphohydrolase (EC 3.1.3.4) from guinea pig cerebral cortex, 1-O-alkyl-rac-glycerol 3-phosphate was found to be accepted as a substrate. This substrate analog was tritium-labeled in order to serve in a rapid sensitive assay for the enzyme, in which labeled 1-alkyl glycerol is released. Heat denaturation and enzyme activity dependence on pH indicated that 1-O-alkyl-rac-glycerol 3-phosphate phosphohydrolase and phosphatidate phosphohydrolase activities in the preparation are attributable to the same enzyme. 1-O-Alkyl-rac-glycerol 3-phosphate was hydrolyzed with a V_max of 1.7 nmol min^?1 mg^?1 of protein and a K_m of 270 μm.     

Stimulation and inhibition of the activity of rat liver cytosolic phosphatidate phosphohydrolase by various phospholipids.

The influence of phospholipids on the activity of the soluble phosphatidate phosphohydrolase from rat liver was studied. Phosphatidylethanolamine stimulated the enzyme activity whereas phosphatidylglycerol, phosphatidylserine, and phosphatidylinositol were inhibitory. At a phospholipid concentration of 0.7 mg/ml, phosphatidylglycerol inhibited phosphatidate phosphohydrolase activity by 75%, while the enzyme activity was stimulated twofold in the presence of phosphatidylethanolamine. Both lysophosphatidylglycerol and lysophosphatidylethanolamine inhibited phosphatidate phosphohydrolase activity as did octylglucoside, sodium cholate, and Tween 20. The finding that phospholipids influence hepatic phosphatidate phosphohydrolase activity indicates that changes in the lipid environment may modulate the enzyme activity.     

Partial purification and properties of microsomal phosphatidate phosphohydrolase from rat liver.

Microsomal phosphatidate phosphohydrolase (phosphatidate phosphatase EC 3.1.3.4) was solubilized and fractionated to yield at least two distinct enzymatically active fractions. One, denoted FA, was non-specific, had a relatively high Km for phosphatidic acid and was insensitive to inhibition by diacylglycerol. The second fraction, FB, was specific for phosphatidates, had a low Km, and was inhibited, non-competitively, by diacylglycerol. FA exhibited a sigmoid substrate-activity curve. The isolated FB aggregated to particles of about 10(6) in the absence of salts and could be dissociated by the addition of monovalent cations at ionic strength 0.4-0.6 to about 2-10(5) daltons and thereby doubled its activity. Dissociation was time- and temperature-dependent. F- was inhibitory. Divalent ions were not required for the activity of FA or FB and inhibited at concentrations exceeding 1 mM.     

The inactivation of rat adipocyte Mg2+-dependent phosphatidate phosphohydrolase by noradrenaline.

The inactivation of rat adipocyte Mg2+-dependent phosphatidate phosphohydrolase by noradrenaline [Cheng & Saggerson (1978) FEBS Lett. 87, 65--68; Cheng & Saggerson (1978) FEBS Lett. 93, 120--124] persists for at least 40 min in crude defatted homogenates kept at 0 degrees C or 20 degrees C, but is diminished at 37 degrees C. The effect of noradrenaline persists through the isolation of post-105000 g supernatants and is then stable in these preparations at 0 degrees C and 37 degrees C. Inclusion of albumin (10--20 mg/ml) in homogenization buffers abolishes the effect of noradrenaline. The effect of noradrenaline is not removed by dialysis of extracts or by raising the concentrations of Mg2+ or phosphatidate in assays.     

Purification of Mg2+-dependent phosphatidate phosphohydrolase from rat liver: new steps and aspects

A new procedure for the partial purification of Mg2+-dependent, N-ethylmaleimide-sensitive phosphatidate phosphohydrolase (Mg2+-PAP; EC 3.1.3.4) from rat liver cytosol is described, using protein precipitation with MgCl2, gel filtration on Sephacryl S-400, chromatography on DEAE-cellulose and affinity chromatography on calmodulin-agarose. From the parallel change in staining intensity and in the level of the specific activity of enzyme fractions, a relationship between a 90-kDa SDS gel band, identified as the beta-isoform of the 90-kDa heat shock protein, and Mg2+-PAP could be detected.     

A sensitive assay for phosphatidate phosphohydrolase in mouse liver microsomes

A technique is described for the assay of phosphatidate phosphohydrolase using 1,2-[9,10-3H]dioleoyl-sn-glycero-3-phosphate as a substrate. This substrate was prepared enzymatically using mouse liver microsomes washed with 0.5 M NaCl, which synthesize minimal amounts of neutral lipids at high enzyme concentrations. Measurement of the product, 1,2-[9,10-3H]dioleoylglycerol, was 10-fold more sensitive than the usual colorimetric assay for inorganic phosphate release. In addition, the assay provides information about the relative contribution of other activities which limit the availability of diacylglycerols for further esterification to triacylglycerols and/or phospholipids.     

Problems encountered in measuring the activity of phosphatidate phosphohydrolase.

The measurement of phosphate release from phosphatidate overestimates the microsomal activity of phosphatidate phosphohydrolase from rat liver, since phosphate is also produced via the glycerol phosphate that results from the deacylation of phosphatidate. The determination of phosphate production can be a reliable assay for the soluble phosphatidate phosphohydrolase in rat liver, because the glycerol phosphate formed is not hydrolysed under the conditions used.     

The effects of Mg2+ on rat liver microsomal Ca2+ sequestration

The effects of Mg2+ on the hepatic microsomal Ca2(+)-sequestering system was tested. Ca2(+)-ATPase activity and Ca2+ uptake were both dependent on the concentration of free Mg2+, reaching maximum levels at 2 mM. The effects of Mg-ATP were also influenced by the concentration of free Mg2+, being maximally effective at a ratio of 1:1. The results suggest that Mg2+ influences Ca2+ sequestration at various steps, namely in addition to forming the substrate of the Ca2(+)-ATPase reaction, Mg-ATP, Mg2+ stimulates the reaction at an additional step, as indicated by its stimulatory effect on the Ca2(+)-ATPase reaction and on Ca2+ uptake, even at optimal Mg-ATP levels. The stimulatory effect of Mg2+ was evident at various pH levels tested, and it was nucleotide specific. The stimulatory effect of Mg2+ might be exerted at the dephosphorylation step of the enzymatic reaction or at an other, yet undefined, site. The results demonstrate a plural effect of Mg2+ on the hepatic microsomal sequestration system. This indicates that, depending on its magnitude, changes in Mg2+ distribution might influence cytosolic Ca2+ levels.     

Liver cytosolic non-dialysable factor(s) can counteract GTP-dependent Ca2+ release in rat liver microsomal fractions

Readdition to rat liver microsomes of dialysed liver post-microsomal supernatant resulted in an almost complete inhibition of the Ca2+-releasing effect of GTP. Such inhibition was heat-labile, and was associated with non-ultrafiltrable supernatant components with a molecular weight higher than 30,000 D. A preliminary fractionation of liver supernatant showed that the inhibitory effect is recovered in the 40-50% ammonium sulfate-precipitated proteins, with an approx. 10-fold enrichment. The active ammonium sulfate fraction did not modify the GTP-induced Ca2+ increase of passive Ca2+ efflux from microsomes, nor did it affect microsomal GTP hydrolysis, which is likely required for its Ca2+ releasing effect. The active ammonium sulfate fraction appears to markedly favour the translocation of GTP-released Ca2+ into a microsomal GTP-insensitive pool. Separation of liver microsomes in smooth and rough fractions revealed that such GTP-insensitive Ca2+ pool is almost completely associated with smooth microsomes.     

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 °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 Mg2+-dependent phosphatidate phosphohydrolase. Control of activity and subcellular distribution in vitro and in vivo.

The subcellular distribution of Mg2+-dependent phosphatidate phosphohydrolase in rat adipocytes between a soluble and a membrane-bound fraction was measured by using both centrifugal fractionation and a novel Millipore-filtration method. The relative proportion of the phosphohydrolase associated with the particulate fraction was increased on incubation of cells with noradrenaline or palmitate. Insulin on its own decreased the proportion of the phosphohydrolase that was particulate and abolished the effect of noradrenaline, but not that of palmitate. The effect of noradrenaline on phosphohydrolase distribution was rapid, the effect being maximal within 10 min. Noradrenaline exerted this effect with a similar concentration-dependence to its lipolytic effect. Inclusion of albumin in homogenization buffers decreased the proportion of the phosphohydrolase that was particulate, but did not abolish the effect of noradrenaline. There was limited correlation between the proportion of the phosphohydrolase that was particulate and the measured rate of triacylglycerol synthesis in adipocytes incubated under a variety of conditions. Starvation, streptozotocin-diabetes and hypothyroidism decreased the specific activities of the phosphohydrolase and glycerolphosphate acyltransferase in homogenates from epididymal fat-pads. Restoration of these activities in the diabetic state was seen after administration of insulin over 2 days or, in the short term, within 2 h after a single administration of insulin. Administration of thyroxine over 3 days caused restoration of these activities in the hypothyroid state. Starvation and diabetes increased the proportion of the phosphohydrolase found in the microsomal fraction. This change was not seen when albumin was present in homogenization buffers. The possible role of fatty acids as regulators of the intracellular translocation of the phosphohydrolase, together with the role of this enzyme in the regulation of triacylglycerol synthesis in adipose tissue, is discussed.     

Translocation to rat liver mitochondria of phosphatidate phosphohydrolase.

When a particle-free supernatant fraction from rat liver was incubated at 37 degrees C with mitochondria and oleate, some of the enzyme phosphatidate phosphohydrolase (PAP), initially present in the particle-free supernatant, was recovered, after the incubation, bound to mitochondria. This translocation of PAP from cytosol to mitochondria was stimulated by oleate or palmitate in a similar fashion to the stimulation of translocation of PAP to endoplasmic reticulum [Martin-Sanz, Hopewell & Brindley (1984) FEBS Lett. 175, 284-288]. Translocation of PAP from particle-free supernatant to a partially purified mitochondrial-outer-membrane preparation was also stimulated by oleate. More PAP was bound to a mitochondrial-outer-membrane fraction washed in 0.5 M-NaCl before resuspension in sucrose than to a sucrose-washed mitochondrial-outer-membrane preparation. In contrast, washing of microsomal membranes in 0.5 M-NaCl did not enhance the binding of PAP to these membranes. PAP also binds to phosphatidate-loaded mitochondria or microsomes (microsomal fractions). In the experimental system employed, more PAP bound to mitochondria loaded with phosphatidate than to microsomes loaded with phosphatidate. The results are discussed in relation to the role of mitochondrial phosphatidate in liver lipid metabolism.     

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.     

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.     

Long-chain fatty acids and their acyl-CoA esters cause the translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction of rat liver

A translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction was promoted in cell-free extracts of rat liver by oleate and palmitate and their CoA esters. Oleate was more potent in this respect than palmitate and the CoA esters were more effective than the unesterified acids. Octanoate, octanoyl-CoA and CoA did not cause the translocation. It is proposed that the interaction of phosphatidate phosphohydrolase with the membranes that synthesize glycerolipids causes it to become metabolically active. This enables the liver to increase its capacity for triacylglycerol synthesis in response to an increased supply of fatty acids.     

Effect of spermine on the translocation of Mg2+-dependent phosphatidate phosphohydrolase

Adipose cytosol treated with spermine showed an aggregation of a cytosolic component which was isolated by centrifugation at 16,000 X g for 20 min. The resultant pellet contained 10% of protein, 40% of lipid and over 75-97% of Mg2+-dependent phosphatidate phosphohydrolase and CTP:phosphocholine cytidylyltransferase activities present in the original cytosol. The specific activities of these enzymes increased 4-fold by the spermine treatment. Characterization of lipids in this component indicated the presence of mainly phospholipids. These studies suggest that the interaction between spermine, the cytosolic component and microsomal membranes may be involved in the translocation of Mg2+-dependent phosphatidate phosphohydrolase.     

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.     

Age-related changes in the translocation of phosphatidate phosphohydrolase from the cytosol to microsomal membranes in rat liver

The effects of oleate, spermine and chlorpromazine were assayed in the presence or absence of 0.15 M KCl on the translocation of phosphatidate phosphohydrolase activity from cytosol to endoplasmic reticulum membranes in liver homogenates obtained from rats aged 1, 30, 60, 180 and 360 days. Marked age-associated decreases in phosphatidate phosphohydrolase distribution onto the membranes were demonstrated under nearly all conditions. In liver homogenates taken from 1-day-old rats and incubated with 0.15 M KCl, most of the enzyme was active (associated with the membranes). Physiological salt concentration (0.15 M KCl) produced a 2-fold increase of oleate-induced translocation of phosphatidate phosphohydrolase activity in liver homogenates from 1-day-old rats; it had no effect on those from 60-day-old rats, and produced a notable decline in liver homogenates obtained from 180- and 360-day-old rats. The promoting effect of spermine on oleate-induced translocation of this enzyme activity was higher in younger rats when incubated in the absence of 0.15 M KCl. Chlorpromazine did not show its usual antagonizing effect on oleate-induced translocation of phosphatidate phosphohydrolase when added to homogenates taken from 1-day-old rats. The antagonizing effect was slightly apparent in liver homogenates from 30-day-old rats and was more pronounced in those from 60-day-old rats in which the values diminished to one-half and to one-third either in the presence or absence of 0.15 M KCl.     

Isolation and characterization of multiple forms of Mg2+-dependent phosphatidate phosphohydrolase from rat adipose cytosol

In the present studies, we have made several unique observations. First, we have shown that cytosolic phosphatidate phosphohydrolase from adipose tissue subjected to butyl-agarose chromatography was resolved into four different components. These components, designated as passthrough (PT), D150, D250 and E, were present in the proportions of 51:7:24:16, respectively, in the rat adipose cytosol. Comparison of the properties of these components revealed some similar properties, and also several differences. These components showed the same pH optimum, required Mg2+ for activity and were inhibited by N-ethylmaleimide, indicating a requirement of active sulfhydryl groups for activity. These components differed from one another with respect to hydrophobicity, sedimentation behavior, Stokes diameter, Km values, thermolability and susceptibility to proteinase treatment. Second, we have shown that each component of this system was associated with lipids which were found to be essential for the catalytic activity. Perturbation of this association by organic solvent or by adding excess amounts of exogenous lipids resulted in the loss of enzyme activity. Finally, we analyzed lipid composition of individual components. These studies suggest that the multi-component system of Mg2+-dependent phosphatidate phosphohydrolase may be a part of the cytomembrane network.     

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.