Phosphatidate phosphohydrolase and palmitoyl-coenzyme A hydrolase in cardiac subcellular fractions of hyperthyroid rabbits and cardiomyopathic hamsters

Activities of phosphatidate phosphohydrolase and palmitoyl-CoA hydrolase were determined in cardiac subcellular fractions prepared from rabbits which has received tri-iodothyronine and from hamsters with hereditary cardiomyopathy (strain BIO 14.6). 1. Both mitochondrial and microsomal fractions of hyperthyroid rabbit hearts produced 4-5 times as much diacylglycerol 3-phosphate from glycerol 3-phosphate and palmitate as did those of euthyroid hearts. 2. Phosphatidate phosphohydrolase, measured with phosphatidate emulsion, was activated by 1mm-Mg(2+) in all but the mitochondrial fraction of euthyroid rabbit hearts. The activation was more pronounced in subcellular fractions isolated from hyperthyroid hearts, so that the measured activities were significantly increased above those of the controls. The highest activity was found in the microsomal and lysosomal fractions. 3. In the absence of Mg(2+) during incubation, the difference in phosphohydrolase activities between eu- and hyper-thyroid states was not significant. 4. The phosphohydrolase of subcellular fractions of control hamsters did not respond to addition of 0.5-8.0mm-Mg(2+). The enzyme from cardiomyopathic hearts was slightly inhibited by this bivalent cation and therefore significant increases in activity were observed only in the absence of Mg(2+) from the assay system. 5. The rate of reaction by soluble phosphatidate phosphohydrolase was similar regardless of the nature of the substrate. Both when microsomal-bound phosphatidate was used as the substrate and when phosphatidate suspension was used, the activity of soluble enzyme was lower than that of the microsomal and lysosomal enzymes measured with phosphatidate suspension; this was especially so when the assay was carried out in the absence of Mg(2+). Neither tri-iodothyronine nor cardiomyopathy influenced the soluble phosphohydrolase activity in the two species. 6. Neither tri-iodothyronine nor cardiomyopathy significantly changed palmitoyl-CoA hydrolase activities in subcellular fractions. 7. Microsomal diacylglycerol acyltransferase and myocardial triacylglycerol content were also unchanged in the hyperthyroid state.     

Relationship between the displacement of phosphatidate phosphohydrolase from the membrane-associated compartment by chlorpromazine and the inhibition of the synthesis of triacylglycerol and phosphatidylcholine in rat hepatocytes

Glycerolipid synthesis was studied in isolated hepatocytes by using 177 microM [14C]oleate and 1 mM [3H]glycerol. Chlorpromazine (25-400 microM) inhibited the synthesis of phosphatidylcholine and triacylglycerol. This was accompanied by an average increase of 12-fold in the accumulation of the labelled precursors in phosphatidate at 200 microM chlorpromazine and a decrease in the conversion of phosphatidate to diacylglycerol of 76%. These results indicate that part of the inhibition of the synthesis of phosphatidylcholine and triacylglycerol occurs at the level of phosphatidate phosphohydrolase. The relative rate of triacylglycerol synthesis at different concentrations of chlorpromazine was approximately proportional to the rate of conversion of phosphatidate to diacylglycerol. Phosphatidylcholine synthesis increased at higher rates of conversion of phosphatidate to diacylglycerol, but it was relatively independent of the latter rate when this was inhibited by more than about 30% with chlorpromazine. The addition of oleate to the hepatocytes caused a translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Chlorpromazine had the opposite effect and displaced the phosphohydrolase from the membranes in the presence or absence of oleate. There was a highly significant correlation between the activity of phosphatidate phosphohydrolase that was associated with the membranes of the hepatocytes and the calculated conversion of [3H]phosphatidate to diacylglycerol. Chlorpromazine also antagonized the association of the phosphohydrolase with microsomal membranes when cell-free preparations were incubated with combinations of oleate and spermine. Furthermore, it inhibited the transfer of the soluble phosphohydrolase to microsomal membranes that were labelled with [14C]phosphatidate and thereby decreased diacylglycerol production. It is concluded that part of the action of chlorpromazine in inhibiting the synthesis of triacylglycerol and phosphatidylcholine occurs because it prevents the interaction of the soluble phosphatidate phosphohydrolase with the membranes on which glycerolipid synthesis occurs. This in turn prevents the conversion of phosphatidate to diacylglycerol.     

A rapid assay for measuring the activity and the Mg2+ and Ca2+ requirements of phosphatidate phosphohydrolase in cytosolic and microsomal fractions of rat liver.

1. A rapid extraction and purification scheme was designed for the recovery of [3H]diacylglycerol formed during the assay of phosphatidate phosphohydrolase. 2. The importance of removing polyvalent cations, particularly Ca2+, from the phosphatidate and other reagents used in the assay of the phosphohydrolase activity was demonstrated. This was achieved mainly by treating the phosphatidate with a chelating resin and by adding 1 mM-EGTA and 1 mM-EDTA to the assays. 3. The activity of the phosphohydrolase in dialysed samples of the soluble and microsomal fractions of rat liver was very low. 4. Addition of optimum concentrations of MgCl2 resulted in a 110-167-fold stimulation in activity. 5. CaCl2 was also able to stimulate phosphohydrolase activity, but to a much smaller extent than MgCl2. 6. Chlorpromazine, an amphiphilic cation, inhibited the reaction when it was measured in these experiments by using a mixed emulsion of phosphatidylcholine and phosphatidate at pH 7.4. 7. Microsomal fractions that were preincubated with albumin contained very low activities of the Mg2+-dependent phosphohydrolase. When these were then incubated with the soluble fraction in the presence of oleate, the soluble phosphohydrolase attached to the microsomal membranes, and it retained its high dependency on Mg2+.     

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.     

Effect of hydrazine exposure on hepatic triacylglycerol biosynthesis.

Acute hydrazine exposure elevated rat liver triacylglycerol content and produced a rapid rise in triacylglycerol production from sn-[1,3-14C]glycerol 3-phosphate by liver homogenate and microsomal fractions. Hydrazine treatment also increased the incorporation of [1,3-14C]glycerol into hepatic triacylglycerol by the intact animal. Homogenates of hepatocyte monolayers exposed to hydrazine in vitro also exhibited an increased capacity to form triacylglycerol from sn-[1,3-14C]glycerol 3-phosphate. Hydrazine-dependent increases in hepatic triacylglycerol production measured in vitro correlated well with an increase in microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) activity. Therefore, the fatty liver associated with hydrazine exposure may be explained in part by a rise in the enzymatic capacity of hepatic triacylglycerol biosynthesis.     

Effects of phenobarbital upon triacylglycerol metabolism in the rabbit.

1. The association between hepatic microsomal enzyme induction and triacylglycerol metabolism was examined in fasting male rabbits (2kg body wt.) injected intra-peritoneally with 50 mg of phenobarbital per kg for 10 days. 2. Occurrence of enzyme induction was established by a significant increase in hepatic aminopyrine N-demethylase activity and cytochrome P-450 content, as well as a doubling of microsomal protein per g of liver and a 54% increase in liver weight. Parallel increments in hepatic gamma-glutamyltransferase (EC 2.3.2.2) activity occurred; these were more pronounced in the whole homogenate than in the microsomes, which only accounted for 12.5% of the total enzyme activity in the controls and 17.0% in the animals given phenobarbital. Increased activity of gamma-glutamyltransferase activity was also observed in the blood serum of the test animals. 3. The rabbits given phenobarbital manifested increased hepatic triacylglycerol content and the triacylglycerol concentration of blood serum was also elevated. These changes were accompanied by a significantly enhanced ability of cell-free fractions of liver from the test animals (postmitochondrial supernatant and microsomal fractions) to synthesize glycerolipids in vitro from sn-[14C] glycerol 3-phosphate and fatty acids, when expressed per whole liver. Relative to the protein content of the fraction, glycerolipid synthesis in vitro was significantly decreased in the microsomes, presumably consequent upon the dramatic increase in their total protein content, whereas no change occurred in the postmitochondrial supernatant, possibly due to the protective effect of cytosolic factors present in this fraction and known to enhance glycerolipid synthesis. 4. Microsomal phosphatidate phosphohydrolase accounted for 85% of the total liver activity of this enzyme and its specific activity was 20-fold higher than that of the cytosolic phosphatidate phosphohydrolase (EC 3.1.3.4), when each was measured under optimal conditions. A significant increase in the activity of both enzymes per whole liver occurred in the rabbits given phenobarbital. A closer correlation between hepatic triacylglycerol content and and microsomal phosphatidate phosphohydrolase, as well as the above observation, suggest that this, rather than the cytosolic enzyme, may be rate-limiting for triacylglycerol synthesis in rabbit liver. 5. Significant correlations were observed between the various factors of hepatic microsomal-enzyme induction (aminopyrine N-demethylase and gamma-glutamyltransferase activity as well as cytochrome P-450 content) and hepatic triacylglycerol content, suggesting that that microsomal enzyme induction may promote hepatic triacylglycerol synthesis and consequently hypertriglyceridaemia in the rabbit.     

The effect of dietary carbohydrate and fat on the activities of some enzymes responsible for glycerolipid synthesis in rat liver.

1. Male rats were fed for 14 days on diets containing (by wt.) 53% of starch, or on diets in which 20% of the starch was replaced by sucrose, corn oil or lard. 2. The hepatic activities of the microsomal glycerol phosphate acyltransferase, dihydroxyacetone phosphate acyltransferase, phosphatidate cytidylyltransferase, diacylglycerol acyltransferase and choline phosphotransferase, and of the soluble phosphatidate phosphohydrolase, were measured. 3. The soluble phosphatidate phosphohydrolase activity was higher in those rats fed on lard than in those fed on the starch diet. Choline phosphotransferase activity was higher in the rats fed on corn oil than in those fed on the starch diet. 4. The rate of hepatic glycerolipid synthesis was measured in vivo 1 min after injection of [1,3-3H]glycerol and [1-14C]palmitate into the portal veins. 5. The relative rate of phosphatidylcholine synthesis in vivo was increased after feeding with corn oil and the higher specific activity of choline phosphotransferase may contribute to this result. The equivalent rate of triacylglycerol synthesis was increased by feeding with lard rather than corn oil, and the increased activity of phosphatidate phosphohydrolase may partly explain this. The latter changes probably contribute to the increased concentration of triacylglycerol which other authors have observed in the livers and sera of animals fed on saturated and monounsaturated fats.     

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.     

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.     

Ontogeny of glycerolipid biosynthetic enzymes in swine liver and adipose tissue.

Enzymes associated with glycerolipid biosynthesis were examined in microsomal fractions of liver and adipose tissue obtained from swine of various ages. Generally, liver glycerophosphate acyltransferase, phosphatidate phosphohydrolase, diglyceride acyltransferase, and choline phosphotransferase activities were substantial at birth but increased 2- to 3-fold by day 14 postpartum, decreased at day 25, then increased at the oldest ages studied (up to 155 days postpartum). In adipose tissue, enzyme activities were low at birth and developed through day 25 in a pattern generally similar to that observed in liver. In contrast to liver, the adipose enzymes were depressed immediately postweaning (day 32) with subsequent recovery. The observed decline in adipose tissue enzyme activities expressed on a tissue basis at older ages was primarily the result of increased adipocyte size, since the activities expressed on a cell basis did not decline as rapidly. In both liver and adipose tissue, phosphatidate was the major glycerolipid synthesized by the microsomal glycerophosphate acyltransferase enzymes at all ages (generally greater than 75%). The ratio of neutral lipids to phospholipids produced by acylation of glycerophosphate was increased when a microsomal--cytosolic preparation was used as a source of enzyme in contrast to a microsomal preparation.     

Oleic acid promotes the activation and translocation of phosphatidate phosphohydrolase from the cytosol to particulate fractions of isolated rat hepatocytes

The incubation of hepatocytes with 1-4mM-oleate increased the total activity of phosphatidate phosphohydrolase that was measured in the presence of Mg2+ to about 2-fold. This was accompanied by an increase in the proportion of the enzyme that was isolated with the particulate fractions. Conversely, the addition of up to 4mM-oleate decreased the recovery of phosphatidate phosphohydrolase in the cytosolic fraction from about 70% to 3% when hepatocytes were lysed with digitonin. Most of the increase in the membrane-associated phosphohydrolase activity was isolated after cell fractionation in the microsomal fraction that was enriched with the endoplasmic-reticulum marker arylesterase. It is proposed that the translocation of phosphatidate phosphohydrolase facilitates the increased synthesis of triacylglycerols in the liver when it is presented with an increased supply of fatty acids.     

The involvement of phosphatidate phosphohydrolase and phospholipase A activities in the control of hepatic glycerolipid synthesis. Effects of acute feeding with glucose, fructose, sorbitol, glycerol and ethanol.

Rats were fed by stomach tube with a single dose of glucose, sorbitol, fructose, glycerol or ethanol of equivalent energy contents or with 0.15 M-NaCl. They were killed 6 h later and the relative rates of phosphatidate deacylation and dephosphorylation measured in the microsomal and supernatant fractions of the livers. Treatment with sorbitol, fructose, glycerol and ethanol increased phosphohydrolase activities in the microsomal and supernatant fractions. The only significant change in deacylase activity was an increase in the microsomal fraction produced by ethanol. It is proposed that hepatic triacylglycerol synthesis is partly controlled by the balance between phosphatidate phosphohydrolase and phospholipase A-type activities.     

Regulation of the translocation of phosphatidate phosphohydrolase between the cytosol and the endoplasmic reticulum of rat liver. Effects of unsaturated fatty acids, spermine, nucleotides, albumin and chlorpromazine.

The translocation of phosphatidate phosphohydrolase between the cytosol and the microsomal membranes was investigated by using a cell-free system from rat liver. Linoleate, alpha-linolenate, arachidonate and eicosapentenoate promoted the translocation to membranes with a similar potency to that of oleate. The phosphohydrolase that associated with the membranes in the presence of [14C]oleate or 1mM-spermine coincided on Percoll gradients with the peak of rotenone-insensitive NADH-cytochrome c reductase, and in the former case with a peak of 14C. Microsomal membranes were enriched with the phosphohydrolase activity by incubation with [14C]oleate or spermine and then incubated with albumin. The phosphohydrolase activity was displaced from the membranes by albumin, and this paralleled the removal of [14C]oleate from the membranes when this acid was present. Chlorpromazine also displaced phosphatidate phosphohydrolase from the membranes, but it did not displace [14C]oleate. The effects of spermine in promoting the association of the phosphohydrolase with the membranes was inhibited by ATP, GTP, CTP, AMP and phosphate. ATP at the same concentration did not antagonize the translocating effect of oleate. From these results and previous work, it was concluded that the binding of long-chain fatty acids and their CoA esters to the endoplasmic reticulum acts as a signal for more phosphatidate phosphohydrolase to associate with these membranes and thereby to enhance the synthesis of glycerolipids, especially triacylglycerol. The translocation of the phosphohydrolase probably depends on the increased negative charge on the membranes, which could also be donated by the accumulation of phosphatidate. Chlorpromazine could oppose the translocation by donating a positive charge to the membranes.     

Properties of phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities in the isolated rat heart. Effect of glucagon, ischaemia and diabetes.

Myocardial triacylglycerol hydrolysis is subject to product inhibition. After hydrolysis of endogenous triacylglycerols, the main proportion of the liberated fatty acids is re-esterified to triacylglycerol, indicating the importance of fatty acid re-esterification in the regulation of myocardial triacylglycerol homoeostasis. Therefore, we characterized phosphatidate phosphohydrolase (PAP) and diacylglycerol acyltransferase (DGAT) activities, enzymes catalysing the final steps in the re-esterification of fatty acids to triacylglycerols in the isolated rat heart. The PAP activity was mainly recovered in the microsomal and soluble cell fractions, with an apparent Km of 0.14 mM for both the microsomal and the soluble enzyme. PAP was stimulated by Mg2+ and oleic acid. Oleic acid, like a high concentration of KCl, stimulated the translocation of PAP activity from the soluble to the particulate (microsomal) fraction. Myocardial DGAT had an apparent Km of 3.8 microM and was predominantly recovered in the particulate (microsomal) fraction. Both enzyme activities were significantly increased after acute streptozotocin-induced diabetes, PAP from 15.6 +/- 1.1 to 28.1 +/- 3.6 m-units/g wet wt. (P less than 0.01) and DGAT from 2.23 +/- 0.11 to 3.01 +/- 0.11 m-units/g wet wt. (P less than 0.01). In contrast with diabetes, low-flow ischaemia during 30 min did not affect PAP and DGAT activity in rat hearts. Perfusion with glucagon (0.1 microM) during 30 min did not affect total PAP activity, but changed the subcellular distribution. More PAP activity was recovered in the particulate fraction. DGAT activity was lowered by glucagon treatment from 0.37 +/- 0.03 to 0.23 +/- 0.02 m-unit/mg of microsomal protein (P less than 0.05). The role of PAP and DGAT activity and PAP distribution in the myocardial glucose/fatty acid cycle is discussed.     

Comparative effects of dietary fish oil and carbohydrate on plasma lipids and hepatic activities of phosphatidate phosphohydrolase, diacylglycerol acyltransferase and neutral lipase activities in the rat

In rats fed a fish oil-enriched diet, plasma triacylglycerols were lowered 51%. At the same time there was a mean 45% reduction in Mg2+-dependent phosphatidate phosphohydrolase activity in liver microsomes and a mean 20% decrease in microsomal triacylglycerol (neutral) and diacylglycerol hydrolase activities, but not of diacylglycerol acyltransferase. These observations support the hypothesis that decreases in the activities of phosphatidate phosphohydrolase and of both lipases are involved in the expression of the inhibitory effects of fish oil feeding on hepatic lipoprotein triacylglycerol secretion. Conversely, the feeding of a sucrose-enriched diet resulted in a mean 39% rise in plasma triacylglycerols, a 19% increase in triacylglycerol hydrolase and a mean 45% increase in Mg2+-dependent microsomal phosphohydrolase activity. The effects of the two nutritional interventions on phosphatidate phosphohydrolase activity confirm a key function for this enzyme in triacylglycerol formation.     

Translocation of Mg2+-dependent phosphatidate phosphohydrolase between cytosol and endoplasmic reticulum in a permanent cell line from human lung

Incubation of A549 cells with digitonin for 4 min resulted in the release of over 90% of the lactate dehydrogenase activity into the medium. Approximately 80% of the Mg2+-dependent but only 7% of the Mg2+-independent phosphatidate phosphohydrolase activity was released in the presence of digitonin. Pretreatment of the cells with oleate reduced the efflux of the Mg2+-dependent phosphatidate phosphohydrolase activity to approximately 5% of total. Oleate did not affect the release of lactate dehydrogenase or the release of the Mg2+-independent phosphohydrolase activity. Incubation of A549 cells with [3H]oleate for 60 min led to incorporation of the label into phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, diacylglycerol, monoacylglycerol, and triacylglycerol, in ascending order. When the level of exogenous oleate was increased to over 2.0 mM, there was a marked increase in the incorporation into monoacylglycerol and diacylglycerol. Only small amounts of radioactivity were associated with phosphatidic acid. Time course studies revealed that the amount of radioactive phosphatidate remained low throughout the incubation period. These investigations were interpreted to indicate that free fatty acids can promote the translocation of the Mg2+-dependent phosphatidate phosphohydrolase activity from cytosol to membrane fractions. This translocation could, at least theoretically, function to facilitate the metabolism of increased amounts of phosphatidate.     

Changes in activities of some enzymes of glycerolipid synthesis in brown adipose tissue of cold-acclimated rats.

1. Measurements were made of the activities of the following enzymes of glycerolipid synthesis in homogenates of interscapsular brown adipose tissue obtained from rats subjected to a 4 degrees C environment for time periods of 6 h up to 12 days: fatty acyl-CoA synthetase (FAS), mitochondrial and microsomal forms of glycerolphosphate acyltransferase (GPAT), monoacylglycerolphosphate acyltransferase (MGPAT) and Mg2+-dependent phosphatidate phosphohydrolase (PPH). 2. Relative to tissue DNA content, the activities of mitochondrial GPAT, MGPAT and Mg2+-dependent PPH were significantly increased after 1 day of exposure to cold, and continued to increase thereafter. By contrast, FAS and microsomal GPAT activities were unchanged relative to tissue DNA. 3. The time profile of the increase in MGPAT activity correlated well with a concomitant increase in the microsomal marker NADP+-cytochrome c reductase. Changes in mitochondrial GPAT and in Mg2+-dependent PPH activities were larger in amplitude than that of MGPAT. 4. It is proposed that these selective changes in enzyme activity may be associated with the onset of brown-adipose-tissue hyperplasia or possibly with an increase in triacylglycerol synthesis during cold-acclimation.     

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.     

The effects of cortisol, corticotropin and thyroxine on the synthesis of glycerolipids and on the phosphatidate phosphohydrolase activity in rat liver

1. Male rats were injected daily for 5 days with 0.15m-NaCl, corticotropin, cortisol or l-thyroxine and the rates of glycerolipid synthesis were measured in the livers after intraportal injection of [(14)C]palmitate and [(3)H]glycerol. 2. Injection of all three hormones decreased the rates of body-weight gain. 3. Cortisol treatment increased the weight of the liver relative to body weight. 4. Thyroxine treatment increased the relative rate of triacylglycerol synthesis from [(3)H]glycerol and decreased the relative accumulation of (3)H and (14)C in diacylglycerol. It did not significantly alter the accumulation of these isotopes in phosphatidate nor the activity of the soluble phosphatidate phosphohydrolase in the total liver. However, this activity increased by 1.5-fold when expressed relative to the soluble protein of the liver. The increased triacylglycerol synthesis appears to be related to a general increase in the turnover of fatty acids in the liver. 5. Treatment with cortisol and corticotropin increased the relative rate of triacylglycerol synthesis from [(3)H]glycerol, decreased the accumulation of (3)H in phosphatidate and increased the flux of both isotopes from phosphatidate to diacylglycerol. This appeared to be caused by the increased activity of the soluble phosphatidate phosphohydrolase that was observed in the livers of the cortisol-treated rats. 6. It is proposed that cortisol could be directly or indirectly involved in increasing the activity of hepatic phosphatidate phosphohydrolase in starvation, diabetes, laparotomy, subtotal hepatectomy, liver damage, ethanol feeding and in obesity. This enzyme adaptation could contribute to the potential of the liver to increase its synthesis and accumulation of triacylglycerols or to secrete very-low-density lipoproteins.     

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.