Substrate specificity of lysophosphatidic acid acyltransferase beta -- evidence from membrane and whole cell assays

Membranes of mammalian cells contain lysophosphatidic acid acyltransferase (LPAAT) activities that catalyze the acylation of sn-1-acyl lysophosphatidic acid (lysoPA) to form phosphatidic acid. As the biological roles and biochemical properties of the six known LPAAT isoforms have yet to be fully elucidated, we have characterized human LPAAT-beta activity using two different assays. In a membrane-based assay, LPAAT-beta used lysoPA and lysophosphatidylmethanol (lysoPM) but not other lysophosphoglycerides as an acyl acceptor, and it preferentially transferred 18:1, 18:0, and 16:0 acyl groups over 12:0, 14:0, 20:0, and 20:4 acyl groups. The fact that lysoPM could traverse cell membranes permitted additional characterization of LPAAT-beta activity in cells: PC-3 and DU145 cells converted exogenously added lysoPM and (14)C-labeled 18:1 into (14)C-labeled phosphatidylmethanol (PM). The rate of PM formation was higher in cells that overexpressed LPAAT-beta and was inhibited by the LPAAT-beta inhibitor CT-32501. In contrast, if lysoPM and (14)C-labeled 20:4 were added to PC-3 or DU145 cells, (14)C-labeled PM was also formed, but the rate was neither higher in cells that overexpressed LPAAT-beta nor inhibited by CT-32501. We propose that LPAAT-beta catalyzes the intracellular transfer of 18:1, 18:0, and 16:0 acyl groups but not 20:4 groups to lysoPA.     

Lysophosphatidic acid can activate platelets without increasing 32P-labelling of phosphatidic acid

Stimulation of platelets by thrombin produced a rise in [32P]phosphatidic acid labelling of platelets which was greater in medium without added calcium than in medium with 2.5 mM calcium. A rise in [32P]lysophosphatidic acid was also seen in platelets stimulated by thrombin in the presence of 2.5 mM extracellular calcium, though it was of lesser magnitude (average 35%) than the rise in phosphatidic acid. In platelets resuspended without added calcium no change in [32P]lysophosphatidic acid was seen in response to thrombin. Lysophosphatidic acid can itself induce platelet aggregation. Similarly to the calcium ionophore A23187, lysophosphatidic acid produced minimal change (in medium with no added calcium) to no change (in medium with 2.5 mM external calcium) in [32P]lysophosphatidic acid. The endoperoxide analog U46619 produced changes in 32P-labelling of platelet phosphatidic and lysophosphatidic acid similar to those produced by thrombin but of lesser magnitude. The results of these studies show that the action of lysophosphatidic acid on platelets differs from the action of thrombin, U46619 and platelet-activating factor, which produce a rapid rise in [32P]phosphatidic acid, and suggests that lysophosphatidic acid, like A23187, largely bypasses the initial receptor-coupled breakdown of phosphoinositides leading to formation of diacylglycerols and phosphatidic acid.     

Swiss 3T3 cells preferentially incorporate sn-2-arachidonoyl monoacylglycerol into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.

The sn-1-stearoyl-2-arachidonoyl phospholipids of animal cells appear to be formed by special mechanisms. To determine whether monoacylglycerol (MG) incorporation pathways are involved we incubated quiescent Swiss 3T3 cells with [3H]glycerol-labeled sn-2-arachidonoyl MG, then analyzed the radioactive cell lipids that accumulated. We also examined cell homogenates to identify enzyme activities that might promote the incorporation of sn-2-arachidonoyl MG into other cell lipids. The cell incubation experiments demonstrated rapid labeling of several lipids, including diacylglycerol, lysophosphatidic acid, phosphatidic acid, and phosphatidylinositol. They also demonstrated selective labeling of sn-1-stearoyl-2-arachidonoyl species of phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. The cell homogenate experiments identified an sn-2-acyl MG acyltransferase activity, an MG kinase activity that phosphorylates sn-2-arachidonoyl MG in preference to sn-2-oleoyl MG, and a stearoyl-specific acyl transferase activity that converts sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidic acid. The results also showed that this stearoyl transferase could act with other enzymes to convert sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol. The combined results indicate that Swiss 3T3 cells incorporate sn-2-arachidonoyl MG into phospholipids by at least two different pathways, including one that specifically forms sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.     

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.     

Sequential actions of phospholipase D and phosphatidic acid phosphohydrolase 2b generate diglyceride in mammalian cells

Phosphatidylcholine (PC) is a major source of lipid-derived second messenger molecules that function as both intracellular and extracellular signals. PC-specific phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) are two pivotal enzymes in this signaling system, and they act in series to generate the biologically active lipids phosphatidic acid (PA) and diglyceride. The identity of the PAP enzyme involved in PLD-mediated signal transduction is unclear. We provide the first evidence for a functional role of a type 2 PAP, PAP2b, in the metabolism of PLD-generated PA. Our data indicate that PAP2b localizes to regions of the cell in which PC hydrolysis by PLD is taking place. Using a newly developed PAP2b-specific antibody, we have characterized the expression, posttranslational modification, and localization of endogenous PAP2b. Glycosylation and localization of PAP2b appear to be cell type and tissue specific. Biochemical fractionation and immunoprecipitation analyses revealed that PAP2b and PLD2 activities are present in caveolin-1-enriched detergent-resistant membrane microdomains. We found that PLD2 and PAP2b act sequentially to generate diglyceride within this specialized membrane compartment. The unique lipid composition of these membranes may provide a selective environment for the regulation and actions of enzymes involved in signaling through PC hydrolysis.     

Decanoyl lysophosphatidic acid induces platelet aggregation through an extracellular action. Evidence against a second messenger role for lysophosphatidic acid.

Platelets rapidly convert 1,2-didecanoyl-sn-glycerol into its corresponding phosphatidic acid and lysophosphatidic acid derivatives, thereby providing a means of introducing these two compounds into platelets. 1-Decanoyl-2-lyso-3-sn-phosphatidic acid, when added directly to platelets, induced platelet aggregation and raised intracellular Ca2+ levels at concentrations of 0.3 microM upwards, but was without effect when formed intracellularly from 1,2-didecanoylglycerol at an estimated concentration of approx. 47 microM. This indicates that the site of platelet activation by lysophosphatidic acid is extracellular. A concentration of thrombin (0.2 unit/ml), which produced maximal platelet aggregation, caused an estimated intracellular formation of 20 microM-lysophosphatidic acid in the presence of 2 mM-Ca2+; however, there was no detectable release of lysophosphatidic acid into the bathing medium. Lysophosphatidic acid, therefore, may not be an intracellular second messenger involved in platelet aggregation by thrombin.     

Studies on the formation by rat brain preparations of CDP-diglyceride from CTP and phosphatidic acids of varying fatty acid compositions.

The enzyme, CTP:phosphatidate cytidylyltransferase (EC2.7.7.41) which catalyses formation of CDP-diglyceride from CTP and phosphatidic acid has been studied in rat brain preparations and other tissues. Improvement, as judged by the higher tissue activities obtained, in the assay method for this enzyme was achieved through use of phosphatidic acids sonicated in buffer-detergent solution saturated with ether and containing bovine serum albumin and use of short incubation times which essentially provided a measure of initial rates. The enzyme of rat brain microsomes yielded with 1,2-dioleolphosphatidic acid as substrate a pH optimum of 6.8 with maleate buffer and optimal concentrations of 60mM for MG2+, 6MM for CTP and 250 mug per 0.8 ml for phosphatidic acid. Enzyme activity was mainly located in the 90,000 X g fraction (microsomal) with small but significant activity in the 12,000 X g fraction. Comparison of activities (nanomoles CTP incorporated per milligram protein per minute) amongst tissues showed the following order: brain, 1.87; liver, 1.32; lung, 1.19; small intestine, 1.00; kidney, 0.69; heart, 0.41; diaphragm, 0.07; skeletal muscle, 0.02. Examination of the effect of varying the fatty acid composition in the phosphatidic acids added exogenously gave the following order (activities in parentheses); 1-stearoyl-2-oleoyl- (5.58), 1-oleoyl-2-stearoyl- (5.37), 1,2-dioleoyl- (4.49) 1-palmitoyl-2-oleoyl-(3.85), 1-stearoyl-2-arachidonoyl-(3.31), 1-arachidonoyl-2-stearoyl-(3.16), 1,2-diarachidonoyl-(0.72), 1,2-dicaproyl-(0.67), 1,2-dipalmitoyl-(0.67) and 1,2-distearoyl-(0.18). The single bis- and lysophosphatidic acids tested were inactive as substrates. Apart from a possible preference for one or more unsaturated fatty acids the transferase enzyme showed no selectivity in respect to the fatty acid distribution of phosphatidic acids.     

Export of mitochondrially synthesized lysophosphatidic acid

We have previously demonstrated that the properties of mitochondrial glycerophosphate acyltransferase are in keeping with the asymmetric distribution of fatty acids found in naturally occurring cell glycerophospholipids. We are now examining if mitochondria can export lysophosphatidic acid and if it is converted to other phospholipids by the microsomes. Rat liver mitochondria were incubated for 3 min with [2-3H]-sn-glycerol 3-phosphate, palmityl-CoA, and N-ethylmaleimide in the acyltransferase assay medium. In the absence of bovine serum albumin in the medium, greater than 80% of the phospholipids sedimented with the mitochondria. In the presence of the albumin, the lysophosphatidic acid was present entirely in the supernatant fluid. The very little phosphatidic acid that was formed sedimented with the mitochondria. Addition of microsomes to the supernatant fluid followed by a further incubation of 5 min converted 61% of the lysophosphatidic acid to phosphatidic acid which sedimented with the microsomes. When mitochondria and microsomes were incubated together in the assay medium containing albumin and N-ethylmaleimide, the product contained more phosphatidic and less lysophosphatidic acid. When the subcellular components were reisolated by differential centrifugation, 70% of the phosphatidic acid sedimented with the microsomes and the lysophosphatidic acid stayed in the postmicrosomal supernatant. Thus, under appropriate conditions mitochondrially produced lysophosphatidic acid can leave the organelles and this phospholipid can be converted to phosphatidic acid by the microsomes.     

Generation of a conditional Ppap2b/Lpp3 null allele

Lpp3, formerly known as Pap2b, is a lipid phosphohydrolase enzyme. Some of its substrates and products are lipids with potent biological and signaling activities such as phosphatidic acid, lysophosphatidic acid, sphingosine-1-phosphate, diacylglycerol, sphingosine, and ceramide. Lpp3 is dynamically expressed during development and is widely distributed in adult tissues. Targeted inactivation of Lpp3 gene (Ppap2b) in the mouse results in embryonic lethality because of defects in extraembryonic vascular development and gastrulation. To study the participation of Lpp3 later in development and in specific cell lineages we generated a conditional allele of Ppap2b. This was accomplished by flanking critical exons, responsible for its catalytic activity with loxP sites. A generalized Cre-mediated recombination of this allele yielded a phenotype fundamentally identical to our previously reported Ppap2b null allele.     

The acylation of 1-acyl-sn-glycero-3-phosphate by neuronal nuclei and microsomal fractions of immature rabbit cerebral cortex

The acylation of 1-acyl-sn-glycero-3-phosphate to form phosphatidic acid was studied using a neuronal nuclear fraction N1 and microsomal fractions P3, R (rough), S (smooth), and P (neuronal microsomes from nerve cell bodies) isolated from cerebral cortices of 15-day-old rabbits. The assays contained this lysophospholipid, ATP, CoA, MgCl2, NaF, dithiothreitol, and radioactive palmitate, oleate, or arachidonate. Of the subfractions, N1 and R had the highest specific activities (expressed per micromole phospholipid in the fraction). The rates with oleate were two to four times the values seen for phosphatidic acid formation from sn-[3H]glycero-3-phosphate and oleoyl-CoA. Using oleate or palmitate, fraction R had superior specific rates to N1 at low lysophosphatidic acid concentrations. With increasing lysophospholipid concentrations the specific rates of N1 and R came closer together and maintained at least a twofold superiority over fraction P. Fraction S had the lowest specific rates of phosphatidic acid formation. Fractions N1, R, and P showed a preference for palmitate and oleate over arachidonate, particularly at low concentrations of lysophosphatidic acid. For N1 and R, the preference was also more marked at higher concentrations of fatty acid. Thus a selectivity for saturated and monounsaturated fatty acids was shown in the formation of phosphatidic acid, as was a concentration of acylating activity in the neuronal nucleus and the rough endoplasmic reticulum.     

Facilitated utilization of endogenously synthesized lysophosphatidic acid by 1-acylglycerophosphate acyltransferase from Escherichia coli

Complete separation of glycerophosphate acyltransferase and 1-acylglycerophosphate acyltransferase from Escherichia coli was obtained by sequential extraction with Triton X-100. Solubilized glycerophosphate acyltransferase was reconstituted by the cholate dispersion and gel filtration method in small unilamellar vesicles. 1-Acylglycerophosphate acyltransferase could not be solubilized from the membranes and was used in endogenous membrane fragments after detergent removal. Mixing of the two preparations and subsequent incubation in the presence of glycerol 3-phosphate, palmitoyl-CoA and oleoyl-CoA resulted in the efficient synthesis of phosphatidic acid. Inclusion of exogenous lysophosphatitic acid in the assay medium resulted in a dilution of the newly synthesized lysophosphatidate. By contrast, the synthesis of phosphatidic acid from glycerol 3-phosphate by the acyltransferases present in native membrane vesicles was barely influenced by the presence of exogenous lysophosphatidic acid. When comparing the utilization of membrane-associated 14C-labeled and newly generated 3H-labeled lysophosphatidic acid, the latter appeared to be the preferred substrate. These results indicate that lysophosphatidic acid, synthesized by glycerophosphate acyltransferase, is utilized by 1-acylglycerophosphate acyltransferase without prior mixing with the total membrane-associated pool of lysophosphatidic acid, and suggest a close proximity of the two enzymes in native E. coli membranes. This property of the acyltransferases is lost upon separation and reconstitution of enzyme activities.     

Coordinated interactions between actin and microtubules through crosslinkers in neurite retraction induced by lysophosphatidic acid

Neurite development requires rearrangement of cytoskeletal elements, which are mechanically and functionally integrated with each other. Although the process of how an extracellular signal induces rearrangement of a single element has been closely examined, the mechanisms by which the signal regulates cytoskeletal integration during cell shape changes are poorly understood. We previously reported that lysophosphatidic acid (LPA) induces actin polymerization-dependent microtubule (MT) rearrangement, leading to neurite retraction in cultured neurons. Here we examined whether the crosslinker proteins were involved in LPA-induced neurite retraction using immortalized mouse neuroblast TR cells. When the MT-binding domains of MACF (MT actin-crosslinking factor) were exogenously expressed in TR cells, MTs were found to be stabilized and become resistant to exposure to LPA. On the other hand, expression of MT-associated protein 2c showed no effect on LPA-induced neurite retraction. These findings suggest that MACF is involved in actin-dependent MT rearrangement during LPA-induced neurite retraction.     

TLR-4 mediated group IVA phospholipase A2 activation is phosphatidic acid phosphohydrolase 1 and protein kinase C dependent

Group IVA phospholipase A₂ (GIVA PLA₂) catalyzes the release of arachidonic acid (AA) from the sn-2 position of glycerophospholipids. AA is then further metabolized into terminal signaling molecules including numerous prostaglandins. We have now demonstrated the involvement of phosphatidic acid phosphohydrolase 1 (PAP-1) and protein kinase C (PKC) in the Toll-like receptor-4 (TLR-4) activation of GIVA PLA₂. We also studied the effect of PAP-1 and PKC on Ca^+ 2 induced and synergy enhanced GIVA PLA₂ activation. We observed that the AA release induced by exposure of RAW 264.7 macrophages to the TLR-4 specific agonist Kdo₂-Lipid A is blocked by the PAP-1 inhibitors bromoenol lactone (BEL) and propranolol as well as the PKC inhibitor Ro 31-8220; however these inhibitors did not reduce AA release stimulated by Ca^+ 2 influx induced by the P2X7 purinergic receptor agonist ATP. Additionally, stimulation of cells with diacylglycerol (DAG), the product of PAP-1 mediated hydrolysis, initiated AA release from unstimulated cells as well as restored normal AA release from cells treated with PAP-1 inhibitors. Finally, neither PAP-1 nor PKC inhibition reduced GIVA PLA₂ synergistic activation by stimulation with Kdo₂-Lipid A and ATP.     

Mitochondrial glycerol phosphate acyltransferase directs the incorporation of exogenous fatty acids into triacylglycerol

The mitochondrial isoform of glycerol-3-phosphate acyltransferase (GPAT), the first step in glycerolipid synthesis, is up-regulated by insulin and by high carbohydrate feeding via SREBP-1c, suggesting that it plays a role in triacylglycerol synthesis. To test this hypothesis, we overexpressed mitochondrial GPAT in Chinese hamster ovary (CHO) cells. When GPAT was overexpressed 3.8-fold, triacylglycerol mass was 2.7-fold higher than in control cells. After incubation with trace [(14)C]oleate ( approximately 3 microm), control cells incorporated 4.7-fold more label into phospholipid than triacylglycerol, but GPAT-overexpressing cells incorporated equal amounts of label into phospholipid and triacylglycerol. In GPAT-overexpressing cells, the incorporation of label into phospholipid, particularly phosphatidylcholine, decreased 30%, despite normal growth rate and phospholipid content, suggesting that exogenous oleate was directed primarily toward triacylglycerol synthesis. Transiently transfected HEK293 cells that expressed a 4.4-fold increase in GPAT activity incorporated 9.7-fold more [(14)C]oleate into triacylglycerol compared with control cells, showing that the effect of GPAT overexpression was similar in two different cell types that had been transfected by different methods. When the stable, GPAT-overexpressing CHO cells were incubated with 100 microm oleate to stimulate triacylglycerol synthesis, they incorporated 1.9-fold more fatty acid into triacylglycerol than did the control cells. Confocal microscopy of CHO and HEK293 cells transfected with the GPAT-FLAG construct showed that GPAT was located correctly in mitochondria and was not present elsewhere in the cell. These studies indicate that overexpressed mitochondrial GPAT directs incorporation of exogenous fatty acid into triacylglycerol rather than phospholipid and imply that (a) mitochondrial GPAT and lysophosphatidic acid acyltransferase produce a separate pool of lysophosphatidic acid and phosphatidic acid that must be transported to the endoplasmic reticulum where the terminal enzymes of triacylglycerol synthesis are located, and (b) this pool remains relatively separate from the pool of lysophosphatidic acid and phosphatidic acid that contributes to the synthesis of the major phospholipid species.     

Lysophosphatidic acid improves corneal endothelial density in tissue culture by stimulating stromal secretion of interleukin‐1β

The short supply of donor corneas is exacerbated by the unsuitability of donors with insufficient endothelial cell density. Few studies have investigated promoting corneal endothelial cell proliferation to increase the endothelial cell density. We hypothesize that pre‐transplantation treatment of proliferative tissue‐cultivated corneas may increase corneal endothelial cell density. We observed that the airlift cultures were superior to immersion cultures with respect to both transparency and thickness. In this tissue culture system, we observed that lysophosphatidic acid increased the rabbit corneal endothelial cell density, number of BrdU‐positive cells and improve wound healing. We also observed an indirect effect of lysophosphatidic acid on corneal endothelial cell proliferation mediated by the stimulation of interleukin‐1β secretion from stromal cells. Human corneal tissues treated with lysophosphatidic acid or interleukin‐1β contained significantly more Ki‐67‐positive cells than untreated group. The lysophosphatidic acid‐ or interleukin‐1β‐treated cultured tissue remained hexagon‐shaped, with ZO‐1 expression and no evidence of the endothelial‐mesenchymal transition. Our novel protocol of tissue culture may be applicable for eye banks to optimize corneal grafting.     

Effects of sphingosine, albumin and unsaturated fatty acids on the activation and translocation of phosphatidate phosphohydrolases in rat hepatocytes.

The activities of two phosphatidate phosphohydrolases were measured in cultured rat hepatocytes incubated with 0.1 mM albumin. The activity, which is inhibited by N-ethylmaleimide (PAP-1) is located in the cytosolic and membrane fractions. PAP-1 activity is stimulated by Mg2+ and it can be translocated from the cytosol to the membranes by relatively low (0.5-1 mM) concentrations of fatty acids. In addition, higher concentrations (1-3 mM) of fatty acids cause an increase in the total PAP-1 activity. Translocation of PAP-1 activity in the hepatocytes is preferentially promoted by unsaturated fatty acids (C18:1, C18:2, C18:3, C20:4 and C20:5), rather than by saturated acids (C14:0, C16:0, C18:0). Increasing the extracellular concentration of albumin from 30 microM to 1 mM displaces PAP-1 activity from the membrane fraction. Sphingosine, but not staurosporine, can inhibit the redistribution of PAP-1 activity induced by oleate. The amphiphilic amines, sphingosine, chlorpromazine and propranolol, also decrease membrane-bound PAP-1 activity in the absence of fatty acids, but they do not alter, significantly, the activity of the cytosolic PAP-1. In the presence of 1 mM oleate, sphingosine, chlorpromazine and propranolol decrease the translocation of PAP-1 from the cytosol to the membranes. The phosphohydrolase activity, which is insensitive to N-ethylmaleimide (PAP-2), is specifically located in the plasma membrane (Jamal, Z., Martin, A., Gomez-Mu?oz, A. and Brindley, D.N. (1991) J. Biol. Chem. 266, 2988-2996) and it is not stimulated by Mg2+. Saturated fatty acids, albumin, sphingosine and propranolol have no significant effects on PAP-2 activity. However, chlorpromazine decreases PAP-2 activity by about 14%. Linolenate, arachidonate and eicosapentaenoate at 1 mM also produced small (7-10%) decreases in PAP-2 activity. It is proposed that both PAP-1 and PAP-2 activities may be involved in signal transduction, although the main function of PAP-1 seems to be involved in the synthesis of glycerolipids.     

Enhancement of lysophosphatidic acid-induced ERK phosphorylation by phospholipase D1 via the formation of phosphatidic acid

We made stable cell lines overexpressing PLD1 (GP-PLD1) from GP+envAm12 cell, a derivative of NIH 3T3 cell. PLD1 activity and extracellular signal-regulated kinase (ERK) phosphorylation were enhanced in GP-PLD1 cells by the treatment of lysophosphatidic acid (LPA). In contrast, these LPA-induced effects were attenuated with the pretreatment of pertussis toxin (PTX) or protein kinase C (PKC) inhibitor. Moreover, accumulation of phosphatidic acid (PA), a product of PLD action, potentiated the LPA-induced ERK activation in GP-PLD1 cells while blocking of PA production with the treatment of 1-butanol attenuated LPA-induced ERK phosphorylation. From these results, we suggest that LPA activate PLD1 through pertussis toxin-sensitive G protein and PKC-dependent pathways, then PA produced from PLD1 activation facilitate ERK phosphorylation.     

Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast

Triacylglycerol is one of the major storage forms of metabolic energy in eukaryotic cells. Biosynthesis of triacylglycerol is known to occur in membranes. We report here the isolation, purification, and characterization of a catalytically active cytosolic 10 S multienzyme complex for triacylglycerol biosynthesis from Rhodotorula glutinis during exponential growth. The complex was characterized and was found to contain lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl-acyl carrier protein synthetase, and acyl carrier protein. The 10 S triacylglycerol biosynthetic complex rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic intermediates. Lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol acyltransferase from the complex were microsequenced. Antibodies were raised against the synthetic peptides corresponding to lysophosphatidic acid acyltransferase and phosphatidic acid phosphatase sequences. Immunoprecipitation and immunolocalization studies show the presence of a cytosolic multienzyme complex for triacylglycerol biosynthesis. Chemical cross-linking studies revealed that the 10 S multienzyme complex was held together by protein-protein interactions. These results demonstrate that the cytosol is one of the sites for triacylglycerol biosynthesis in oleaginous yeast.     

A HPLC-fluorescence detection method for determination of phosphatidic acid phosphohydrolase activity: application in human myocardium

Phosphatidic acid phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) to diacylglycerol, the second messenger responsible for activation of protein kinase C. Despite the crucial role of PAP lipid signaling, there are no data on PAP signaling function in the human heart. Here we present a nonradioactive assay for the investigation of PAP activity in human myocardium using a fluorescent derivative of PA, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate (BODIPY-PA), as substrate in an in vitro PAP-catalyzed reaction. Unreacted BODIPY-PA was resolved from the PAP products by a binary gradient HPLC system and BODIPY-diacylglycerol was detected by fluorimetry. The reaction proceeded at a linear rate for up to 60 min and increased linearly with increasing amounts of cardiac protein in a range of 0.25 to 8.0 μg. This assay proved to be sensitive for accurate quantitation of total PAP activity, PAP-1 activity, and PAP-2 activity in human atrial tissue and right ventricular endomyocardial biopsies. Total PAP activity was approximately fourfold higher in ventricular myocardium than in atrial tissue. There was negligible PAP-1 activity in atrial myocardium compared with ventricular myocardium, indicating regional differences in activities and distribution pattern of PAP-1 and PAP-2 in the human heart.