The molecular species of phosphatidic acid, diacylglycerol and phosphatidylcholine synthesized from sn-glycerol 3-phosphate in rat lung microsomes

The species pattern of phosphatidic acid, diacylglycerol and phosphatidylcholine synthesized from [14C]glycerol 3-phosphate was measured using a newly developed HPLC technique yielding 13 molecular species. A direct comparison of these species patterns presupposes determination of the lipolytic activity of lung microsomes. The lipolytic activity was quantitatively determined by measuring the changes of the endogenous concentration of diacylglycerol, triacylglycerol and free fatty acids. The species pattern of endogenous diacylglycerol measured in the time-course of lipolysis did not show any changes up to an incubation period of 20 min, suggesting that the lipolytic activity showed only a very low selectivity for individual substrate species. Diisopropylfluorophosphate (5 mumol/mg microsomal protein) strongly decreased the lipolytic activities as well as the microsomal phosphatidate phosphohydrolase activity, as measured by means of exogenous phosphatidic acid, and also the generation of phosphatidic acid from [14C]glycerol 3-phosphate. In lung microsomes, labeled phosphatidic acid and diacylglycerols were synthesized from the endogenous free fatty acids and sn-[14C]glycerol 3-phosphate, which had previously been added. By addition of CDPcholine to the prelabeled microsomes the synthesis of phosphatidylcholine was measured. After hydrolysis of phosphatidic acid and phosphatidylcholine with cytoplasmatic phosphatidate phosphohydrolase or phospholipase C, respectively, the de novo synthesized species patterns of these two lipids and of the diacylglycerol were determined. Comparison of the species pattern of de novo synthesized phosphatidic acid with that of diacylglycerol largely showed the same distribution of radioactivity among the individual species, except that the relative proportion of label was higher in the 16:0/16:0 and 16:0/18:0 species of phosphatidic acid and lower in the 16:0/20:4 and 18:0/20:4 species than in the corresponding species of diacylglycerol. The species pattern of de novo-synthesized diacylglycerol showed no differences from that of the phosphatidylcholine synthesized from it. From this result we concluded that the cholinephosphotransferase of lung microsomes is nonselective for individual species of the diacylglycerol substrate. The 16:0/18:1 and 16:0/18:2 species of phosphatidic acid, diacylglycerol and phosphatidylcholine showed a higher synthesis rate than their 18:0 counterparts, whereas the 16:0 or 18:0 analogues of species containing 20:4 and 22:6 fatty acids showed nearly the same synthesis rates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Further evidence for the existence of different diacylglycerol pools of the phosphatidylcholine synthesis in microsomes

Endogenous diacylglycerol and diacylglycerol, synthesized in vitro by glycerol 3-phosphate acylation, are not mixed and represent different substrate pools for the biosynthesis of phosphatidylcholine in microsomes of rat muscle, liver and lung. Freshly isolated lung microsomes contain 12-18 nmol diacylglycerol per mg protein, and incubation with CDPcholine showed a biphasic curve for the synthesis of phosphatidylcholine as lung microsomes enriched in diacylglycerol through the glycerol phosphate pathway. With respect to the synthesis of phosphatidylcholine, a part of this endogenous diacylglycerol (0.4-0.8 nmol/mg) was comparable with diacylglycerol de novo formed in vitro by glycerol 3-phosphate acylation. An increase in the relative proportion of de novo-formed diacylglycerol in the total amount of diacylglycerol caused an increase in phosphatidylcholine synthesis by nearly the same factor. The apparent Km of the de novo-formed diacylglycerol substrate for the choline phosphotransferase was 10-times higher than the pool size of this diacylglycerol substrate in freshly isolated lung microsomes. The results supported the idea that the availability of this substrate type may be rte limiting for the de novo synthesis of phosphatidylcholine. As shown by use of the proteolytic technique measuring the mannose-6-phosphatase as lumenal control activity, the phosphatidylcholine synthesis from de novo-formed diacylglycerol and endogenous as well as exogenous diacylglycerol seems to be located on the cytoplasmic leaflet of the microsomal vesicles isolated from rat lung.     

Characterization of sn-glycerol 3-phosphate acyltransferase from guinea pig harderian gland microsomes

Microsomal sn-glycerol 3-phosphate acyltransferase from the guinea pig Harderian gland was studied. Its specific activity (1.0 nmol/min X mg, with palmitoyl-CoA as a substrate) was almost the same as that of the rat liver microsomal enzyme. The enzyme acted on various types of acyl-CoA, the relative reaction rates being as follows: palmitoyl-CoA, 100(%); stearoyl-CoA, 30; oleoyl-CoA, 50; linoleoyl-CoA, 40; and arachidonoyl-CoA, 20. When assayed in the presence of 1 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the activity on palmitoyl-CoA was inhibited by only 20-30%, whereas those for other acyl-CoAs were completely abolished. The DTNB-resistant activity was inhibited by 0.1 mM dihydroxyacetonephosphate and 0.5 mM dithiothreitol, whereas the DTNB-sensitive activity was not affected. Furthermore, heat treatment at 50 degrees C for 15 min abolished most of the DTNB-sensitive activity, but not the DTNB-resistant activity. These results, taken together, suggested that the microsomal fraction of the guinea pig Harderian gland contained at least two types of sn-glycerol 3-phosphate acyltransferase, and that, in contrast to in the case of rat liver microsomes, a DTNB-resistant enzyme that utilized exclusively palmitoyl-CoA was predominant.     

Cytosol-stimulated remodeling of phosphatidylcholine in rat lung microsomes

When 600 × g supernatants of 10% (w/v) rat lung homogenates were incubated with CDP[Me-¹⁴C]choline, both saturated and unsaturated species of phosphatidylcholine were formed from endogenous diacylglycerols. The percentage radioactivity in the disaturated species of total phosphatidylcholine increased with time from 12% after 5 min to 30% after 60 min incubation. In similar experiments with 20000 × g supernatants, the increase in the disaturated species of microsomal phosphatidylcholine was from 25 to 37% over the same time period. In incubations of isolated microsomes in buffer, the percent of ¹⁴C label in disaturated phosphatidylcholine remained constant at a level of 25%. To investigate a possible role of cytosolic factor(s) in the increase in the percentage of disaturated phosphatidylcholine with time, microsomes were prelabeled by incubation in buffer with CDP[Me-¹⁴C]choline to give a fixed ratio of radioactive saturated and unsaturated phosphatidylcholine species. When the reisolated microsomes were incubated in buffer, the distribution of radioactivity over saturated and unsaturated species remained constant. In contrast, incubation of prelabeled microsomes in the presence of cytosol caused an increase in the percent radioactivity in saturated phosphatidylcholines from a starting value of 18 to 30% after 60 min incubation, while leaving total phosphatidylcholine radioactivity unaffected. These results indicate a remodeling of phosphatidylcholine under the influence of a cytosolic factor(s). Evidence is presented that suggests that Ca²⁺-independent cytosolic phospholipase A₂ activity as well as a microsomal ATP-independent CoA-mediated acyltransferase activity might contribute to this remodeling. The cytosol donates the necessary CoA for this acyl transfer as well as saturated acyl-CoA for the reacylation of lysophosphatidylcholine.     

Ethanolaminephosphotransferase in rat lung: selectivity for endogenous and exogenous diacylglycerol

Ethanolaminephosphotransferase (EC 2.7.8.1) activity was determined in lung microsomes using diacylglycerols generated endogenously from [14C]glycerol 3-phosphate and different mixtures of fatty acids. Ethanolaminephosphotransferase used endogenously generated dipalmitoylglycerol better than dioleoylglycerol. The apparent Km and the reaction rates for four different endogenously generated mixtures were the same (16 nmol/mg microsomal proteins). The apparent Km values for CDP-ethanolamine were the same (0.26 mm) for endogenously generated dipalmitoylglycerol and dioleoylglycerol. The amount of diacylglycerol generated in microsomes was 2-3-times the apparent Km for diacylglycerol. Dipalmitoylglycerol, supplied exogenously as a Tween 20/phosphatidylglycerol emulsion, was nearly twice as active as dioleoylglycerol. Both dipalmitoylglycerol and dioleoylglycerol were more active as substrates when emulsions were made with phosphatidylglycerol/Tween 20 than with Tween 20 alone. The results suggest that ethanolaminephosphotransferase in lung is relatively nonselective for molecular species of diacylglycerol. In addition, the results suggest that the concentration of diacylglycerol and the physical state in which it is presented to the enzyme can affect the apparent selectivity of ethanolaminephosphotransferase for diacylglycerols.     

Inhibition by ozone of the acylation of glycerol 3-phosphate in mitochondria and microsomes from rat lung

The synthesis of lipids from [U-¹⁴C]glycerol 3-phosphate by mitochondrial or microsomal fractions from rat lung was inhibited by ozone. The susceptible reaction was the first acylation of glycerol 3-phosphate. Enzymes unaffected by the ozone exposure included: acyl-CoA thioesterase, acyl-CoA thiokinase, acyl-CoA:acylglycerol 3-phosphate acyltransferase, acyl-CoA:diacylglycerol acyltransferase, and acyl-CoA:acylglycerophosphocholine acyltransferase. The effect of ozone on lipid synthesis is closely comparable to the inhibition by N-ethylmaleimide suggesting that the effect of ozone is the oxidation of enzyme sulfhydryl groups. There was no indication of lipid oxidation caused by ozone and no indication of the production of a stable toxic compound.     

Glycerol 3-phosphate acylation in microsomes of type II cells isolated from adult rat lung

Glycerol 3-phosphate acylation was studied in type II cells isolated from adult rat lung. The process was found to be largely microsomal. In the microsomes phosphatidic acid is the main product of glycerol 3-phosphate acylation. Glycerol-3-phosphate acyltransferase is rate limiting in the phosphatidic acid formation by the microsomes. Type II cell microsomes incorporate palmitoyl and oleoyl residues into phosphatidic acid at an equal rate if palmitoyl-CoA and oleoyl-CoA are added separately. However, if palmitoyl-CoA and oleoyl-CoA are added as an equimolar mixture the unsaturated fatty acyl moiety is incorporated much faster. Under the latter conditions monoenoic species constitute the most abundant products of glycerol 3-phosphate acylation. The microsomes incorporate both palmitoyl and oleoyl residues readily into both the 1- and 2-position of phosphatidic acid, even when palmitoyl-CoA and oleoyl-CoA are added together. Assuming that both phosphatidic acid phosphatase and cholinephosphotransferase do not discriminate against substrates with an unsaturated acyl moiety at the 1-position and a saturated acyl moiety at the 2-position, the last two observations indicate that a considerable percentage of phosphatidylcholine molecules synthesized de novo may have a saturated fatty acid at the 2-position and an unsaturated fatty acid at the 1-position, and that remodeling at the 1-position may be important for the formation of surfactant dipalmitoylphosphatidylcholine. They also indicate that type II cell microsomes are capable of synthesizing the dipalmitoyl species of phosphatidic acid. However, since there is a preference for the acylation of glycerol 3-phosphate with unsaturated fatty acyl residues, the percentage of dipalmitoyl species in the synthesized phosphatidic acid, and thereby the percentage of dipalmitoyl species in the phosphatidylcholine synthesized de novo, will probably depend on the relative availability of the various acyl-CoA species.     

Coenzyme A-mediated transacylation of sn-2 fatty acids from phosphatidylcholine in rat lung microsomes

Evidence was obtained for a CoA-dependent transfer of linoleate from rat lung microsomal phosphatidylcholine to lysophosphatidylethanolamine without the intervention of a Ca2+-requiring phospholipase A2 activity and ATP. To study this CoA-mediated transacylation process, microsomes were prepared in which the endogenous phosphatidylcholine was labeled by protein-catalyzed exchange with phosphatidylcholines containing labeled fatty acids in the sn-2-position. The apparent Km for CoA in the transfer of arachidonate from phosphatidylcholine to 1-acyllysophosphatidylethanolamine was 1.5 microM. At saturating lysophosphatidylethanolamine concentrations, the transacylation was linear with the amount of microsomal protein, i.e., a fixed percentage of the labeled fatty acid was transferred independent of the amount of microsomal protein. A maximal transfer of 12.2% for arachidonate and 2.0% for linoleate from the respective phosphatidylcholines to lysophosphatidylethanolamine was observed in 30 min. With 1-acyl-2-[1-14C]arachidonoylphosphatidylcholine as acyl donor, lysophosphatidylethanolamine was the best acceptor followed by lysophosphatidylglycerol and lysophosphatidylserine. Lysophosphatidate barely functioned as acceptor. These data provide further evidence for the widespread occurrence of CoA-mediated transacylation reactions. The arachidonate transacylation from phosphatidylcholine to other phospholipids in lung tissue may contribute to the low level of arachidonate in pulmonary phosphatidylcholine.     

Phosphatidylethanolamine and phosphatidylcholine are sources of diacylglycerol in ras-transformed NIH 3T3 fibroblasts

Ras-transformation of cells is accompanied by an increase of the level of diacylglycerol (DAG), which participates in the signal transduction pathways. DAG could be generated from phospholipids either by activation of phospholipase C or by a more complex pathway involving phospholipase D and phosphatidate phosphohydrolase. To clarify which phospholipids produce DAG and which pathways are involved, we examined the DAG generating enzyme activities, using phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) as substrates. The study showed that the breakdown of PC and more markedly of PE by phospholipases C and D was stimulated in membranes from ras-transformed cells. Phosphatidate phosphohydrolase activity was also elevated in oncogene-expressing cells. The increase in glycerol uptake was most pronounced in cells given PE, followed by PC. The fatty acid analysis revealed apparent similarities between the acyl chains of PE and DAG only in the transformed cells. These findings suggest that PE is a source of DAG in ras-fibroblasts but does not rule out the role of PC in DAG production, due to the activation of the PC-specific phospholipases C and D.     

Molecular species of phosphatidylcholine and phosphatidylglycerol in rat lung surfactant and different pools of pneumocytes type II.

It is not yet completely understood how a cell is able to export specific phospholipids, like dipalmitoylphosphatidylcholine (dipalmitoyl-PC), which is secreted by pneumocytes type II, into pulmonary surfactant. The acyl species composition of [3H]PC which was synthesized in type II cells in the presence of [2-3H]glycerol resembled the species composition of PC localized in intracellular pneumocyte membranes. This species pattern was different from the pattern of PC of lamellar bodies, i.e., intracellularly stored surfactant, by a higher proportion of dipalmitoyl-PC mainly at expense of 1-palmitoyl-2-oleoyl-PC. Lamellar body PC in turn showed the same species distribution as surfactant PC. The data suggest that subcellular compartmentation and/or intracellular transfer of PC destined to storage in lamellar bodies, but not secretion of lamellar bodies, involves an enrichment of dipalmitoyl-PC and a depletion of 1-palmitoyl-2-oleoyl-PC. In contrast, the acyl species pattern of phosphatidylglycerol does not seem to undergo gross changes on the path from synthesis to secretion.     

Cellular RNA and influenza-virion RNA are synthesized from different pyrimidine-nucleoside-triphosphate pools in chick-embryo cells.

Chick embryo cells infected with an influenza A (fowl plague) virus have been labelled with (3H)-uridine for different lengths of time. Virion RNA and cellular RNA have been separated by specific hybridization with a surplus of unlabelled viral complementary RNA and RNase digestion. The ratio of the specific radioacticity in the UMP and CMP moieties of both types of RNA has been determined. Since the rate of approach to equilibrium of CMP to UMP labelling of both types of RNA is completely different it is concluded that cellular and virion RNA are synthesized using different pyrimidine nucleoside triphosphate pools.     

Utilization of disaturated and unsaturated phosphatidylcholine and diacylglycerols by cholinephosphotransferase in rat lung microsomes

1. Cholinephosphosphotransferase catalyzes the conversion of diacylglycerol and CDPcholine into phosphatidylcholine and CMP. Incubation of rat lung microsomes containing phosphatidyl[Me-14C]choline with CMP resulted in an increase in water-soluble radioactivity, suggesting that also in rat lung microsomes the cholinephosphotransferase reaction is reversible. 2. Microsomes containing 14C-labeled disaturated and 3H-labeled monoenoic phosphatidylcholine were prepared by incubation of these organelles with [1-14C]palmitate and [9,10-3H2]oleate in the presence of 1-palmitoyl-sn-glycero-3-phosphocholine, ATP, coenzyme A and MgCl2. Incubation of these microsomes with CMP resulted in an equal formation of 14C- and 3H-labeled diacylglycerols, indicating that disaturated and monoenoic phosphatidylcholines were used without preference by the backward reaction of the cholinephosphotransferase. When in a similar experiment the phosphatidylcholine was labeled with [9,10-3H2]palmitate and [1-14C]linoleate, somewhat more 14C- than 3H-labeled diacylglycerol was formed. 3. The backward reaction was used to generate membrane-bound mixtures of [1-14C]palmitate- and [9,10-3H2]oleate- or of [9,10-3H2]palmitate- and [1-14C]linoleate-labeled diacylglycerols. When the microsomes containing diacylglycerols were incubated with CDPcholine, both 3H- and 14C-labeled diacylglycerols were used for the formation of phosphatidylcholine, indicating that there is no absolute discrimination against disaturated diacylglycerols. This observation is in line with our previous findings and indicates that also the CDPcholine pathway may contribute to dipalmitoylphosphatidylcholine synthesis in lung.     

Selective changes in enzymes of the sn-glycerol 3-phosphate and dihydroxyacetone-phosphate pathways of triacylglycerol biosynthesis during differentiation of 3T3-L1 preadipocytes

Enzyme activities of the sn-glycerol 3-phosphate (glycerol-P) and of the dihydroxyacetone-phosphte (DHAP) pathway of glycerolipid biosynthesis were investigated during the differentiation of 3T3-L1 preadipocytes into adipocytes. Total particulate glycerol-P and DHAP acyltransferase activities increased 70- and 30-fold, respectively, during differentiation induced with methylisobutylxanthine and dexamethasone. The N-ethylmaleimide-sensitive (microsomal) glycerol-P and DHAP acyltransferase activities were virtually undetectable in nondifferentiated cells, and increased in parallel over 70-fold during differentiation. These and several kinetic observations are consistent with the induction of a single microsomal enzyme having dual activity. During differentiaion, the N-ethylmaleimide-resistant DHAP acyltransferase activity increased 10-fold, suggesting the presence of at least two DHAP acyltransferase isoenzymes. Qualitatively similar changes in microsomal glycerol-P and DHAP acyltransferase activities were observed when cell differentiation was induced with insulin or with insulin plus dexamethasone and methylisobutylxanthine. Acyl-DHAP oxidoreductase (EC 1.1.1.101) specific activity increased only 3- to 5-fold during adipocyte differentiation. Alkyl-DHAP synthase activity was not detected. These data demonstrate that selective changes in enzyme activities of the gycerol-P pathways of glycerolipid synthesis occur during the differentiation of 3T3-L1 preadipocytes.     

The relationship between palmitoyl-coenzyme A synthetase activity and esterification of sn-glycerol 3-phosphate in rat liver mitochondria

1. The specific activities for palmitoyl-CoA synthetase and for sn-glycerol 3-phosphate esterification, with palmitoyl-CoA generated either by the endogenous synthetase or from palmitoyl-(−)-carnitine, CoA and excess of carnitine palmitoyltransferase, were measured with rat liver mitochondria. 2. The mean specific activity of palmitoyl-CoA synthetase was approximately five- and seven-fold the rates of sn-glycerol 3-phosphate esterification from palmitate and palmitoyl-(−)-carnitine respectively. No significant correlation was found in different rats between the activities of palmitoyl-CoA synthetase and sn-glycerol 3-phosphate esterification from either acyl precursor. However, there was a significant correlation (r=0.83, P<0.001) between the rates of glycerolipid synthesis from palmitate and palmitoyl-(−)-carnitine. 3. The mean molar composition of the glycerolipid synthesized from palmitate was 58% lysophosphatidate, 31% phosphatidate and 11% neutral lipid. With palmitoyl-(−)-carnitine the equivalent values were 70, 23 and 7%, which were significantly different. 4. When palmitoyl-CoA synthetase had been inactivated by 60–70% after preincubation of mitochondria at 37°C, it became rate-limiting in glycerolipid biosynthesis. Additions of 1–5mm-ATP prevented inactivation of palmitoyl-CoA synthetase. 5. Preincubation also inhibited the oxidation of palmitate, palmitoyl-CoA, palmitoyl-(−)-carnitine and malate plus glutamate. These inhibitions could not be prevented by addition of ATP. 6. Diversion of palmitoyl-CoA to form palmitoyl-(−)-carnitine did not inhibit sn-glycerol 3-phosphate esterification. 7. The palmitoyl-CoA pool synthesized by the palmitoyl-CoA synthetase was augmented by adding partially purified synthetase or carnitine palmitoyltransferase and palmitoyl-(−)-carnitine. No stimulation of palmitate incorporation into glycerolipids occurred. 8. At low concentrations of Mg²⁺, palmitate, ATP and CoA the velocity with palmitoyl-CoA synthetase decreased more than that of glycerolipid synthesis from palmitate. 9. It is concluded that in the presence of optimum substrate concentrations the activity of sn-glycerol 3-phosphate acyltransferase and not of palmitoyl-CoA synthetase is rate-limiting in the synthesis of phosphatidate and lysophosphatidate in isolated rat liver mitochondria.