Acylation Of Glycerol 3-phosphate Research Articles

Crystallization and preliminary X-ray analysis of the glycerol-3-phosphate 1-acyltransferase from squash (Cucurbita moschata)

Glycerol-3-phosphate 1-acyltransferase (E.C. 2.3.1.15; G3PAT) catalyses the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acylCoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol 3-phosphate. Crystals of squash G3PAT have been obtained by the hanging-drop method of vapour diffusion using PEG 4000 as the precipitant. These crystals are most likely to belong to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 61.1, b = 65.1, c = 103.3 A, alpha = beta = gamma = 90 degrees and a monomer in the asymmetric unit. X-ray diffraction data to 1.9 A resolution have been collected in-house using a MAR 345 imaging-plate system.

Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction.

Alignment of amino acid sequences from various acyltransferases [sn-glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), acyl-CoA:dihydroxyacetone-phosphate acyltransferase (DHAPAT), 2-acylglycerophosphatidylethanolamine acyltransferase (LPEAT)] reveals four regions of strong homology, which we have labeled blocks I-IV. The consensus sequence for each conserved region is as follows: block I, [NX]-H-[RQ]-S-X-[LYIM]-D; block II, G-X-[IF]-F-I-[RD]-R; block III, F-[PLI]-E-G-[TG]-R-[SX]-[RX]; and block IV, [VI]-[PX]-[IVL]-[IV]-P-[VI]. We hypothesize that blocks I-IV and, in particular, the invariant amino acids contained within these regions form a catalytically important site in this family of acyltransferases. Using Escherichia coli GPAT (PlsB) as a model acyltransferase, we examined the role of the highly conserved amino acid residues in blocks I-IV in GPAT activity through chemical modification and site-directed mutagenesis experiments. We found that the histidine and aspartate in block I, the glycine in block III, and the proline in block IV all play a role in E. coli GPAT catalysis. The phenylalanine and arginine in block II and the glutamate and serine in block III appear to be important in binding the glycerol 3-phosphate substrate. Since blocks I-IV are also found in LPAAT, DHAPAT, and LPEAT, we believe that these conserved amino acid motifs are diagnostic for the acyltransferase reaction involving glycerol 3-phosphate, 1-acylglycerol 3-phosphate, and dihydroxyacetone phosphate substrates.

Hepatic phosphatidylcholines: evidence for synthesis in the rat by extensive reutilization of endogenous acylglycerides.

Studies were performed to compare the extent of fatty acid incorporation into liver phosphatidylcholines (PCs) by acyl remodeling and by de novo synthesis. To this end, isolated rat livers were first perfused with palmitoleic acid (16:1) and [2-3H]glycerol and then with 17:1 fatty acid and nonradiolabeled glycerol that resulted in the formation of new molecular species of radiolabeled PCs containing 16:1 and 17:1 acyl groups. The specific activities of newly formed molecular species of the de novo precursors of acylglyceride synthesis, phosphatidic acids (PAs) and diglycerides (DGs), and the products of synthesis, PCs and triglycerides (TGs), were measured at periods during both the labeling period of perfusion with 16:1 (first 15 min) and the more prolonged chase period with 17:1 (up to 120 min). At the end of the labeling period, the specific activity of all the 16:1-containing PAs, DGs, and 16:1-16:1-16:1 and 16:1-16:1-18:2 TGs were the same and were much higher than any molecular species that did not contain 16:1. The specific activities of these molecular species are indicative of the specific activity of molecular species synthesized exclusively by de novo synthesis (i.e., by acylation of glycerol 3-phosphate) during the labeling period. In contrast, the specific activity of 16:1-16:1 PC was only 2/3 that of the other 16:1-16:1 glycerides, and the specific activities of the other 16:1-containing PCs were only about 1/3 that of the corresponding 16:1-containing PAs, DGs, and 16:1-16:1 TGs. After the labeling period and during the chase period with perfusion of 17:1 and nonradiolabeled glycerol, the specific activities of major 16:1 PCs exceeded the specific activities of their corresponding PAs and DGs and remained considerably higher than these precursors of de novo synthesis for the duration of perfusions. However, during this period, the specific activities of major 16:1 PCs were less than their corresponding molecular species of TGs. During the chase period, new 17:1 molecular species of PCs were formed that were also radiolabeled. The specific activity of 16:1-17:1 PC, the 17:1 PC with the highest specific activity, always exceeded its corresponding PA and DG precursors. During the chase period, non-16:1 and non-17:1 molecular species of PCs that comprised the bulk of hepatic PCs were also radiolabeled and the specific activities of these molecular species progressively increased during this period.(ABSTRACT TRUNCATED AT 400 WORDS)

Glycerol 3-phosphate acylation by microsomal fractions from avocado mesocarp.

Glycerol 3-phosphate acylation by microsomal fractions from avocado mesocarp.

11] Diacylglycerol acyltransferase and monoacylglycerol acyltransferase from liver and intestine

The acylation of diacylglycerol catalyzed by the diacylglycerol acyltransferase is the only enzyme reaction unique to triacylglycerol synthesis. This reaction lies at the diacylglycerol branch point of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol synthesis. In most tissues, the major pathway for the biosynthesis of the diacylglycerol substrate proceeds from the acylation of glycerol 3-phosphate. In intestine and liver, however, the monoacylglycerol pathway provides an alternate route for diacylglycerol synthesis. 2-monoacylglycerol—produced by the action of gastric and pancreatic lipases on dietary triacylglycerol—enters the intestinal mucosa where monoacylglycerol acyltransferase plays a major role in resynthesizing triacylglycerol. In the liver of certain species, monoacylglycerol acyltransferase activity is high during early development; however, neither its specific role in liver nor the source of the monoacylglycerol substrate is known. Both diacylglycerol acyltransferase and monoacylglycerol acyltransferase are intrinsic membrane proteins whose active sites face the cytosolic surface of the endoplasmic reticulum.

Utilization of petroselinate (C 18:1 Δ6 ) by glycerol acylation enzymes in microsomal preparations of developing embryos of carrot ( Daucus carota L.), safflower ( Carthamus tinctorius L.) and oil rape ( Brassica napus L.)

Glycerol 3-phosphate acyltransferase (E.C. 2.3.1.15), lysophoshatidic acid acyltransferase (E.C. 2.3.1.51) and lysophosphatidylcholine acyltransferase (E.C. 2.3.1.23) activities with acyl substrates containing petroselinate (18:1 Δ6 ) or oleate (18:1 Δ9 ) were compared in microsomal preparations from developing zygotic embryos of carrot and safflower, microspore-derived embryos from rape and somatic embryos from carrot. In the glycerol 3-phosphate acylation step, 18:1 Δ9 -CoA was used at a higher rate than 18:1 Δ6 -CoA in active preparations from all species but the enzyme from the zygotic carrot embryos showed higher rates with 18:1 Δ6 -CoA substrate than the safflower and rape enzymes. Acylation of sn-1 -18:1 Δ9 -glycerol 3-phosphate (18:1 Δ9 -LPA) or 18:1 Δ6 -LPA with 18:1 Δ9 -CoA showed similar rates with all species studied and was much higher than with 18:1 Δ6 -CoA as acyl donor. The preparations from carrot zygotic embryos acylated 18:1 Δ6 -LPA with 18:1 Δ6 -CoA at a substantial rate whereas this combination of substrates was only poorly utilized in preparations of carrot somatic embryos and not at all in safflower and rape. Microsomes from carrot zygotic embryos acylated both 18:1 Δ6 -CoA and 18:1 Δ9 -CoA to exogenous lysophosphatidylcholine, when presented as single acyl-CoA substrates, but 18:1 Δ9 -CoA utilization was greatly inhibited by the presence of 18:1 Δ6 -CoA. This preparation also acylated sn-[ 14 C] glycerol 3-phosphate with both 18:1 Δ9 -CoA and 18:1 Δ6 -CoA in the formation of radioactive phosphatidic acid, diacylglycerols and triacylglycerols. The amount and distribution of label between different lipid classes was similar with the two acyl-CoAs and with very little [ 14C]glycerol entering phosphatidylcholine. Fatty acid analysis of various lipids in developing carrot seed are also presented. The results of the present study are discussed in terms of specializations of lipid synthesizing enzymes in tissues accumulating high amounts of 18:1 Δ6 .

Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castor-bean (Ricinus communis) endosperm

Microsomal membrane preparations from the developing endosperm of castor bean (Ricinus communis) catalysed the transfer of oleate from [14C]oleoyl-CoA to phosphatidylcholine (PtdCho). In the presence of NADH, radioactive ricinoleate (12-hydroxyoctadec-9-enoate) was synthesized from [14C]oleate, and this was largely recovered in PtdCho and as free fatty acid. The addition of unlabelled ricinoleoyl-CoA to these incubation mixtures did not increase the low [14C]ricinoleate concentration found in the acyl-CoA fraction nor decrease the [14C]ricinoleate concentration in PtdCho and free fatty acid, and thus no evidence was obtained for a hydroxylation with oleoyl-CoA as a substrate. The addition of NADH, necessary for the formation of ricinoleate, caused a decrease of the total radioactivity in PtdCho with a corresponding increase in the amount of label in free ricinoleic acid. This increase was due to the action of a phospholipase A, which released ricinoleic acid but not oleic acid from PtdCho. Such a phospholipase activity, attacking ricinoleoyl-PtdCho but not oleoyl-PtdCho, was also demonstrated in microsomal preparations from developing cotyledons of safflower and oil-seed rape. An analysis of the acyl groups at different positions in microsomal PtdCho of castor bean showed that ricinoleate was almost entirely associated with position sn-2. Likewise the [14C]ricinoleate in [14C]PtdCho formed after incubations with microsomal preparations with NADH and [14C]oleoyl-CoA resided in position sn-2 with none in position sn-1. In contrast, the [14C]linoleate formed by desaturation of [14C]oleoyl-PtdCho was present at both positions. In the presence of ATP, CoA and Mg2+, the ricinoleate acid released from PtdCho was activated to ricinoleoyl-CoA. The ricinoleoyl-CoA was an efficient acyl donor in the acylation of glycerol 3-phosphate (Gro3P) to yield phosphatidic acid and triacylglycerols. In microsomal preparations incubated with an equimolar mixture of [14C]oleoyl-CoA and [14C]ricinoleoyl-CoA in the presence of Gro3P, only a minor amount of [14C]ricinoleate entered PtdCho, and this was believed to be via the exchange of phosphocholine groups between a diacylglycerol pool and the PtdCho. On the basis of our results, a scheme of ricinoleate formation and its incorporation into triacylglycerols in castor-bean endosperm is proposed.

Lipid synthesis in permeabilized cultured rat hepatocytes

Hepatic lipid synthesis was verified and studied in lysolecithin-permeabilized cultured rat hepatocytes and compared to that of intact liver cells. Triacylglycerol synthesis in permeabilized cells incubated in the presence of glycerol 3-phosphate and long chain fatty acids approached that of intact hepatocytes. Similarly, phosphatidylcholine synthesis in permeable cells incubated in the presence of exogenous CDP-choline was similar to that of intact hepatocytes and at the expense of microsomal neutral lipid synthesis. Phosphatidic acid accumulation in lysolecithin-permeabilized liver cells was remarkably increased as compared to that of intact cells, and its synthesis was mostly accounted for by the activity of mitochondrial glycerol-3-phosphate acyltransferase. Mitochondrial-generated phosphatidate was found to migrate to the endoplasmic reticulum, thus establishing a novel lipid esterification pathway which begins in mitochondrial glycerol 3-phosphate acylation and results in microsomal triacylglycerol and phospholipid synthesis. The free access of permeabilized liver cells to substrates and modulators of lipid synthesis, while maintaining an overall synthetic pattern similar to that of intact hepatocytes, makes them a system of choice for studying hepatic lipid synthesis in general and the microsomal/mitochondrial distribution of fluxes in particular.

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

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

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-fonned diacylglycerol in the total amount of diacylglycerol caused an increase in phosphatidylcholine synthesis by nearly the same factor. The apparent K m of the de novo-fonned diacylglycerol substrate for the choline phosphotransf erase 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 rate 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.

Acylation of glycerol 3-phosphate is the sole pathway of de novo phospholipid synthesis in Escherichia coli

The inhibition of phospholipid synthesis engendered by starving glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli (plsB or gpsA) for G3P is incomplete; 5 to 10% of the normal rate of phospholipid synthesis remains, even after prolonged starvation. We report that G3P starvation of a strain having lesions in both the gpsA and plsB genes resulted in essentially complete (greater than 98.5%) inhibition of phospholipid synthesis, indicating that all de novo glycerolipid synthesis in E. coli proceeds by acylation of G3P.

In vitro effects of chlorpromazine on glycerol 3-phosphate acyl transferase and 1-acylglycerol-3-phosphate acyltransferase in rat liver microsomes

The in vitro effect of chlorpromazine on rat liver glycerol-3-phosphate acyltransferase and 1-acylglycerol-3-phosphate acyltransferase was studied. Chlorpromazine decreased glycerol-3-phosphate acyltransferase activity in a concentration-dependent manner. The inhibition was competitive with respect to palmitoyl-CoA, while non-competitive with respect to sn-glycerol-3-phosphate. K i, was determined to be approximately 0.15mM with respect to both palmitoyl-CoA and sn-glycerol-3-phosphate. From these results, together with the inhibitory effect of amphiphilic anions and neutral detergents on the enzyme demonstrated by others, it was proposed that the hydrophobic moiety of chlorpromazine competes with acyl-CoA. The activity of 1-acylglycerol-3-phosphate acyltransferase was inhibited by excess of 1-acyl- sn-glycerol-3-phosphate. In the acceptor concentration range where the substrate inhibition was observed, chlorpromazine showed stimulatory effect on the enzyme activity. At lower concentrations of the acceptor, however, chlorpromazine produced marked inhibition of the enzyme activity. From the kinetic analysis, the inhibition was found to be uncompetitive with respect to acyl-CoA. It was found that the enzyme was more susceptible to the inhibitory action of chlorpromazine with unsaturated acyl-CoAs than with the saturated species, raising the possibility that chlorpromazine alters the molecular species composition of phosphatidic acid produced by the acylation reaction.

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.

The effect of coronary artery occlusion and reperfusion on the activities of triglyceride lipase and glycerol 3-phosphate acyl transferase in the isolated perfused rat heart

Glycerol 3-phosphate acyltransferase (GPAT) and Triglyceride lipase (TGL) were measured in homogenates from non-ischaemic and ischaemic tissue from the isolated perfused rat heart. Ischaemia was produced by occlusion of the left descending coronary artery for 10 min. Compared to activities measured in tissue from normally perfused hearts, GPAT activity measured in tissue from the ischaemic area was considerably reduced. TGL activity in the ischaemic area was markedly increased compared to activity measured in normally perfused hearts. No change was seen in GPAT or TGL activity measured in tissue from the non-ischaemic area. The change in activities produced by ischaemia were prevented by pre-perfusion with the cardio-selective beta-antagonist Atenolol. Reperfusion of the ischaemic area resulted in TGL activity returning to the value measured in tissue from normally perfused hearts. However, GPAT activity, after 1 min of reperfusion, fell to a value lower than after 10 min ischaemia. The reperfusion-induced fall in GPAT activity was prevented by pre-perfusion with the alpha 1 antagonist Doxasozin. Pre-perfusion of the alpha 2 antagonist Yohimbine resulted in a prolongation of the increased TGL activity in the ischaemic area during reperfusion. All changes in enzyme activities were prevented by injection of 6 OH-dopamine 24 h before hearts were removed. These changes in enzyme activities show that during ischaemia there is an increased beta-adrenergic drive. On reperfusion the beta-adrenergic drive is removed but an alpha 1 adrenergic drive becomes apparent.

The synthesis of linoleate and phosphatidic acid and its relationship to oil production in the microsomes of developing seeds of safflower ( Carthamus tinctorius, L. var. Gila)

Microsomes from the developing cotyledons of safflower seeds (var. Gila) were incubated, under desaturating conditions, with [1- 14C]oleoyl-CoA in the presence of glycerol 3-phosphate. The mass and specific radioactivity of oleate and linoleate in acyl-CoA, phosphatidylcholine and phosphatidic acid showed that (i) the oleate in oleoyl-CoA was selectively transferred to position 2 of phosphatidylcholine by acyl exchange where it underwent desaturation to linoleate; (ii) the newly formed linoleate was returned by further acyl exchange, to the acyl-CoA pool from which it was selectively utilised in the acylation of glycerol 3-phosphate to yield phosphatidic acid, a precursor of triacylglycerol. The results confirm that acyl exchange between acyl-CoA and phosphatidylcholine plays a major role in regulating the quality of C18 unsaturated fatty acids in the acyl-CoA pool for oil synthesis.

The role of the acyl-CoA pool in the synthesis of polyunsaturated 18-carbon fatty acids and triacylglycerol production in the microsomes of developing safflower seeds

Microsomes isolated from the developing cotyledons of the seeds of the safflower varieties, very-high-linoleate, Gila and high-oleate, were capable of exchanging the acyl groups in acyl-CoA with the fatty acids in position 2 of phosphatidylcholine. The specificity of the ‘acyl-exchange’ towards the acyl moiety in acyl-CoA was selective in the order: oleate > linoleate > linolenate. Stearoyl-CoA was completely selected against when presented in a mixed substrate with unsaturated 18-carbon acyl-CoAs. Microsomes, of the very-high-linoleate safflower variety, rapidly desaturated in situ-labelled [ 14C]oleoylphosphatidylcholine in the presence of NADH. Little oleate desaturation, however, was observed in the microsomes of the high-oleate variety. Microsomes of the Gila and high-oleate varieties of safflower rapidly synthesised phosphatidic acid by the acylation of glycerol 3-phosphate with acyl-CoA. The phosphatidic acid was metabolised to diacylglycerol, which was further acylated to triacylglycerol. A strong selectivity for linoleoyl-CoA was found for the acylation of glycerol 3-phosphate in both the Gila and high-oleate microsomes. On the basis of these results, we propose that the pattern of 18-carbon unsaturated fatty acids in the triacylglycerols of all ‘oil’-producing seeds is a direct reflection of the fatty acids in the acyl-CoA pool. This, in turn, is governed by: A, the rate and specificity of the acyl exchange between acyl-CoA and phosphatidylcholine; B, the rate of oleate (and linoleate) desaturation in phosphatidylcholine; and C, the rate and specificity of the glycerophosphate acyltransferase.

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.

Positional Specificity and Fatty Acid Selectivity of Purified sn-Glycerol 3-Phosphate Acyltransferases from Chloroplasts

Soluble acyl-CoA:sn-glycerol 3-phosphate acyltransferases (EC 2.3.1.15) which are localized in chloroplasts were purified from leaves of Pisum sativum and Spinacia oleracea and obtained free from interfering activities. The purification raised the specific activities by factors of about 1,000 for pea and 200 for spinach preparations. In pea chloroplasts, acyltransferase activity occurs in two soluble forms with apparent isoelectric points of 6.3 and 6.6. For both forms, the same molecular weight of about 42,000 was determined. The enzyme from spinach chloroplasts showed a slightly higher molecular weight and a lower isoelectric point of 5.2.The enriched enzyme fractions possessed a specificity for glycerol 3-phosphate as acyl acceptor and did not use dihydroxyacetone phosphate. Besides acyl-CoA, acyl-acyl carrier protein also can function as acyl donor. With acyl-CoA as acyl donor, the enzyme shows a high positional specificity, since the predominant product is 1-acylglycerol 3-phosphate.Different acyl-CoAs, when offered separately, were all accepted as substrates, whereas incubations with mixtures of palmitoyl-, stearoyl-, and oleoyl-CoA demonstrated a preference for oleic acid. The acyltransferase from spinach displays higher selectivity than does the enzyme from pea and, therefore, may be responsible for the preferred esterification of oleic acid at the C-1 position in chloroplast lipids and the exclusion of palmitic acid from this position as observed during in vivo labeling experiments.

Biosynthesis of neutral lipids by mitochondria and microsomes during development of insects

1. 1. The stereospecific distribution of fatty acids in triacylglycerols was very similar in larvae and pharate adults of Ceratitis capitata. Palmitate accounted for about 70% of the fatty acids located at the sn-1 position whereas oleate was mainly restricted to the sn-2 position. 2. 2. The esterification of labelled 2-oleoyl- sn-[2 n- 3H]glycerol by mitochondrial and microsomal preparations from either larvae or pharate adults was higher than that of 2-palmitoyl- sn-glycerol. However, the capacity of both cellular preparations of the larvae for the acylation of the 2-acyl- sn-glycerols was higher than that of the pharate adult preparations. 3. 3. Double-label experiments using 2-oleoyl- sn-[2 n- 3H]glycerol and sn-[U- 14C]glycerol 3-phosphate were carried out to test the relative utilization of both substrates by cellular fractions of different stages of development of the insect. The monoacylglycerol pathway had a greater quantitative importance than the acylation of glycerol 3-phosphate by either mitochondrial or microsomal preparations. 4. 4. In vivo incorporation of 2-oleoyl- sn-[2 n- 3H]glycerol and [1- 14C]palmitate showed that both labelled precursors were maximally incorporated by the insect at the larval pupal apolysis stage. The labelling contributions of both moieties agreed with the important and different participation of the triacylglycerol acyltransferase activity in the mechanisms of lipid synthesis according to the stage of development. The fatty acid distribution of in vivo synthesized triacylglycerols showed a decrease of the de novo pathway of synthesis when development proceeds.

Triacylglycerol synthesis in lipid particles from baker's yeast ( Saccharomyces cerevisiae)

Triacylglycerol synthesis has been studied in a lipid particle preparation of baker's yeast ( Saccharomyces cerevisiae), and compared with the synthesis in other subcellular fractions. Fatty acid-CoA ligase (AMP) (EC 6.2.1.3) activity and sn-glycerol 3-phosphate acyltransferase activity (EC 2.3.1.15) were present in all the subcellular fractions tested but the highest specific activities of both enzymes were observed with the lipid particle fraction. The products of the glycerol 3-phosphate acylation indicate that triacylglycerol synthesis proceeds through the phosphatidic acid pathway. However, only a small and nearly constant amount of lysophosphatidic acid was found with the lipid particle fraction while the other subcellular fraction produced lysophosphatidic and phosphatidic acid with a more pronounced precursor/product relationship. Triacylglycerol synthesis from endogenous diacylglycerol present in the lipid particle was also demonstrated.