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.     

Oil biosynthesis in microsomal membrane preparations from Mortierella alpina

Microsomal membrane preparations from Mortierella alpina catalysed the conversion of sn-glycerol 3-phosphate and [(14)C]oleoyl-CoA to radioactive phosphatidic acid, diacylglycerol and triacylglycerol. Experiments with lysophosphatidic acid and [(14)C]oleoyl-CoA gave a similar pattern of radioactivity in the complex lipids. The specific activity of lysophosphatidate acyltransferase was almost eight times greater than sn-glycerol-3-phosphate acyltransferase, indicating that the first acylation step was limiting in oil assembly in the microsomal membranes. Little conversion of radioactive oleate into phosphatidylcholine occurred, suggesting that triacylglycerol assembly and its relationship to phosphatidylcholine metabolism differed to that found in oilseeds.     

The acylation of sn-glycerol 3-phosphate and the metabolism of phosphatidate in microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius L.) seed.

Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalysed the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. The resulting phosphatidate was further utilized in the synthesis of diacyl- and tri-acylglycerol by the reactions of the so-called 'Kennedy pathway' [Kennedy (1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940]. Diacylglycerol equilibrated with the phosphatidylcholine pool when glycerol backbone, with the associated acyl groups, flowed from phosphatidate to triacylglycerol. The formation of diacylglycerol from phosphatidate through the action of a phosphatidate phosphohydrolase (phosphatidase) was substantially inhibited by EDTA and, under these conditions, phosphatidate accumulated in the microsomal membranes. The inhibition of the phosphatidase by EDTA was alleviated by Mg2+. The presence of Mg2+ in all incubation mixtures stimulated quite considerably the synthesis of triacylglycerol in vitro. Microsomal preparations incubated with acyl-CoA, sn-glycerol 3-phosphate and EDTA synthesized sufficient phosphatidate for the reliable analysis of its intramolecular fatty acid distribution. In the presence of mixed acyl-CoA substrates the sn-glycerol 3-phosphate was acylated exclusively in position 1 with the saturated fatty acids, palmitate and stearate. The polyunsaturated fatty acid linoleate was, however, utilized largely in the acylation of position 2 of sn-glycerol 3-phosphate. The affinity of the enzymes involved in the acylation of positions 1 and 2 of sn-glycerol 3-phosphate for specific species of acyl-CoA therefore governs the non-random distribution of the different acyl groups in the seed triacylglycerols. The acylation of sn-glycerol 3-phosphate in position 1 with saturated acyl components also accounts for the presence of these groups in position 1 of sn-phosphatidylcholine through the equilibration of diacylglycerol with the phosphatidylcholine pool, which occurs when phosphatidate is utilized in the synthesis of triacylglycerol. These results add further credence to our previous proposals for the regulation of the acyl quality of the triacylglycerols that accumulate in developing oil seeds [Stymne & Stobart (1984) Biochem. J. 220, 481-488; Stobart & Stymne (1985) Planta 163, 119-125].     

Comparison of glycerolipid biosynthesis in non-green plastids from sycamore (Acer pseudoplatanus) cells and cauliflower (Brassica oleracea) buds.

The availability of methods to fractionate non-green plastids and to prepare their limiting envelope membranes [Alban, Joyard & Douce (1988) Plant Physiol. 88, 709-717] allowed a detailed analysis of the biosynthesis of lysophosphatidic acid, phosphatidic acid, diacylglycerol and monogalactosyl-diacylglycerol (MGDG) in two different types of non-green starch-containing plastids: plastids isolated from cauliflower buds and amyloplasts isolated from sycamore cells. An enzyme [acyl-ACP (acyl carrier protein):sn-glycerol 3-phosphate acyltransferase) recovered in the soluble fraction of non-green plastids transfers oleic acid from oleoyl-ACP to the sn-1 position of sn-glycerol 3-phosphate to form lysophosphatidic acid. Then a membrane-bound enzyme (acyl-ACP:monoacyl-sn-glycerol 3-phosphate acyltransferase), localized in the envelope membrane, catalyses the acylation of the available sn-2 position of 1-oleoyl-sn-glycerol 3-phosphate by palmitic acid from palmitoyl-ACP. Therefore both the soluble phase and the envelope membranes are necessary for acylation of sn-glycerol 3-phosphate. The major difference between cauliflower (Brassica oleracea) and sycamore (Acer pseudoplatanus) membranes is the very low level of phosphatidate phosphatase activity in sycamore envelope membrane. Therefore, very little diacylglycerol is available for MGDG synthesis in sycamore, compared with cauliflower. These findings are consistent with the similarities and differences described in lipid metabolism of mature chloroplasts from 'C18:3' and 'C16:3' plants (those with MGDG containing C18:3 and C16:3 fatty acids). Sycamore contains only C18 fatty acids in MGDG, and the envelope membranes from sycamore amyloplasts have a low phosphatidate phosphatase activity and therefore the enzymes of the Kornberg-Pricer pathway have a low efficiency of incorporation of sn-glycerol 3-phosphate into MGDG. By contrast, cauliflower contains MGDG with C16:3 fatty acid, and the incorporation of sn-glycerol 3-phosphate into MGDG by the enzymes associated with envelope membranes is not limited by the phosphatidate phosphatase. These results demonstrate that: (1) non-green plastids employ the same biosynthetic pathway as that previously established for chloroplasts (the formation of glycerolipids is a general property of all plastids, chloroplasts as well as non-green plastids), (2) the envelope membranes are the major structure responsible for the biosynthesis of phosphatidic acid, diacylglycerol and MGDG, and (3) the enzymes of the envelope Kornberg-Pricer pathway have the same properties in non-green starch-containing plastids as in mature chloroplasts from C16:3 and C18:3 plants.     

Comparison of the acylation of sn-glycerol 3-phosphate and membrane-bound lipid in the microsomal fraction from rabbit brain throughout maturation.

1. Age-related changes in the specific activity of palmitoyl-CoA synthetase, sn-glycerol 3-phosphate acyltransferase (EC 2.3.1.15) and the esterification of [3H]palmitate into endogenous lipid in the microsomal fraction from rabbit brain have been determined throughout development. 2. The increased specific activity of sn-glycerol 3-phosphate acyltransferase at the onset of myelination (rising in parallel with other lipogenic enzymes) is consistent with a direct role of the acyltransferase in promoting the accumulation of cerebral lipid. In adult brain microsomes, although the specific activity was low, the total activity was only 20% lower than during active myelination. 3. Palmitoyl-CoA, synthesized by the palmitoyl-CoA synthetase in the microsomal membrane, was the preferred substrate for the esterification of sn-glycerol 3-phosphate. There was no evidence for a pool of palmitoyl-CoA formed from palmitate. 4. The esterification of [3H]palmitate into membrane-bound lipid remained high throughout development and may be part of an acyl-exchange cycle via lysophospholipids. [3H]palmitate was incorporated into both neutral lipids and phospholipids, while phosphatidic acid was the major product of sn-[1(3)-3H]-glycerol-3-phosphate esterification. 5. The microsomal fraction contained a pool of unesterified fatty acid, which was activated and esterified into sn-glycerol 3-phosphate.     

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.     

Phosphatidic acid metabolism in rat liver microsomes.

Rat liver microsomes contain phosphatidate phosphatases which split phosphatidic acid into inorganic phosphate and diacylglycerol and a system of phospholipases and lipases, which split phosphatidic acid into free fatty acids, glycerol and inorganic phosphate. In the presence of ATP,CoA and [1-14C]palmitate, part of the monoacyl-sn-glycerol 3-phosphate formed by phospholipase action is reesterified, yielding radioactive phosphatidic acid. The sum of di- and triacylglycerols formed from phosphatidic acid in the presence of ATP and CoA exceeded the amount of diacylglycerol formed in their absence. The yield of neutral lipids from sn-glycerol 3-phosphate and monoacyl-sn-glycerol 3-phosphate markedly exceeded that from phosphatidic acid. Comparison of the yields of di- and triacylglcerols from glycerol-labelled and fatty-acid-labelled phosphatidic acid was used to establish the extent of deacylation and reacylation. About 60% of the diacylglycerol was formed by direct dephosphorylation. The triacylglycerols, on the other hand, were formed almost exclusively from recycled phosphatidic acid.     

Triacylglycerol biosynthesis in the adipose tissue of the obese-hyperglycaemic mouse.

Obesity in obese-hyperglycaemic mouse is associated with an increase in number and size of adipocytes. Adipocytes from the obese mouse showed increased incorporation of [14C]acetate and[14C]glucose into triacylglycerol. This increased capacity of triacylglycerol formation was correlated with increased activities of various triacylglycerol-forming enzymes measured in the microsomal fraction of adipose tissue from obese mice. Microsomal fractions from lean and obese mice contained sn-glycerol 3-phosphate acyltransferase, phosphatidate phosphohydrolase and diacylglycerol acyltransferase. Phosphatidate phosphohydrolase was also detected in the soluble fraction. In the presence of Mg2+, the phosphatidate phsophohydrolase from the soluble and the microsomal fractions was active towards membrane-bound phosphatidate. Among the three enzymes studied here, the increase in Mg2+-dependent phosphatidate phosphohydrolase was most prominent in adipose tissue of obese mice.     

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.     

Studies of rat liver microsomal diglyceride acyltransferase and cholinephosphotransferase using microsomal-bound substrate: effects of high fructose intake.

Radiolabeled phosphatidate and diglyceride were prepared bound to rat liver microsomes. These compounds were used as substrates in studies of diglyceride acyltransferase, cholinephosphotransferase, and CTP:phosphatidic acid cytidylyltransferase. Optimum incubation conditions for these reactions in microsomes from normal male rats are described. High fructose diets were fed to rats for 11 days; this resulted in an increased rate of neutral lipid formation from sn-glycerol-3-phosphate by liver microsomal preparations. This was attributed, in part, to a previously reported increase in liver phosphatidate phosphatase activity. The significance of this increase is supported by the finding of a fall in microsomal phosphatidate content and a doubling in microsomal diglyceride. In addition, diglyceride acyltransferase measured with microsomal-bound diglyceride was increased twofold with no equivalent change in cholinephosphotransferase activity. Such a change should result in preferential triglyceride formation from the increased microsomal diglyceride pool. CTP:phosphatidic acid cytidylytransferase activity was depressed by the high fructose diet. These combined alterations would lead to an accelerated hepatic triglyceride formation, a result found in vivo during high fructose feeding. The high fructose diet decreased slightly the total microsomal phospholipid content and markedly depressed phosphatidylethanolamine levels.     

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 triacyglycerol 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.     

Characteristics of mitochondrial and microsomal monoacyl- and diacyl-glycerol 3-phosphate biosynthesis in rabbit heart

Acylation of sn-glycerol 3-phosphate by heart subcellular fractions was characterized. The enzyme kinetics revealed that the rate of reaction of acylation by mitochondria was slower, but constant for a longer period (up to 20min), than that by the microsomal fraction. The range of palmitate, oleate and linoleate concentrations yielding optimal sn-glycerol 3-phosphate acylation was broader for mitochondria than for the microsomal fraction, the latter showing a preference for linoleate. The mitochondrial fraction synthesized a relatively large quantity of monoacyl-sn-glycerol 3-phosphate, reaching 135% of the microsomal biosynthesis during an assay period of 15min. By contrast, the microsomal fraction formed considerably more diacyl- than monoacyl-sn-glycerol 3-phosphate, except with linoleate as the acyl donor, in which case approximately equal quantities of the two products were produced. The biosynthesis of monoacyl-sn-glycerol 3-phosphate was also observed in experiments in which hepatic subcellular fractions were used to provide supporting evidence. Cardiac mitochondrial diacyl-sn-glycerol 3-phosphate formation was less than 17% of the microsomal formation. However, evidence is presented to exclude the possibility that monoacyl-sn-glycerol 3-phosphate in the mitochondrial fraction is formed by deacylation of the contaminating microsomal diacyl-sn-glycerol 3-phosphate. The participation of the dihydroxyacetone phosphate pathway in the biosynthesis of these substances was minimal. The addition of CTP and the fatty acid specificity of the reaction both provided results that reinforced the postulate that mitochondrial differs from microsomal acylation. Thus our findings demonstrate that the characteristics of acyl-CoA-sn-glycerol 3-phosphate O-acyltransferase (EC 2.3.1.15) in rabbit heart mitochondria are distinct from those of cardiac microsomal enzyme and hepatic enzymes.     

Enzymatic aspects of the reduction of microsomal glycerolipid biosynthesis after perfusion of the liver with dibutyryl adenosine-3',5'-monophosphate.

Livers from fed male rats were perfused in a nonrecycling system for 60 min with a medium containing 100 mg/dl glucose, 3 g/dl bovine serum albumin, and ~0.5 mm oleic acid, with or without 20 μm dibutyryl cyclic adenosine-3′,5′-monophosphate (Bt₂cAMP). At the termination of the experiment, microsomes were isolated from these livers. In agreement with data reported previously, Bt₂cAMP decreased output of triacylglycerol, but stimulated ketogenesis and output of glucose; uptake of free fatty acid was unaffected by the nucleotide. Perfusion with Bt₂AMP decreased the biosynthesis of triacylglycerol, diacylglycerol, and phosphatidate from sn-[U-¹⁴C]glycerol-3-phosphate by microsomes isolated from these livers. Perfusion with Bt₂cAMP also decreased incorporation of sn-glycerol-3-phosphate into phosphatidate by microsomes isolated from the livers, when the microsomes were incubated with NaF to inhibit phosphatidate phosphohydrolase, and when fatty acid, coenzyme A and ATP were replaced by the acyl coenzyme A derivative; the formation of phosphatidate under these conditions was used as an estimate of the activity of sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15). However, the activities of microsomal phosphatidate phosphohydrolase (EC 3.1.3.4) and diacylglycerol acyltransferase (EC 2.3.1.20), measured with microsomal bound substrate, were increased by Bt₂cAMP. These data have been interpreted to mean that Bt₂cAMP inhibits hepatic microsomal synthesis of triacylglycerol at a step prior to the formation of phosphatidate, presumably at the glycerophosphate acyltransferase (EC 2.3.1.15) step(s).     

Biosynthesis of D-alanyl-lipoteichoic acid: role of diglyceride kinase in the synthesis of phosphatidylglycerol for chain elongation.

Lipophilic and hydrophilic D-alanyl-lipoteichoic acids are elongated in Lactobacillus casei by the transfer of sn-glycerol 1-phosphate units from phosphatidylglycerol to the poly(glycerophosphate) moiety of the polymer. These sn-glycerol 1-phosphate units are added to the end of the poly(glycerophosphate) which is distal to the glycolipid anchor; 1,2-diglyceride results from this addition. The presence of a diglyceride kinase was suggested by the ATP-dependent phosphorylation of 1,2-diglyceride to phosphatidic acid. Inorganic phosphate was used to initiate the synthesis of lipophilic lipoteichoic acid (LTA) and the elongation of both lipophilic and hydrophilic LTA. Three observations suggest that phosphate and other anions play a role in the in vitro synthesis of LTA and its precursors. First, the conversion of 1,2-diglyceride to phosphatidic acid by diglyceride kinase was stimulated. Second, the synthesis of phosphatidylglycerol was increased. Third, the elongation of lipophilic and hydrophilic LTA was enhanced. These observations indicated that one effect of phosphate might be to enhance the utilization of 1,2-diglyceride for the synthesis of phosphatidic acid. This phospholipid is a precursor of phosphatidylglycerol, the donor of sn-glycerol 1-phosphate for elongation of LTA.     

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)     

Glycerolipid labelling kinetics in isolated intact chloroplasts.

Glycerolipid synthesis was studied in intact chloroplasts isolated from three different plant species. The sequential acylation of sn-glycerol 3-phosphate and lysophosphatidate (1-acyl-sn-glycerol 3-phosphate) was confirmed by monitoring the incorporation of oleate synthesized in situ into lysophosphatidate, phosphatidate and diacylglycerol. Lysophosphatidate was not only readily detected in these experiments, but was also present in the chloroplasts at the beginning of the time courses. The rate of glycerolipid synthesis depended primarily on sn-glycerol 3-phosphate supply, and given adequate sn-glycerol 3-phosphate, the proportion of newly synthesized fatty acids diverted into glycerolipids appeared to be determined by differing acyltransferase activities in the chloroplasts isolated from different plant species.     

The interconversion of diacylglycerol and phosphatidylcholine during triacylglycerol production in microsomal preparations of developing cotyledons of safflower (Carthamus tinctorius L.).

Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalyse the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. Under these conditions the radioactive glycerol in sn-glycerol 3-phosphate accumulates in phosphatidic acid, phosphatidylcholine, diacyl- and tri-acylglycerol. The incorporation of glycerol into phosphatidylcholine is via diacylglycerol and probably involves a cholinephosphotransferase. The results show that the glycerol moiety and the acyl components in phosphatidylcholine exchange with the diacylglycerol during the biosynthesis of diacylglycerol from phosphatidic acid. The continuous reversible transfer of diacylglycerol with phosphatidylcholine, which operates during active triacylglycerol synthesis, will control in part the polyunsaturated-fatty-acid quality of the final seed oil.     

The stimulation of rat liver microsomal CDP-diacylglycerol formation by guanosine triphosphate

GTP has been found to markedly enhance the formation of CDP-diacylglycerol in rat liver microsomes. Neither GDP, GMP nor the nonhydrolyzable analogues of GTP increased the synthesis of the liponucleotide. The GTP stimulation of phosphatidate cytidylyltransferase activity is inhibited by EDTA and NaF. GTP enhances the activity of the enzyme in a concentration-, time-, and temperature-dependent manner and preincubation of rat liver microsomes with GTP produces a persistently activated phosphatidate cytidylyltransferase. GTP reduces the Km for phosphatidic acid, but has no effect on either the Km for CTP or the Vmax of the reaction. GTP, by stimulating the activity of the phosphatidate cytidylyltransferase, enhances the formation of phosphatidylinositol from CTP, phosphatidic acid, and inositol. Evidence is presented suggesting that the mechanism by which GTP stimulates the activity of the phosphatidate cytidylyltransferase involves a covalent modification of the enzyme itself or a protein intimately associated with the phosphatidate cytidylyltransferase.     

Biogenesis of mitochondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions

The ability in vitro of yeast mitochondrial and microsomal fractions to synthesize lipid de novo was measured. The major phospholipids synthesized from sn-[2-(3)H]glycerol 3-phosphate by the two microsomal fractions were phosphatidylserine, phosphatidylinositol and phosphatidic acid. The mitochondrial fraction, which had a higher specific activity for total glycerolipid synthesis, synthesized phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid, together with smaller amounts of neutral lipids and diphosphatidylglycerol. Phosphatidylcholine synthesis from both S-adenosyl[Me-(14)C]methionine and CDP-[Me-(14)C]choline appeared to be localized in the microsomal fraction.     

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.