Phosphatidylglycerol synthesis in spinach chloroplasts: characterization of the newly synthesized molecule

Intact chloroplasts from spinach (Spinacia oleracea L., hybrid 424) readily incorporate [¹⁴C]glycerol-3-phosphate and [¹⁴C]acetate into diacylglycerol, monoacylglycerol, diacylglycrol, free fatty acids (only when acetate is the precursor), phosphatidic acid, phosphatidylcholine, and most notably phosphatidylglycerol. The fraction of phosphatidylglycerol synthesized is greatly increased by the presence of manganese chloride in the reaction mixture. Glycerol-3-phosphate-labeled phosphatidylglycerol is equally labeled in the two glycerol moieties of the molecule. Acetate-labeled phosphatidylglycerol is equally labeled in both acyl groups. Position one contains primarily oleate, linoleate and small amounts of palmitate. Position two contains primarily palmitate. No radioactive trans-Δ³-hexadecenoate was detected. The labeling patterns indicate that the radioactive phosphatidylglycerol is the product of de novo chloroplast lipid biosynthesis and furthermore, phosphatidylglycerol may be a substrate for fatty acid desaturation.     

Control of plastidic glycolipid synthesis and its relation to chlorophyll formation

Mechanisms restricting the accumulation of chloroplast glycolipids in achlorophyllous etiolated or heat-treated 70S ribosome-deficient rye leaves (Secale cereale L. cv “Halo”) and thereby coupling glycolipid formation to the availability of chlorophyll, were investigated by comparing [¹⁴C]acetate incorporation by leaf segments of different age and subsequent chase experiments. In green leaves [¹⁴C]acetate incorporation into all major glycerolipids increased with age. In etiolated leaves glycerolipid synthesis developed much more slowly. In light-grown, heat-bleached leaves [¹⁴C]acetate incorporation into glycolipids was high at the youngest stage but declined with age. In green leaves [¹⁴C]acetate incorporation into unesterified fatty acids and all major glycerolipids was immediately and strongly diminished after application of an inhibitor of chlorophyll synthesis, 4,6-dioxoheptanoic acid. The turnover of glyco- or phospholipids did not differ markedly in green, etiolated, or heat-bleached leaves. The total capacity of isolated ribosome-deficient plastids for fatty acid synthesis was not much lower than that of isolated chloroplasts. However, the main products synthesized from [¹⁴C]acetate by chloroplasts were unesterified fatty acids, phosphatidic acid, and diacylglycerol, while those produced by ribosome-deficient plastids were unesterified fatty acids, phosphatidic acid, and phosphatidylglycerol. Isolated heat-bleached plastids exhibited a strikingly lower galactosyltransferase activity than chloroplasts, suggesting that this reaction was rate-limiting, and lacked phosphatidate phosphatase activity.     

In situ incorporation of Fatty acids into lipids of the outer and inner envelope membranes of pea chloroplasts

When incubated with [1-¹⁴C]acetate and cofactors (ATP, Coenzyme A, sn-glycerol-3-phosphate, UDPgalactose, and NADH), intact chloroplasts synthesized fatty acids that were subsequently incorporated into most of the lipid classes. To study lipid synthesis at the chloroplast envelope membrane level, ¹⁴C-labeled pea (Pisum sativum) chloroplasts were subfractionated using a single flotation gradient. The different envelope membrane fractions were characterized by their density, lipid and polypeptide composition, and the localization of enzymic activities (UDPgalactose-1,2 diacylglycerol galactosyltransferase, Mg²⁺-dependent ATPase). They were identified as very pure outer membranes (light fraction) and strongly enriched inner membranes (heavy fraction). A fraction of intermediate density, which probably contained double membranes, was also isolated. Labeled glycerolipids recovered in the inner envelope membrane were phosphatidic acid, phosphatidyl-glycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol. Their ¹⁴C-fatty acid composition indicated that a biosynthetic pathway similar to the prokaryotic pathway present in cyanobacteria occurred in the inner membrane. In the outer membrane, phosphatidylcholine was the most labeled glycerolipid. Phosphatidic acid, phosphatidylglycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol were also labeled. The ¹⁴C-fatty acid composition of these lipids showed a higher proportion of oleate than palmitate. This labeling, different from that of the inner membrane, could result either from transacylation activities or from a biosynthetic pathway not yet described in pea and occurring partly in the outer chloroplast envelope membrane. This metabolism would work on an oleate-rich pool of fatty acids, possibly due to the export of oleate from chloroplast toward the extrachloroplastic medium. The respective roles of each membrane for chloroplast lipid synthesis are emphasized.     

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.     

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli. In vitro and in vivo incorporation into phospholipids

sn-Glycerol 3-phosphorothioate, a bacteriocidal analog of sn-glycerol 3-phosphate in strains of Escherichia coli with a functioning glycerol phosphate transport system, was investigated for its ability to be incorporated into phospholipid under in vitro and in vivo conditions. A cell-free particulate fraction from E. coli strain 8 catalyzes the transfer of sn-[3H]glycerol 3-phosphoro[35S]thioate to chloroform-soluble material in the presence of either CDP-diglyceride or palmitoyl coenzyme A. With CDP-diglyceride as the co-substrate, the product of the reaction was tentatively identified as phosphatidylglycerol phosphorothioate. No formation of phosphatidylglycerol was observed, suggesting that the specific phosphatase required for the synthesis of phosphatidylglycerol does not catalyze, or else at a greatly reduced rate, the hydrolysis of the phosphorothioate monoester linkage. The kinetics of incorporation of sn-[3H]glycerol 3-phosphate and phosphorothioate into chloroform-soluble material in the presence of CDP-diglyceride are almost identical. In the presence of palmitoyl coenzyme A, sn-[3H]glycerol 3-phosphoro[35S]thioate was converted to the phosphorothioate analog of phosphatidic acid. Kinetic analysis showed that the apparent Km values for the incorporation of the phosphate and the phosphorothioate derivatives into phospholipid were 0.4 and 0.8 mM, respectively. The Vmax for the phosphorothioate analog was approximately half that for the phosphate derivative. Chemically synthesized thiophosphatidic acid was not a substrate for CTP:phosphatidic acid cytidylyltransferase. sn-[3H]Glycerol 3-phosphoro[35S]thioate was incorporated into phospholipid by cultures of E. coli strain 8. The major phosphorothioate-containing phospholipid synthesized in vivo was identified as 1,2-diacyl-sn-[3H]glycerol 3-phosphoro[35S]thioate. The phosphorothioate analog of phosphatidylglycerol phosphate was not observed despite our observations that this analog can be synthesized in vitro. Our results indicate that the phosphorothioate analog is an effective sn-glycerol 3-phosphate surrogate and suggest that a major reason for its toxicity toward E. coli strain 8 may be due to a total blockade of endogenous phospholipid biosynthesis.     

Rates and products of long-chain Fatty Acid synthesis from [1-C]acetate in chloroplasts isolated from leaves of 16:3 and 18:3 plants

Chloroplasts highly active in the synthesis of long-chain fatty acids from [1-¹⁴C]acetate were prepared from leaves of Solanum nodiflorum, Chenopodium quinoa, Carthamus tinctorius, and Pisum sativum. These preparations were used to test whether the various additions to incubation media found to stimulate the synthesis of particular lipid classes in vitro by Spinacia oleracea chloroplasts were applicable generally. Chloroplasts from 18:3 plants incorporated a greater proportion of radioactivity into unesterified fatty acids under control conditions than did those from 16:3 plants. Supplying exogenous sn-glycerol 3-phosphate or Triton X-100 to chloroplasts increased the synthesis of glycerolipids in all cases and accentuated the capacity of chloroplasts from 18:3 plants to accumulate phosphatidic acid rather than the diacylglycerol accumulated by chloroplasts from 16:3 plants. The UDP-galactose-dependent synthesis of labeled diacylgalactosylglycerol was much less active in incubations of chloroplasts from 18:3 plants also containing sn-glycerol 3-phosphate and Triton X-100 compared with similar incubations from 16:3 plants. Exogenous CoA stimulated total fatty acid synthesis in all chloroplast preparations and the further addition of ATP diverted radioactivity from the unesterified fatty acid to acyl-CoA. The results have been discussed in terms of the two pathway hypothesis for lipid synthesis in leaves.     

Studies on the antibacterial activity of dodecylglycerol. Its limited metabolism and inhibition of glycerolipid and lipoteichoic acid biosynthesis in Streptococcus mutans BHT

Growth-inhibitory concentrations of racemic sn-1(3)-dodecylglycerol inhibit the incorporation of [14C] glycerol into lipids and lipoteichoic acid of Streptococcus mutans BHT and alter the per cent composition of the glycerolipids. Increases in phosphatidic acid and diphosphatidylglycerol (at the expense of phosphatidylglycerol) contribute the most to the change in lipid composition. No cellular lysis occurs under these conditions. Radioactive racemic sn-1(3)-dodecylglycerol is readily taken up by the cell and is metabolized primarily to lysophosphatidic acid and phosphatidic acid with smaller amounts converted to phosphatidylglycerol and diacylglycerol. The accumulation of phosphatidic acid and the loss of viability respond in parallel to different concentrations of dodecylglycerol. An increase in CTP is also observed which together with the increase in phosphatidic acid suggests a possible impairment in the synthesis of CDP-diacylglycerol.     

Synthesis of phosphatidylglycerol by chloroplasts from leaves of Spinacia oleracea L. (spinach)

Purified chloroplasts from leaves of Spinacia oleracea L. (spinach) incorporated glycerol 3-phosphate into diacylglycerol, monoacylglycerol, phosphatidylglycerol, phosphatidic acid, and lysophosphatidic acid. The omission of ATP or CTP, CoA or illumination decreased the incorporation markedly. The fraction of incorporated glycerol 3-phosphate found in phosphatidylglycerol was greatly reduced by the omission of bicarbonate, acetate, and ATP, or in darkness, low-osmolarity medium, or high magnesium ion concentration (10 mM). Incorporation of glycerol 3-phosphate into lipid and specifically into phosphatidylglycerol was optimal at a $\text{Mg}{}^{\mathrm{2+}}\text{CTP}$ ratio of 1, whereas the optimal ratio for $\text{Mg}{}^{\mathrm{2+}}\text{ATP}$ was closer to 2. The $\text{Mg}{}^{\mathrm{2+}}\text{CTP}$ gave lower total incorporation but a higher fraction of incorporation in phosphatidylglycerol. Triton X-100 inhibited incorporation of glycerol 3-phosphate into lipid, especially into phosphatidylglycerol.     

Manipulating the incorporation of [1-C]acetate into different leaf glycerolipids in several plant species

During short term labeling of expanding leaves of seven plant species with [1-¹⁴C]acetate, 35 to 64% of the label incorporated into lipids was found in phosphatidylcholine and 5 to 24% in phosphatidylglycerol. In pumpkin, sunflower, broad bean, and maize, only 4 to 12% of the label was found in diacylgalactosylglycerol, but in tomato, parsley, and spinach, the proportion was 17 to 31%. The latter group was further distinguished by having diacylgalactosylglycerol containing C16:3.

The proportions of total incorporated [1-¹⁴C]acetate entering the lipids could be manipulated in a predictable manner. Phosphatidylcholine labeling was depressed by treating intact leaves with glycerol or ethylene glycol monomethyl ether or by incubating leaf discs in vitro. An associated increase in phosphatidylglycerol labeling occurred within the first group of plants, whereas an increase in labeling of either diacylgalactosylglycerol, phosphatidylglycerol, or sulfolipid occurred within the second group. Treating intact leaves with glycerol or incubating leaf discs in vitro was shown to elevate cellular concentrations of sn-glycerol 3-phosphate.

These results have been interpreted in terms of the two-pathway hypothesis for glycerolipid biosynthesis, in which it is proposed that phosphatidylcholine is synthesized via a different pathway (eukaryotic) to that for synthesis of phosphatidylglycerol (prokaryotic). Both pathways may contribute toward the synthesis of diacylgalactosylglycerol, with the contribution of each being assessed from the proportion of hexadecatrienoic acid found in the particular plant.

     

Glycerolipid metabolism in lung from ventilated and unventilated rabbits

The incorporation of [14C]-glycerol 3-phosphate and [3H]-palmitate into phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and triacylglycerols by lung microsomes from ventilated and unventilated rabbits was measured. Unventilated lung microsomes showed an impairment of the "de novo" synthesis of phosphatidic acid and, therefore, a general decrease of glycerolipids synthesized from glycerol 3-phosphate. The incorporation of [3H]-palmitate into phosphatidic acid was considerably lower than the incorporation of [14C]-glycerol 3-phosphate by lung microsomes from both ventilated and unventilated rabbits, and the 3H/14C molar ratio did not change during incubation time. These observations suggest the preferential utilization of endogenous fatty acids by acyltransferases involved in the formation of phosphatidic acid. The activities of the enzymes implicated in the synthesis of phosphatidylcholine from lysophosphatidylcholine remained unchanged in lung from both ventilated and unventilated rabbits.     

Species pattern of phosphatidic acid, diacylglycerol, CDP-diacylglycerol and phosphatidylglycerol synthesized de novo in rat liver mitochondria

Rat liver mitochondria were incubated with [3H]glycerol 3-phosphate, ATP, CTP and coenzyme A allowing acylatin of glycerophosphate with endogenous fatty acids and the further conversion of labelled phosphatidic acid (PA) to diacylglycerol (DG), CDP-diacylglycerol (CDP-DG) and phosphatidylglycerol (PG). In these glycerolipids, the distribution of label among the individual molecular species was found to be similar, with 16:0-18:1, 16:0-18:2 and 18:0-18:2/16:0-16:0 being the main species. It was concluded that mitochondrial enzymes involved in the de novo synthesis of these glycerolipids exhibited no acyl selectivity for their substrates. The pattern of molecular species of mitochondrial PA, DG and CDP-DG closely approached that of the same glycerolipids synthesized de novo in isolated rat liver microsomes.     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

Final step of phosphatidic Acid synthesis in pea chloroplasts occurs in the inner envelope membrane

The second enzyme of phosphatidic acid synthesis from glycerol-3-phosphate, 1-acylglycerophospate acyltransferase, was localized to the inner envelope membrane of pea chloroplasts. The activity of this enzyme was measured by both a coupled enzyme assay and a direct enzyme assay. Using the coupled enzyme assay, phosphatidic acid phosphatase was also localized to the inner envelope membrane, although this enzyme has very low activity in pea chloroplasts. The addition of UDP-galactose to unfractionated pea chloroplast envelope preparations did not result in significant conversion of newly synthesized diacylglycerol to monogalactosyldiacylglycerol. Thus, the envelope synthesized phosphatidic acid may not be involved in galactolipid synthesis in pea chloroplasts.     

Synthesis of saturated phosphatidylcholine and phosphatidylglycerol by freshly isolated rat alveolar type II cells

Saturated phosphatidylcholine and phosphatidylglycerol are important components of pulmonary surface active material, but the relative contributions of different pathways for the synthesis of these two classes of phospholipids by alveolar type II cells are not established. We purified freshly isolated rat type II cells by centrifugal elutriation and incubated them with [1-14C]palmitate as the sole exogenous fatty acid in one series of experiments or with [9,10-3H]palmitate, mixed fatty acids (16:0, 18:1 and 18:2), and [U-14C]glucose in another series of experiments. Type II cells readily incorporated [1-14C]palmitate into saturated phosphatidic acid (55-59% of total phosphatidic acid), saturated diacylglycerol (82-87% of total diacylglycerol), saturated phosphatidylcholine (69-76% of total phosphatidylcholine), and saturated phosphatidylglycerol (55-59% of total phosphatidylglycerol). Saturated phosphatidic acid, diacylglycerol and phosphatidylglycerol were nearly equally labeled in the sn-1 and sn-2 positions, whereas saturated phosphatidylcholine was preferentially labeled in the sn-2 position. With [9,10-3H]palmitate and [U-14C]glucose, the labeling patterns of phosphatidic acid, diacylglycerol and phosphatidylglycerol were similar to each other but different from that of phosphatidylcholine. The glucose label was found predominantly in the unsaturated phosphatidylcholines at early times (3-10 min) and in the saturated phosphatidylcholines at later times (30-90 min). Similarly, the 3H/14C ratio was very high in saturated phosphatidylcholine and always above that in saturated diacylglycerol. We conclude that freshly isolated type II cells synthesize saturated phosphatidic acid, diacylglycerol, phosphatidylcholine and phosphatidylglycerol and that under our in vitro conditions the deacylation-reacylation pathway is important for the synthesis of saturated phosphatidylcholine but is less important for the synthesis of saturated phosphatidylglycerol. By the assumptions stated in the text during the pulse chase experiment de novo synthesis of saturated phosphatidylcholine from saturated diacylglycerol accounted for 25% of the total synthesis of saturated phosphatidylcholine.     

beta-Carotene Synthesis in Isolated Spinach Chloroplasts : Its Tight Linkage to Photosynthetic Carbon Metabolism

Carefully isolated intact spinach chloroplasts virtually free of contamination of other organelles effectively form β-carotene from NaH¹⁴CO₃ or [U-¹⁴C]-3-phosphoglycerate (PGA) under photosynthetic conditions. The photosynthate pool formed in chloroplasts from 1 to 2 millimolar [U-¹⁴C]-3-PGA or 3 to 6 millimolar NaH¹⁴CO₃ was fully sufficient to supply β-carotene synthesis with intermediates for about 1 hour at maximal rates of about 20 nanomoles ¹⁴C incorporated per milligram chlorophyll per hour. Fatty acid synthesis remains, under these circumstances, in linear dependence to substrate concentrations with far lower activity. Isotopic dilution of the β-carotene synthesis by adding unlabeled glyceraldehyde 3-phosphate, dihydroxyacetone-P, 3-PGA, 2-PGA, phosphoenolpyruvate, pyruvate, respectively, may be interpreted as a direct substrate flow from photosynthetically fixed CO₂ to isopentenyl pyrophosphate synthesizing system. Unlabeled acetate did not dilute β-carotene synthesis. Fatty acid synthesis acted similarly with unlabeled substrates; but it also was diluted by unlabeled acetate. These results indicate a tight linkage of photosynthetic carbon fixation and plastid isoprenoid synthesis.     

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.     

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)     

The role of chloroplasts and microsomal fractions in polar-lipid synthesis from [1-14C]acetate by cell-free preparations from spinach (Spinacia oleracea) leaves.

1. Isolated spinach (Spinacia oleracea) chloroplasts were incapable of accumulating polar lipids when incubated with [1-14C]acetate in a cofactor-free medium. When CoA, ATP and glycerol 3-phosphate were added to incubation media, the accumulated products were non-esterified fatty acids, acyl-CoA and 1,2-diacylglycerol, all intermediates of lipid metabolism. 2. Chloroplast acyl-CoA was used to synthesize phosphatidylcholine only when a microsomal fraction was added back to the incubation medium. 3. The 1,2-diacylglycerol synthesized by isolated chloroplasts was converted almost quantitatively into diacylgalactosylglycerol when exogenous UDP-galactose was available. 4. Stereospecific analyses of the isolated lipids suggested that the diacylglycerol synthesized by isolated chloroplasts may be an important precursor for the synthesis in vivo of diacylgalactosylglycerol and phosphatidylglycerol but was unlikely to be a precursor of phosphatidylcholine. 5. A scheme for plant-lipid biosynthesis is presented that integrates the functions of chloroplasts, the cytoplasm and the endoplasmic reticulum.     

Synthesis of fluorescent and radiolabeled analogues of phosphatidic acid

Procedures for the synthesis of fluorescent and radiolabeled analogues of phosphatidic acid are described. The fluorophore 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was coupled to 6-amino-caproic acid and 12-aminododecanoic acid by reaction of NBD-chloride with the amino acids under mild alkaline conditions at room temperature. 1,2-Dioleoyl-sn-[U-14C]glycerol 3-phosphate was prepared by acylation of sn-[U-14C]glycerol 3-phosphate with oleic acid anhydride using dimethylaminopyridine as the catalyst. This compound was converted to 1-oleoyl-sn-[U-14C]glycerol 3-phosphate by hydrolysis with phospholipase A2. The lysophosphatidic acid was reacylated with NBD-aminocaproyl imidazole or NBD-aminododecanoyl imidazole to form the fluorescent, radiolabeled analogue of phosphatidic acid. Fluorescent, non-radiolabeled analogues of phosphatidic acid were prepared by phospholipase D hydrolysis of fluorescent phosphatidylcholine.     

The role of lipoteichoic acid biosynthesis in membrane lipid metabolism of growing Staphylococcus aureus

Pulse-chase experiments with [2-3H]glycerol and [14C]acetate revealed that in Staphylococcus aureus lipoteichoic acid biosynthesis plays a dominant role in membrane lipid metabolism. In the chase, 90% of the glycerophosphate moiety of phosphatidylglycerol was incorporated into the polymer: 25 phosphatidylglycerol + diglucosyldiacylglycerol leads to (glycerophospho)25-diglucosyldiacylglycerol + 25 diacylglycerol. Glycerophosphodiglucosyldiacylglycerol was shown to be an intermediate, confirming that the hydrophilic chain is polymerized on the final lipid anchor. Total phosphatidylglycerol served as the precursor pool and was estimated to turn over more than twice for lipoteichoic acid synthesis in one bacterial doubling. Of the resulting diacylglycerol approximately 10% was used for the synthesis of glycolipids and the lipid anchor of lipoteichoic acid. The majority of diacylglycerol recycled via phosphatidic acid to phosphatidylglycerol. Synthesis of bisphosphatidylglycerol was negligible and only a minor fraction of phosphatidylglycerol passed through the metabolically labile lysyl derivative. In contrast to normal growth, energy deprivation caused an immediate switch-over from the synthesis of lipoteichoic acid to the synthesis of bisphosphatidylglycerol.