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.     

Possible relationship of the hepatic microsomal ATP-dependent calcium pump to sex differences in triacylglycerol synthesis

Microsomes were isolated from livers of fed male and female rats and the rates of incorporation of sn-[¹⁴C]-glycerol-3-phosphate into phosphatidate, diacylglycerol and triacylglycerol by the microsomes were measured. Simultaneously, microsomal ATP-dependent uptake of calcium was evaluated and correlated with synthesis of phosphatidate from sn-glycerol-3-phosphate. The rate of glycerolipid synthesis by hepatic microsomes from female rats was greater than that of microsomes from male rats. By contrast, the active accumulation of calcium and subsequent inhibition of synthesis of phosphatidate from glycerol-3-phosphate was lower in microsomes from livers of female rats than from male animals. This reciprocal relationship between uptake of calcium and incorporation of sn-glycerol-3-phosphate into phosphatidate as reported earlier (Biochem. Biophys. Res. Commun. $\text{78}$, 1053–1059 (1977)) may, in part, be responsible for the differences in the rates of hepatic triacylglycerol synthesis between livers from male and female rats.     

Alterations in lipid turnover in developing muscle

The incorporation and turnover of [³H] glycerol into skeletal muscle cell cultures derived from embryonic chickens was studied. Both rates of incorporation and turnover of specific lipids were dependent on culture age and lipid species. The pattern of glycerol incorporation showed that prefusion myoblasts primarily synthesized both phosphatidylcholine and triglycerides whereas postfusion myotubes primarily synthesized phosphatidyl choline. This pattern could be modified in postfusion but not prefusion cells by briefly incubating the cells with unilammelar phosphatidyl choline vesicles. Analysis of major lipid species revealed that muscle triglycerides and phospholipids turned over at a higher rate in prefusion cultures compared to the postfusion state. These findings are discussed in light of the marked shift in lipid metabolism which occurs during myogenesis.     

Acyltransferases and the biosynthesis of pulmonary surfactant lipid in adenoma alveolar type II cells.

Acyltransferases are present in microsomes from alveolar type II cell adenomas (produced by urethan injections) that transfer palmitic acid in the presence of CoA, ATP, and Mg⁺⁺ to $\text{sn}$-glycerol-3-P to form phosphatidic acid, to dihydroxyacetone-P to form acyldihydroxyacetone-P, and to 1-acyl-$\text{sn}$-glycero-3-phosphocholine to form 3-$\text{sn}$-phosphatidylcholine. The data clearly demonstrate that the microsomal preparations can catalyze significant incorporation of palmitic acid into the 2-position of the disaturated species of 3-sn-phosphatidylcholine independently of phosphatidic acid formation as evidenced by the fact that $\text{sn}$-glycerol-3-P and calcium ions (which inhibit choline phosphotransferase) did not influence the incorporation of palmitic acid into the main surfactant lipid. Thus, a deacylation-acylation reaction involving 2-lysophosphatidylcholine appears to be an important pathway for the synthesis of surfactant lipid in alveolar type II cells; the control of acyl specificity at the 2-position is determined by the relative concentrations of the coparticipating substrates, l-palmitoyl-$\text{sn}$-glycero-3-phosphocholine and palmitoyl-CoA.     

The cerulenin-induced formation of 1-acyl-lysophosphatidyl glycerol in Bacillus megaterium.

When a culture of $\text{Bacillus megaterium}$ ATCC 14581, growing at 20° and treated with the fatty acid synthesis inhibitor, cerulenin, was incubated with [U-¹⁴C]palmitate, 50% of the incorporated label was found in 1-palmitoyl-lysophosphatidyl glycerol within 5 min. Most of the remaining ¹⁴C appeared in free fatty acid and phosphatidyl glycerol. By 45 min almost all of the lyso compound had disappeared and 80% of the incorporated label was found in phosphatidyl glycerol. At 20°, in the absence of cerulenin or at 35° in either its presence or absence, no labeled lysophosphatidyl glycerol could be found at any time after [U-¹⁴C]palmitate addition. The major radioactive lipid, in these cases, was always phosphatidyl glycerol. At 20°, the palmitate of phosphatidyl glycerol but not of lysophosphatidyl glycerol was readily desaturated.     

The temperature-mediated metabolism of 1-acyl-lysophosphatidyl glycerol in cerulenin-treated cultures of Bacillus megaterium.

When [U-¹⁴C]palmitate was added to a culture of $\text{B. megaterium}$ that had been grown at 35°, transferred to 20° and treated with cerulenin, label was initially incorporated into lysophosphatidyl glycerol. The labeled lyso derivative, in turn, was converted to phosphatidyl glycerol, apparently by esterification of the 2-position with endogenous acyl groups. Labeled lysophosphatidyl glycerol synthesis at 20° was observed only when a culture was treated with cerulenin prior to the addition of [U-¹⁴C]palmitate. When [U-¹⁴C]palmitate was added before cerulenin, labeled lysophosphatidyl glycerol formation was not detected. When chloramphenicol was added with cerulenin at the time of culture transfer from 35° to 20°, the synthesis of lysophosphatidyl glycerol was unaffected but the rate of its esterification to phosphatidyl glycerol was significantly retarded. Transfer of such a culture back to 35° resulted in a marked acceleration in the rate of conversion of lysophosphatidyl glycerol to phosphatidyl glycerol.     

Lysophosphatidylserine dependent incorporation of acyl CoA into phospholipids in rat brain microsomes

[¹⁴C]OleoylCoA was incorporated into phosphatidylinositol 4$\text{1}\text{2}$ times more efficiently than into phosphatidylserine in rat brain and liver microsomes when incubated with various levels of 1-acyl-sn-glycero-3-phosphoserine. In contrast, 1-acyl-sn-glycero-3-phosphocholine dependent incorporation of oleoylCoA was only into phosphatidylcholine. When [l-³H]serine labeled 1-acyl-sn-glycero-3-phosphoserine was used as the labeled substrate, no phosphatidylserine synthesis could be detected in rat brain microsomes. OleoylCoA incorporation in phospholipids in the presence of lysophosphatidylserine was primarily at the 2-position while stearoylCoA was incorporated at the 1-position. These results are interpreted to suggest that there is no acylCoA:1-acyl-sn-glycero-3-phosphoserine acyltransferase in rat brain microsomes and the lysophosphatidylserine dependent position-specific incorporation of acylCoA into various phospholipids may be due to an exchange reaction. A simple highly reproducible one dimensional thin-layer chromatographic system is described for the separation of all the major phospholipids of brain and liver.     

Lysophosphatidylcholine uptake and metabolism in the adult rabbit lung

Tracer quantities of 3H-labeled lysoPC and 32P-labeled natural rabbit surfactant were given intratracheally via a bronchoscope and [14C]palmitate was given intravenously to 25 rabbits with labeled PC and lysoPC measured in the alveolar wash, lung homogenate, lamellar bodies and microsomes at five times from 10 min to 6 h after tracheal injection. Surprisingly, only 31% of the administered lysoPC remained in its original form in the total lungs (alveolar wash + lung homogenate) by 10 min, of which 77% was in the alveolar wash. Meanwhile, by 10 min an additional 37% was already converted to PC, of which more than 98% was in the lung homogenate. LysoPC continued to be rapidly and efficiently converted to PC, with 62% conversion measured at 3 h. The converted lysoPC initially appeared with high specific activity in microsomes, then in lamellar bodies, and finally in the alveolar wash. The intravascular palmitate labeled lung PC had similar specific activity-time profiles in the subcellular fractions, while intratracheally administered natural rabbit surfactant had a constantly low specific activity in microsomes and much higher specific activities in lamellar bodies and alveolar wash. Another 25 rabbits received intratracheal lysoPC labeled in both the choline and palmitate moieties and then were studied from 1 to 24 h after tracheal injection. The ratio of the palmitate to choline labels indicated uptake and conversion to PC primarily by direct acylation rather than transacylation and by intact reuptake and conversion rather than breakdown and resynthesis. LysoPC is an attractive 'metabolic probe' of surfactant metabolism which undergoes very rapid and efficient intracellular conversion to PC via a subcellular pathway that parallels the remodeling and de novo synthetic pathways.     

Effect of temperature of imbibition on phospholipid metabolism in pea embryonic axes

Pea seeds were imbibed in radioactive choline and then germinated. Treatments were either at 5° or 25° and the seeds were imbibed for 5 hr at one temperature and then transferred to the other. [$\text{Me}$-¹⁴C]Choline incorporation into phosphatidyl choline in the ER and the plasma membrane obtained from the embryonic axes after germination was measured. Seeds kept constantly at 25° had a very rapid initial incorporation of choline followed by a loss of label. Seeds kept at 5° had a very much lower rate of incorporation. However, seeds transferred from 5 to 25° behaved for at least 48 hr as if continuously kept at 5°, while in seeds transferred from 25 to 5° incorporation stopped after 15 hr. The seeds apparently respond to transient exposure to temperature by a changed metabolism of phospholipid. Data are also given for the choline content of the seeds under the different treatments and for the changes in total phospholipid.     

Effects of γ-aminobutyric acid on 33Pi incorporation into rat brain phospholipids in vivo

The effects of γ-aminobutyric acid (GABA), bicuculline and strychnine on the incorporation $\text{in vivo}$ of ³³Pi into phospholipids of rat brain were studied at 10 and 30 minutes after intracisternal injection of the radionuclide. GABA inhibited labeling of phospholipids in the three brain regions studied at both times. Bicuculline by itself had no significant effect on ³³Pi incorporation, but totally blocked the inhibitory effect of GABA in all three brain regions. Strychnine by itself inhibited phospholipid labeling in the brain stem and forebrain, had no significant effect on GABA inhibition of ³³Pi incorporation in the cerebellum and forebrain, and partially blocked the GABA effect in the brain stem. GABA inhibited ³³Pi incorporation into phosphatidic acid, phosphatidylinositol, phosphatidyl choline and phosphatidyl ethanolamine but had no effect on phosphatidyl serine. The data suggest that the inhibitory effects of GABA on CNS phospholipid labeling are mediated specifically through GABA receptor sites.     

Role of lamellar inclusions in surfactant production: studies on phospholipid composition and biosynthesis in rat and rabbit lung subcellular fractions.

Lamellar inclusion bodies in the type II alveolar epithelial cell are believed to be involved in pulmonary surfactant production. However, it is not clear whether their role is that of synthesis, storage, or secretion. We have examined the phospholipid composition and fatty acid content of rabbit lung wash, lamellar bodies, mitochondria, and microsomes. Phosphatidylcholine and phosphatidylglycerol, the surface-active components of pulmonary surfactant, accounted for over 80% of the total phospholipid in lung wash and lamellar bodies but for only about 50% in mitochondria and microsomes. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin accounted for over 40% of the total in mitochondria and microsomes but for only 6% in lung wash and 15% in lamellar bodies. The fatty acid composition of lamellar body phosphatidylcholine was similar to that of lung wash, but different from that of mitochondria and microsomes, in containing palmitic acid as a major component with little stearic acid and few fatty acids of chain length greater than 18 carbon atoms. The biosynthesis of phosphatidylcholine and phosphatidylglycerol was examined in the mitochondrial, microsomal, and lamellar body fractions from rat lung. Cholinephosphotransferase was largely microsomal. The activity in the lamellar body fraction could be attributed to microsomal contamination. The activity of glycerolphosphate phosphatidyltransferase, however, was high in the lamellar body fraction, although it was highest in the mitochondria and was also active in the microsomes. These data suggest that the lamellar bodies are involved both in the storage of the lipid components of surfactant and in the synthesis of at least one of those components, phosphatidylglycerol.     

Transfer of phospholipids between the endoplasmic reticulum and mitochondria in rat hepatocytes in vivo

The transfer of phospholipids from the endoplasmic reticulum to the inner mitochondrial membrane was investigated by pulse labeling $\text{in}\text{vivo}$. With [³H]glycerol microsomal phosphatidylethanolamine and phosphatidylcholine were rapidly labeled during the first 30 min; while maximum incorporation into the inner mitochondrial membrane occurred only after about 5 hours. It appears that the $\text{in}\text{vivo}$ transfer of these phospholipids between the two membrane compartments is a relatively slow process.     

Enzymatic transfer of the acyl group from acyl dihydroyxacetone phosphate to different substrates

The acyl group of acyl dihydroxyacetone phosphate was shown to be enzymatically transferred in guinea pig liver mitochondria to various acceptors such as lysolecithin, lysophosphatidyl ethanolamine and $\text{sn}$-glycerol-3-phosphate to form lecithin, phosphatidyl ethanolamine and phosphatidate, respectively. Coenzyme A and Mg⁺⁺, but not ATP, were required for this reaction. A rapid exchange of acyl group between acyl dihydroxyacetone phosphate and dihydroxyacetone phosphate was also observed.     

Phospholipid activation of Lactobacillus plantarum undecaprenyl pyrophosphate synthetase

The ability of a number of phospholipids to stimulate $\text{Lactobacillus}$$\text{plantarum}$ undecaprenyl pyrophosphate synthetase was investigated. The detergent Triton X-100, which is added to stabilize the enzyme during purification and is required for $\text{in}$$\text{vitro}$ activity, was removed with the non-ionic resin XAD-2. The effects of cardiolipin, phosphatidyl ethanolamine, phosphatidyl choline, and phosphatidyl glycerol on the activity of XAD-2 treated undecaprenyl pyrophosphate synthetase were determined. Of the phospholipids studied only cardiolipin stimulated $\text{in}$$\text{vitro}$ enzymic activity as effectively as Triton X-100.     

Glucagon and epinephrine-stimulated phospholipid methylation in hepatic microsomes

Phospholipid methylation by hepatic microsomes was measured following glucagon or epinephrine administration either to intact rats or to the isolated perfused liver. Both hormones stimulated the methylation measured as the incorporation of S-adenosyl-L-[methyl-³H]methionine into phospholipids. The labeled products were identified by thin layer chromatography and most of the counts were found to be incorporated into phosphatidylcholine. The stimulatory effects of the hormones were evident already 5 minutes following hormone administration both in $\text{in vivo}$ and in $\text{in vitro}$. The observed stimulation of the methylation process by glucagon and epinephrine might be related to the previously reported stimulatory effect of these hormones on the microsomal Ca²⁺-ATPase, and indicate that methylation process(es) might mediate some of the effects of these hormones.     

Dependence of Escherichiacoli pyridine nucleotide transhydrogenase on phospholipids and its sensitivity to N-ethylmaleimide

A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of $\text{Escherichia}\text{coli}$ by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of $\text{E.}\text{coli}$, was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD⁺ and NADP⁺, but NADPH served to accelerate the rate of inhibition.     

DIFFERENTIAL DISTRIBUTION OF [U-14C]GLUCOSE AND [U-14C]GLYCEROL AMONG MOLECULAR SPECIES OF PHOSPHATIDYL CHOLINE,PHOSPHATIDYL ETHANOLAMINE AND 1,2-DIACYLGLYCEROL IN RABBIT BRAIN12

Specific radioactivities of molecular species of phosphatidyl choline(PC), phosphatidyl ethanolamine(PE) and 1,2-diacylglycerol were determined in rabbit brain 15 and 30 min after intraventricular injection of 10OpCi of either [U-¹⁴C]glucose or [U-¹⁴C]glycerol. The rate of de nouo synthesis of glycerophospholipids and their molecular species could be determined after glycerol labelling, since 94.0–99.7% of ¹⁴C activity was recovered in glyceryl moieties of brain lipids. After injection of glucose radioactivity was measured in both glyccrol and acyl residues of lipids. High incorporation rates were measured in species of PC, PE and 1,2-diacylglycerol with oleic acid in position 2 and with palmitic, stearic or oleic acids in position 1. The conclusion may therefore be drawn that these molecular species were preferably synthesized de novo by selective acylation of glycerol 3-phosphate. The lowest specific activities were observed for 1,2-dipalmitoyl- and l-stearoyl-2- arachidonoyl-glycerol, -PC and -PE. These turnover rates point to incorporation of arachidonate, and probably also of palmitate in dipalmitoyl-PC, amounting to 20% of total PC, via deacylation-acylation- cycle.     

Precursor supply and hepatic enzyme activities as regulators of triacylglycerol synthesis in isolated hepatocytes and perfused liver

The relative significance of alterations in precursor supply and enzyme activities for the rate of triacylglycerol synthesis was studied in isolated hepatocytes and perfused livers. Precursor availability was varied in vitro by changing the fatty acid concentration in the incubation medium or adding ethanol to the perfusion medium in order to increase the cellular glycerol 3-phosphate concentration. The rate of glycerolipid synthesis in hepatocytes, measured in terms of the label incorporated into the various lipid classes from tritiated glycerol, was strongly dependent on the fatty acid concentration up to 2 mm of oleate (fatty acid/albumin molar ratio $\text{7}\text{1}$). Ethanol in vitro increased the incorporation of labeled oleate into phosphatidic acid and diacylglycerol in the isolated perfused liver, but its effect on the incorporation into triacylglycerol was small. Ethanol in vitro increased the label incorporation into both diacylglycerol and triacylglycerol in the livers from cortisol-treated rats. Although cortisol treatment increased the soluble phosphatidate phosphohydrolase activity 4.4-fold in the hepatocytes, it had no effect on the rate of triacylglycerol synthesis, whereas fasting increased this rate about 3-fold, although only a moderate concomitant increase in soluble phosphatidate phosphohydrolase activity was observed. Neither cortisol treatment nor fasting affected the microsomal glycerol-3-phoshate acyltransferase activity. The results demonstrate that substrate availability can override enzyme modulations in the regulation of triacylglycerol synthesis and that phosphatidate phosphohydrolase is not the main regulator of triacylglycerol synthesis.     

Effects of neurotransmitters and other pharmacological agents on 32Pi incorporation into phospholipids of the iris muscle of the rabbit

The effects of norepinephrine, other catecholamines, α- and β- adrenergic receptor blocking agents and acetylcholine on the incorporation of ³²Pi into phospholipids of the iris muscle of the rabbit were studied $\text{in vitro}$. There was a marked stimulation of ³²Pi into phosphatidic acid (PhA), phosphatidyl inositol (PhI) and to a much lesser extent phosphatidyl choline but not into phosphatidyl ethanolamine. The increase in the ³²P labeling of PhA and PhI in the presence of norepinephrine or acetylcholine, which ranged from 2- to 6-fold, was found to be time- and concerntration-dependent. Under our experimental conditions, several adrenergic drugs, including DL-propranolol, phentolamine, isoproterenol, phenylephrine, but not sotalol, increased markedly (nearly up to 5-fold) the ³²Pi incorporation into PhA and PhI of the iris. In contrast, phenoxybenzamine, an α-receptor blocker, blocked completely the stimulatory effects of norepinephrine on phospholipid synthesis. The stimulation of phospholipid synthesis by acetylcholine was completely abolished by atropine. Incorporation of ³²Pi into PhA and PhI was significantly increased in the presence of serotonin, dopamine, epinephrine or histamine. Addition of γ-aminobutyric acid or cyclic AMP was ineffective. These observations suggest that in the iris muscle of the rabbit, which is innervated by cholinergic and adrenergic fibers, the phospholipid effect is probably a membrane effect that is not associated with synaptic transmission.     

Biosynthesis of phosphatidyl glycerophosphate in Escherichia coli

An enzyme (L-glycerol 3-phosphate: CMP phosphatidyltransferase) catalyzing the synthesis of phosphatidyl glycerophosphate from CDP-diglyceride and L-glycerol 3-phosphate has been rendered soluble by treatment of the particulate, membrane-containing fraction of E. coli with Triton X-100 and has been partially purified. The enzyme, devoid of phosphatidyl glycerophosphatase activity, is specific for L-glycerol 3-phosphate and is completely dependent upon added Mg(++) or Mn(++) for activity. It has high affinity for CDP-diglyceride and can be used for the assay of this nucleotide. Other properties of the enzyme are also described.