Membrane lipids composition and metabolism during early embryonic development. Phospholipid subcellular distribution and 32P labeling

Phospholipid composition and 32P metabolism were studied in oocytes and early developing embryos of the toad, Bufo arenarum, Hensel. The content and distribution of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, sphingomyelin, phosphatidylserine, and diphosphatidylglycerol in embryos, whole oocytes, and the subcellular fractions of both were determined. Phosphatidylcholine and phosphatidylethanolamine were the major constituents of yolk platelet. Diphosphatidylglycerol was confined to the mitochondrial fraction, where it represented about 7% of the total phosphoacylglycerols. Relatively large amounts of sphingomyelin were found in microsomal and postmicrosomal supernatants. After in vivo labeling with 32P, the early development of individual phospholipids in subcellular fractions and in whole eggs was followed. The greatest uptake was found in mitochondrial and yolk platelet fractions. A steady increase in the amount of 32P present in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol was seen in the whole embryo from oocyte to late gastrula stage and in all subcellular fractions. Phosphatidic acid exhibited a slight decrease in specific activity, except in the yolk platelet fraction. This high 32P incorporation would indicate a rapid and uneven polar headgroup turnover determined by phospholipid class and subcellular fraction. At the same time, the phospholipid content of the subcellular fractions studied remained unchanged during early embryogenesis. Moreover, 32P was actively incorporated into the individual phospholipids in the absence of measurable net synthesis.     

Investigation of phospholipids of the pulmonary extracellular lining by electron paramagnetic resonance. The effects of phosphatidylglycerol and unsaturated phosphatidylcholines on the fluidity of dipalmitoyl phosphatidylcholine.

The membranous structures of the pulmonary extracellular lining were removed from the lungs of rabbits by pulmonary lavage and isolated by differential centrifugation. This membranous fraction contained 93% of the total extracellular phospholipids present in lavage effluents and consisted of membranous vesicles, membrane fragments, tubular myelin and secreted lamellar bodies. The fraction was rich in phosphatidylcholine (79.4%) containing 85.2% palmitic acid in the 1-position and 57.4% palmitic acid in the 2-position. Phosphatidylglycerol was the next most abundant phospholipid, accounting for 9.4% of the total. E.p.r. spectra, obtained by using 5-doxylmethylstearate as a probe, showed that the extracellular phospholipids of the pulmonary lining were organized into structures which were much more fluid than erythrocyte-ghost membranes. The fluidity of phosphatidylcholine isolated from the membranous fraction was similar to that of the fraction itself, indicating that the minor phospholipids had very little influence on the fluidity of the major phospholipid. At physiological temperature, the fluidity of dipalmitoyl phosphatidylcholine was relatively low, but could be markedly increased by the presence of 1-palmitoyl-2-oleoyl phosphatidylcholine or phosphatidylglycerol (10%). Protein present in the extracellular phospholipid fraction did not affect the fluidity of the fraction. These studies indicate that the unsaturated phosphatidylcholines could play a major role in determining the fluidity of the important surface-tension-lowering phospholipids such as dipalmitoyl phosphatidylcholine.     

The incorporation of radioactive fatty acids and of radioactive derivatives of glucose into the phospholipids of subsynaptosomal fractions of cerebral cortex.

1. Crude synaptosomal fractions (P2) from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium containing labelled fatty acids and [3H]glucose. After the shortest incubation period (7.5 min) a high percentage (50-80%) of the total radioactive fatty acids was found in the P2 fractions. 2. After the incubation, the synaptosomal fractions were submitted to hypo-osmotic disruption and subsynaptosomal fractionation was carried out by using discontinuous-sucrose-gradient centrifugation. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomal preparation) and H (disrupted synaptosomes), as were the specific activities of a number of marker enzymes and the distribution of acetylcholine. 3. By using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose, the order to specific radioactivities in fraction D was found to be: phosphatidylinositol greater than phosphatidylcholine greater than phosphatidylserine greater than phosphatidylethanolamine. 4. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were always higher in fraction D than in fraction E. As fraction E had higher specific activities of several membrane marker enzymes, the enhanced labelling found in fraction D was considered to be localized in the synaptic vesicles. In this fraction, phosphatidylinositol made particularly large contributions to the total phospholipid labelling derived from [14C]arachidonate and [3H]glucose. 5. The similar labelling ratios of fatty acid/glucose in the phospholipids of fractions D and E, and the high specific radioactivities in the total phospholipid of the soluble fraction O, suggested intrasynaptosomal phospholipid transport.     

Phospholipid exchange reactions within the liver cell

1. Isolated rat liver mitochondria do not synthesize labelled phosphatidylcholine from CDP-[(14)C]choline or any phospholipid other than phosphatidic acid from [(32)P]phosphate. The minimal labelling of phosphatidylcholine and other phosphoglycerides can be attributed to microsomal contamination. However, when mitochondria and microsomes are incubated together with [(32)P]phosphate, the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of the reisolated mitochondria become labelled, suggesting a transfer of phospholipids between the two fractions. 2. When liver microsomes or mitochondria containing labelled phosphatidylcholine are independently incubated with the opposite un-labelled fraction, there is a substantial and rapid exchange of the phospholipid between the two membranes. Exchange of phosphatidylinositol also occurs rapidly, whereas phosphatidylethanolamine and phosphatidic acid exchange only slowly. There is no corresponding transfer of marker enzymes. The transfer of phosphatidylcholine does not occur at 0 degrees , and there is no requirement for added substrate, ATP or Mg(2+), but the omission of a heat-labile supernatant fraction markedly decreases the exchange. 3. After intravenous injection of [(32)P]phosphate, short-period labelling experiments of the individual phospholipids of rat liver microsomes and mitochondria in vivo give no evidence for a similar exchange process. However, the incubation of isolated microsomes and mitochondria with [(32)P]phosphate also fails on reisolation of the fractions to demonstrate a precursor-product relationship between the individual phospholipids of the two membranes. 4. The intraperitoneal injection of [(32)P]phosphate results in a far greater proportion of the dose entering the liver than does intravenous administration. After intraperitoneal administration of [(32)P]phosphate the specific radioactivities of the individual phospholipids are in the order microsomes > outer mitochondrial membrane > inner mitochondrial membrane. 5. The incorporation of (32)P into cardiolipin is very slow both in vivo and in vitro. After labelling in vivo the radioactivity in the cardiolipin persists compared with that of the other phospholipids, whose specific radioactivities in the microsomes and mitochondrial fragments decay at a similar rate to that of the acid-soluble phosphate pool. 6. The possibility of phospholipid exchange processes occurring in the liver cell in vivo is discussed, and it is suggested that only a small but highly labelled part of the endoplasmic-reticulum lipoprotein pool is involved in the transfer.     

Turnover of phosphatidylcholine in cell membranes of adult rat brain

—Incorporation of radioactive choline into subcellular fractions of adult rat brains, after intracranial injection of the precursor, was studied at different times. Maximum labelling of phosphatidylcholine occurred at 5 h in all fractions, the highest specific radio-activity being found in mitochondria. The specific radioactivity diminished rapidly for a few hours after the peak of maximum incorporation and continued to decrease thereafter at a much slower rate. The type of decay curve found in all fractions except myelin, indicated the possible presence of two populations of phosphatidylcholine with quite different turnover rates. The significance of these findings is discussed.     

The metabolism of phospholipids in mouse brain slices

1. Slices of mouse brain grey matter were incubated with [³²P]phosphate and [1-¹⁴C]acetate. Doubly labelled phospholipids were extracted from subcellular fractions prepared from the slices in a mixture of metabolic inhibitors, under conditions where there was negligible change in radioactive labelling during the preparation. Two tissue fractions were studied in detail; one contained a high proportion of mitochondria and the other was mainly microsomal. 2. In all tissue fractions the highest incorporations of both [³²P]phosphate and [1-¹⁴C]acetate occurred into phosphatidylcholine. 3. After incubation for 1hr., the ³²P/¹⁴C ratios for phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid in the mitochondrial fraction were similar to those in the microsomal fraction. 4. The ³²P/¹⁴C ratios were similar in phosphatidylcholine and phosphatidylethanolamine and much lower than those in phosphatidic acid and phosphatidylinositol.     

The interaction of phospholipase A2 with phospholipid analogues and inhibitors

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation by phorbol 12-myristate 13-acetate of arachidonic acid release from rat basophilic leukemia cells stimulated with A23187

Rat basophilic leukemia (RBL-2H3) cells were cultured in medium containing [3H]arachidonic acid and labelling of the different lipid fractions was followed with time. After up to 4 h of culture, the label was found mostly in phosphatidylcholine. After 8 h, labelling of phosphatidylethanolamine gradually exceeded that of phosphatidylcholine, until at 24 h, approximate equilibrium labelling of the lipid fractions was attained and 45% of the label was found in phosphatidylethanolamine, 35% in phosphatidylcholine, 18% in the phosphatidylserine/inositide fraction and the remainder in the neutral lipid fraction. Stimulation of cells with A23187 after 30 min of labelling caused release of [3H]arachidonic acid which was accountable by a decrease in radioactivity of phosphatidylcholine, whereas stimulation of cells after 24 h of labelling caused the release of radioactive arachidonic acid, which was accompanied by a decrease of label in both phosphatidylcholine and phosphatidylethanolamine. Incubation of the labelled cells with phorbol 12-myristate 13-acetate prior to ionophore addition enhanced both the release of [3H]arachidonic acid and its metabolites and the decrease in label of the same phospholipids as those affected by ionophore alone. Under our conditions, the enhancement effects of phorbol ester were greatest after 2-5 min of preincubation, prior to ionophore addition. The results suggest that in basophilic leukemia cells, arachidonic acid release proceeds from several pools of phospholipids and that the activity of the phospholipase(s) involved is modulated by protein kinase C.     

The rat retina is a useful in vivo model to study membrane lipid synthesis: Rates of biosynthesis of neutral glycerides and phospholipids

The phospholipid composition was studied in the whole rat retina, as well as in its subcellular fractions. A relative enrichment of phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine was observed in rod outer segments (ROS) in comparison with entire retina: nuclear-photoreceptor inner segmentssynaptic bodies (P₁) and synaptosomal-mitochondrial (P₂) fractions. Phosphatidylcholine was the predominant phospholipid class found in all subcellular fractions analyzed. The microsomal fraction was relatively enriched in phosphatidic acid and in phosphatidylinositol. In addition, the rat eye has been used as an in vivo system to study membrane lipid synthesis. After intravitreal injections of [2-³H]glycerol a rapid labeling of retinal glycerolipids took place. Up to 120 min after injection only the glycerol backbone of lipids was labeled. Phosphatidic acid and diacylglycerol displayed rapid rates of synthesis and breakdown. Fastest rates of labeling were attained by phosphatidylcholine followed by phosphatidylinositol. Differences were found when in vitro labeling by [2-³H]glycerol was compared with intravitreal injections. Labeling of phospholipids of subcellular fractions by intravitreally injected [2-³H]glycerol showed that most of the label accumulated in microsomal phosphatidylcholine and phosphatidylinositol. Diacylglycerols and phosphatidylethanolamine also took up 10 and 20% respectively of the precursor. It is concluded that the rat eye is a useful experimental model to study synthesis and metabolism of membrane lipids in the retina.     

Abnormal membrane phospholipid content in subcellular fractions from the Morris 7777 hepatoma.

1. Mitochondrial and microsomal fractions were prepared from normal rat liver and the Morris 7777 hepatoma and characterized by the use of the marker enzymes, succinate dehydrogenase and rotenone-insensitive NADPH-cytochrome c reductase. 2. The phospholipid content per mg membrane protein of Morris 7777 hepatoma mitochondria was increased by 75% as compared with mitochondria from normal rat liver. Microsomes from this poorly-differentiated tumor were found to have a 45% decrease in the content of phospholipid. These abnormalities were independent of tumor size or age. 3. The percent phospholipid content of the subcellular fractions was determined, and revealed an increase in the percent sphingomyelin in both the microsomal and mitochondrial fractions of the tumor. Decreases in the percent phosphatidylcholine and phosphatidylethanolamine were noted in tumor microsomes as compared with normal liver. Diphosphatidylglycerol was not found in significant quantities in the microsomal fraction of this hepatoma line. 4. The content of the various phospholipid classes per mg protein in the respective mitochondrial and microsomal fractions was determined. Large increases in nearly all the major phospholipid classes were found in tumor mitochondria; tumor microsomes were characterized by an increased content of sphingomyelin but the content of nearly all other phospholipids was significantly decreased. These findings suggest the presence of disturbances in the regulation of phospholipid metabolism in subcellular organelle membranes of the Morris 7777 hepatoma.     

Phospholipids in chromatin: incorporation of [32P]O4(2-) in different subcellular fractions of hepatocytes

Nuclear, chromatin and microsomal fractions were isolated from hepatocytes prepared from rats injected with [32P]O4(2-) and killed subsequently at times between 1 and 48 h. Specific activities of the total phospholipids (PL) were determined for each subcellular fraction. The major points noted were the initial specific activity of the chromatin PL was half that of both nuclear and microsomal PL at 1 h; the first peak of labelling occurred at 6 h in both nuclear and microsomal PL, but was 3 h later (9h) in the chromatin PL; and a second peak of labelling occurred in the chromatin and microsomal PL, but not in those of the nuclei. On fractionation of the PL, the major and most metabolically active components were phosphatidylcholine + phosphatidylethanolamine, whilst sphingomyelin accounted for only about 8 per cent of the total PL. The chromatin and microsomal fractions were somewhat similar in their labelling patterns though with a delayed peaking of activity in the chromatin. This is indicative of a synthesis and transport of PL from the microsomes to the chromatin.     

Time course for labeling of brain membrane phosphoinositides and other phospholipids after intracerebral injection of [32P]-ATP. Evaluation by an improved HPTLC procedure

An improved two-dimensional HPTLC procedure was developed for separating phospholipids including individual phosphoinositides, phosphatidic acids and plasmalogens. This procedure was used to examine the time course for uptake of label by phospholipids in brain subcellular membranes after intracerebral injection of [gamma-32P]-ATP. There were considerable differences in the phospholipid labeling pattern among different subcellular fractions. In particular, a high proportion of labeled phosphatidylinositol 4,5-bisphosphates and phosphatidic acids was found in the myelin fraction during the initial 4 hr after injection. In other subcellular fractions, labeling of phosphoinositides was maximum at 2 hr, but with prolonged time, poly-phosphoinositides started to show a decline in radioactivity whereas labeling of other phospholipids continued to show a steady increase instead. Results indicate at least two different modes for the uptake of label by brain membrane phospholipids after intracerebral injection of [32P]-ATP.     

Co-ordination between membrane phospholipid synthesis and accelerated biosynthesis of cytoplasmic ribonucleic acid and protein

1. The rate of synthesis of membrane phospholipid was studied in rat liver and seminal vesicles by following the incorporation of [(32)P]orthophosphate, [(14)C]choline and [(14)C]glycerol. Particular emphasis was laid on the endoplasmic reticulum, which was fractionated into smooth microsomal membranes, heavy rough membranes, light rough membranes and free polyribosomes. 2. Phospholipid labelling patterns suggested a heterogeneity in the synthesis and turnover of the different lipid moieties of smooth and rough endoplasmic membranes. The major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were labelled relatively rapidly with (32)P over a short period of time whereas incorporation of radioisotope into the minor phospholipids, sphingomyelin, lysolecithin and phosphatidylinositol proceeded slowly but over a longer period of time. 3. The incorporation of orotic acid into RNA and labelled amino acids into protein of the four submicrosomal fractions was also studied. 4. Rapid growth of the liver was induced by the administration of growth hormone and tri-iodothyronine to hypophysectomized and thyroidectomized rats and by partial hepatectomy. Growth of seminal vesicles of castrated rats was stimulated with testosterone propionate. 5. The rate of labelling of membrane phospholipids was enhanced in all major subcellular particulate fractions (nuclear, mitochondrial and microsomal) during induced growth. However, it was in the rough endoplasmic reticulum that the accumulation of phospholipids, RNA and protein was most marked. The effect of hormone administration was also to accelerate preferentially the labelling with (32)P of sphingomyelin relative to that of phosphatidylcholine or phosphatidylethanolamine. 6. Time-course analyses showed that, in all four growth systems studied, the enhancement of the rate of membrane phospholipid synthesis coincided with the rather abrupt increase in the synthesis of RNA and protein of the rough endoplasmic reticulum. Growth hormone and tri-iodothyronine administered to hypophysectomized rats had additive effects in all the biosynthetic processes. The latent period of action of each hormone was maintained so that two waves of proliferation of endoplasmic reticulum occurred if the hormones were administered simultaneously. 7. It is concluded that there is some mechanism in the cell that tightly co-ordinates the formation of membranes, especially those of the endoplasmic reticulum, when an increased demand is made for protein synthesis.     

Effects of glucose transport on the incorporation of 32P-inorganic phosphate into phospholipids in Hymenolepis diminuta (Cestoda)

1. The absorption of glucose and alpha-methyl-D-glucoside by Hymenolepis diminuta was concentration dependent. 2. alpha-Methyl-D-glucoside competitively inhibited glucose absorption and was not metabolised by the parasite. 3. alpha-Methyl-D-glucoside significantly lowered (P less than 0.05) the incorporation of 32Pi into all phospholipid classes. 4. Glucose inhibited (P less than 0.01) 32Pi incorporation into phosphatidylcholine only. 5. Phlorizin did not affect 32Pi labelling of phospholipids. 6. Serotonin and histamine stimulated (P less than 0.01) 32Pi labelling of all phospholipid classes. 7. Radioactivities in water soluble fractions were increased (P less than 0.01) in the presence of glucose, serotonin and histamine.     

Ratio and composition of the plasmalogen and diacylated forms of phospholipids in subcellular fractions of the avian brain

Studies have been made on the specific content of plasmalogen and diacylated forms of phosphatidylethanolamine and phosphatidylcholine in subcellular fractions (myelin, nuclei, microsomes, mitochondria, synaptosomes) from the brain of pigeons, as well as in the myelin fraction from the brain of the crow Corvus cornix and the hawk Accipiter gentelis. Fatty acid composition and fatty aldehyde composition of these two main phospholipids of the brain were studied in the subcellular fractions obtained. It was shown that plasmalogen forms of phospholipids are localized in birds mainly in the myelin fraction which exhibits the highest plasmalogen concentration as compared to the same fraction of all the vertebrates investigated. With respect to fatty acid and fatty aldehyde composition, as well as to the degree of their unsaturation, myelin plasmalogens from birds are similar to those from other cold-blooded and warm-blooded animals. This fact indicates that high relative content of plasmalogens together with their high unsaturation account for normal functional activity of myelin membranes in all vertebrates.     

Phospholipids and fatty acids of Neisseria gonorrhoeae.

The phospholipids and fatty acids of two strains of Neisseria gonorrhoeae of different penicillin susceptibilities were examined. The phospholipids, which comprise about 8% of the dry weight of the cells, consisted of phosphatidylethanolamine (70%) and phosphatidylglycerol (20%); small amounts of phosphatidylcholine and traces of cardiolipin were also present. Growing and stationary-phase cells were similar in content and composition of phospholipids except for phosphatidylcholine, which increased two- to fivefold in the stationary-phase cells. The fatty acids of the phospholipids were characterized by two major acids, palmitic and a C16:1, with myristic and a C18:1 acid present in smaller amounts. The fatty acids present in purified phospholipid fractions varied considerably in relative proportions from fraction to fraction. No significant difference in the composition of phospholipids from the two strains was evident. Large amounts of beta-hydroxy lauric acid were detected only after saponification of the organisms. Differences in the lipid composition between the gonococcus and other gram-negative bacteria are discussed.     

Phospholipids and glycolipids in subcellular fractions of mosquitoAedes aegypti cells

Summary Thin layer chromatography of [¹⁴C]palmitate labeled phospholipids of mosquitoAedes aegypti cells reveals that phosphatidylethanolamine is the major phospholipid, and ceramide phosphorylcholine the major sphingolipid. Glycolipids of these cells contain mannose in addition to glucose. The distribution of phospholipids in subcellular membrane fractions shows an enrichment in sphingomyelin and phosphatidyl serine in plasma membranes and other smooth membrane fractions. Cardiolipin is located predominantly in fractions, rich in mitochondrial membranes.     

Differential effect of L-thyroxine on phospholipid biosynthesis in mitochondria and microsomal fraction.

1. The action of L-thyroxine on the incorporation of radioactive choline or CDP-choline into phosphatidylcholine in vitro was explored in liver and brain microsomal fraction and mitochondria obtained from young adult rats. 2. In liver mitochondria isolated from animals treated with L-thyroxine (40 mg/kg body wt. during 6 days), the incorporation of both radioactive precursors into phosphatidylcholine was significantly decreased compared with normal controls, whereas in the total homogenate and in the microsomal fraction the incorporation was similar in the experimental and control groups. In subcellular fractions isolated from brain, the incorporation of precursors was similar in L-thyroxine-treated and normal animals. 3. Liver mitochondria isolated from normal animals incubated in vitro with CDP-choline, in the presence of different concentrations of L-thyroxine, showed also a marked decrease in the incorporation of label into phosphatidylcholine, whereas no significant changes were found in the total homogenate and in the microsomal fraction compared with control experiments. 4. The differential effect of L-thyroxine on the incorporation of radioactive precursors into phosphatidylcholine of isolated liver subcellular fractions gives further support to the hypothesis that liver mitochondria can independently synthesize part of their own phospholipids. 5. Possible mechanisms of the action of the hormone at the mitochondrial level are discussed.     

Phospholipid composition and acyltransferase activity of lamellar bodies isolated from rat lung.

The role of the lamellar body of the type II pneumocyte in the synthesis and storage of the phospholipids of the surfactant lipoprotein lining the alveolar surface has been investigated. Electron microscopy has been used to establish the purity of the isolated lamellar body, microsomal, and mitochondrial fractions. Additional proof of lamellar body purity was obtained by enzyme marker studies. The phospholipid:protein ratio of each of the above fractions was determined as well as that of surfactant lipoprotein isolated from rat lung. Lamellar body phospholipid:protein ratio was highest, 3.7 μmol of lipid phosphorus/mg of lung protein. The phospholipid composition of the lamellar body fraction was found to be similar to that of the isolated surfactant lipoprotein. Lamellar body phosphatidylcholine and phosphatidylglycerol each contained over 90% saturated fatty acids. The lamellar body fraction was found to possess significant acyltransferase activity between [1-¹⁴C]palmitoyl-CoA and phosphatidylcholine. This activity was somewhat higher than in the microsomal fraction and much greater than in the mitochondrial fraction. The activity in all fractions was stimulated by Ca²⁺ and Mg²⁺. [1-¹⁴C]oleoyl-CoA did not serve as an effective acyl donor. When 1-palmitoyl-2-lysophosphatidylcholine was used as the acceptor molecule and [1-¹⁴C]palmitoyl-CoA the donor, acyltransferase activity was increased over that found with phosphatidylcholine as donor in all fractions. The microsomal fraction had the greatest activity and the lamellar body fraction the least. The data obtained support the hypothesis that the lamellar body is involved in the synthesis and storage of the phospholipids of the surfactant lipoprotein complex.     

Stimulation of cholinephosphate cytidylyltransferase activity by estrogen in fetal rabbit lung is mediated by phospholipids

We have investigated the mechanism by which estrogen stimulates phosphatidylcholine synthesis in fetal rabbit lung. The hormone increased the activity of cholinephosphate cytidylyltransferase in the 105 000 X g supernatant fraction but had no effect on the activities of this enzyme in the homogenate or other subcellular fractions. Although microsomal cytidylyltransferase has been reported to regulate phosphatidylcholine synthesis in other systems, and translocation of the enzyme from cytosol to microsomes has been reported in association with increased phosphatidylcholine synthesis, we found no evidence of this in the case of estrogen-stimulated phosphatidylcholine synthesis in the fetal lung. Cytosolic cytidylyltransferase activity was dependent on phospholipids. Extraction with acetone/butanol drastically reduced its activity as well as the stimulatory effect of estrogen. The activity and the effect of estrogen were restored on re-addition of lipids extracted with chloroform/methanol from additional supernatants. Fractionation of the total lipids revealed that the stimulatory effect was entirely associated with the phospholipids; neutral lipids and glycolipids did not stimulate. Treatment of the phospholipid fraction with phospholipase C abolished the stimulatory effect. The stimulatory effect of estrogen, however, could not be attributed to any individual phospholipid species but appeared to require the entire phospholipid mixture. We conclude that estrogen stimulates fetal lung phosphatidylcholine synthesis by increasing the activity of cytosolic cytidylyltransferase and this activation in turn is mediated by cytosolic phospholipids.