Fatty acid metabolism in sn-glycerol-3-phosphate acyltransferase (plsB) mutants.

Fatty acid metabolism was examined in Escherichia coli plsB mutants that were conditionally defective in sn-glycerol-3-phosphate acyltransferase activity. The fatty acids synthesized when acyl transfer to glycerol-3-phosphate was inhibited were preferentially transferred to phosphatidylglycerol. A comparison of the ratio of phospholipid species labeled with 32Pi and [3H]acetate in the presence and absence of glycerol-3-phosphate indicated that [3H]acetate incorporation into phosphatidylglycerol was due to fatty acid turnover. A significant contraction of the acetyl coenzyme A pool after glycerol-3-phosphate starvation of the plsB mutant precluded the quantitative assessment of the rate of phosphatidylglycerol fatty acid labeling. Fatty acid chain length in membrane phospholipids increased as the concentration of the glycerol-3-phosphate growth supplement decreased, and after the abrupt cessation of phospholipid biosynthesis abnormally long chain fatty acids were excreted into the growth medium. These data suggest that the acyl moieties of phosphatidylglycerol are metabolically active, and that competition between fatty acid elongation and acyl transfer is an important determinant of the acyl chain length in membrane phospholipids.     

Phospholipid aliphatic chain composition modulates lipid class composition, but not lipid asymmetry in Clostridium butyricum

The phospholipid composition of the butyric acid-producing clostridia is responsive to the degree of enrichment of the lipids with cis-unsaturated fatty acids. When Clostridium butyricum and Clostridium beijerinckii are grown on oleic acid in media devoid of biotin, the acyl and alk-1-enyl chains of the phospholipids become highly enriched with 18:1 and C19-cyclopropane. Under these conditions there is a marked increase in the glycerol acetals of the major plasmalogens of these organisms. We have grown both species on mixtures of palmitate and oleate in the absence of biotin. The alk-1-enyl chains were highly enriched with C18-unsaturated and C19-cyclopropane residues at all but the highest ratios of palmitate to oleate (80:20, w/w) added to the medium. At ratios of palmitate to oleate greater than or equal to 40:60, the saturated acid was incorporated predominantly into the phospholipid acyl chains in both organisms. The effects of increasing unsaturation of the acyl chains as the ratio of oleate to palmitate was increased was examined in C. butyricum. In cells grown on mixtures of palmitate and oleate equal to or exceeding 40% palmitate, the ratio of glycerol acetal lipid to total phosphatidylethanolamine (PE) was relatively constant. As the proportion of oleic acid added to the medium was increased, the ratio of glycerol acetal lipid to PE increased from 0.7 to 2.0. Thus the ratio of the polar lipids appears to respond to the content of phospholipids that contain two unsaturated chains. The fraction of PE present as plasmalogen remained relatively stable (0.82 +/- 0.05) at varying ratios of medium oleic and palmitic acids. Both the glycerol acetal of ethanolamine plasmalogen, and ethanolamine plasmalogen, are shown to be 80% or more in the outer monolayer of the cell membrane. These two polar lipids represent approx. 50% of the phospholipids in cells grown on exogenous fatty acid. The bulk of the remainder is polyglycerol phosphatides. We suggest that the ability of both species to grow with highly unsaturated membranes is related to their ability to modulate their polar lipid composition.     

Replacement of acyl and alk-1-enyl groups in Clostridium butyricum phospholipids by exogenous fatty acids.

The effect of exogenous unsaturated fatty acids on the acyl and alk-1-enyl group composition of the phospholipids of Clostridium butyricum has been examined. Unsaturated fatty acids support the growth of this organism in the absence of biotin. When cells were grown at 37 degrees in media containing oleate or linoleate and a Casamino acid mixture containing traces of biotin, the exogenous fatty acids were found mainly in the alk-1-enyl chains of the plasmalogens with less pronounced incorporation into the acyl chains. However, at 25 degrees in this medium, both the acyl and alk-1-enyl chains contained substantial amounts of the 18:1 supplement plus the C19-cyclopropane chains derived from it. Ak-1-enyl chains in all the major phosphatide classes showed a uniformly high substitution by the oleate supplement in cells grown at 37 degrees. The oleate and C19-cyclopropane content of the acyl chains was more variable among the phosphatide classes. At 37 degrees, trans-9-octadecenoic acid (elaidic acid) also supported growth and was incorporated into both acyl and alk-1-enyl chains at a high level. When cells were grown on oleate at 37 degrees in media containing biotin-free Casamino acids, both the acyl and alk-1-enyl chains had a high level of 18:1 plus C19-cyclopropane chains. In the cells grown at 37 degrees with oleate substantial changes were seen in the phospholipid class composition. There was a large decrease in the ethanolamine plus N-methylethanolamine plasmalogens with a corresponding increase in the glycerol acetals of these plasmalogens. The glycerol phosphoglycerides were also significantly lower with the appearance of an unknown, relatively nonpolar phospholipid fraction.     

Turnover of phospholipid fatty acyl chains in cultured neuroblastoma cells: involvement of deacylation-reacylation and de novo synthesis in plasma membranes

Cultured neuroblastoma cells (NIE-115) rapidly incorporated the essential fatty acid, linoleic acid (18:2 (n = 6), into membrane phospholipids. Fatty acid label appeared rapidly (2-10 min) in plasma membrane phospholipids without evidence of an initial lag. Specific activity (nmol fatty acid/mumol phospholipid) was 1.5-2-fold higher in microsomes than in plasma membrane. In these membrane fractions phosphatidylcholine had at least 2-fold higher specific activity than other phospholipids. With 32P as radioactive precursor, the specific activity of phosphatidylinositol was 2-fold higher compared to other phospholipids in both plasma membrane and microsomes. Thus a differential turnover of fatty acyl and head group moieties of both phospholipids was suggested. This was confirmed in dual-label (3H fatty acid and 32P), pulse-chase studies that showed a relatively rapid loss of fatty acyl chains compared to the head group of phosphatidylcholine; the opposite occurred with phosphatidylinositol. A high loss of fatty acyl chain relative to phosphorus indicated involvement of deacylation-reacylation in fatty acyl chain turnover. The patterns of label loss in pulse-chase experiments at 37 and 10 degrees C indicated some independent synthesis and modification of plasma membrane phospholipids at the plasma membrane. Lysophosphatidylcholine acyltransferase and choline phosphotransferase activities were demonstrated in isolated plasma membrane in vitro. Thus, studies with intact cells and with isolated membrane fractions suggested that neuroblastoma plasma membranes possess enzyme activities capable of altering phospholipid fatty acyl chain composition by deacylation-reacylation and de novo synthesis at the plasma membrane itself.     

Fatty acid synthesis and metabolism of phospholipid acyl groups in strain L mouse fibroblasts

Strain L mouse fibroblasts grown in medium supplemented with 2.5% delipidized horse serum were found capable of desaturating oleic and linoleic acid to dienoic and trienoic acid(s), respectively. Although 40-60% of de novo fatty acid synthesis from [2-3H]acetate was inhibited by the administration of exogenous oleic or linoleic acid, sterole synthesis was only slightly affected. Within 24-48 h after incorporation, phospholipid fatty acyl groups could undergo active exchange between phospholipids. After this dynamic transition period was over, not only were the phospholipid acyls retained but some vicinal fatty acyl pairs of phospholipid also appeared to be stable and remained together throughout the depletion period. At any time in the experiment, however, introduction of exogenous fatty acid perturbed this phospholipid acyl retention, delayed the time at which the phospholipid acyl groups no longer moved between phospholipids and also decreased the ultimate number of phospholipid acyl groups retained by strain L mouse fibroblasts.     

Metabolism of extracellular phospholipids in Tetrahymena pyriformis

We studied the metabolism of phospholipids exogenously added to cultures of the protozoan, Tetrahymena pyriformis. Tetrahymena cells were found to metabolize the extracellular phospholipids and the fatty acyl chains of the latter were accumulated predominantly as a form of triacylglycerol in the cells. This metabolism was considered to be initiated via endocytosis of phospholipid vesicles, as judged from the following facts: Cytochalasin B, an inhibitor of endocytosis, suppressed the metabolism almost completely. Phospholipid vesicles were incorporated into a phagosome-like structure in Tetrahymena cells, as observed under an electron microscope. When phospholipids doubly labeled with 14C and 3H at the glycerol moiety and fatty acyl chain, respectively, were incubated with Tetrahymena cells, the glycerol moiety and fatty acyl chain at the sn-2-position of the exogenous phospholipids were incorporated into the cellular triacylglycerol fraction in a 1 to 1 ratio. Monoacylglycerol acyltransferase activity was detected in the microsomal fraction of Tetrahymena cells. From these results, together with those of our previous study on lysosomal phospholipid hydrolysis in Tetrahymena (J. Biochem. 99, 125-133 (1986)), it is suggested that the extracellular phospholipids which were taken up by the cells via endocytosis were hydrolyzed through the action of lysosomal phospholipases A1 and C, and also that one of the products, sn-2-monoacylglycerol, served as an acyl acceptor for the synthesis of triacylglycerol via the microsomal "monoacylglycerol pathway."     

Mechanism of the apparent regulation of Escherichia coli unsaturated fatty acid synthesis by exogenous oleic acid.

Starvation of strains of Escherichia coli which are glycerol auxotrophs and are also defective in beta oxidation results in the accumulation of large amounts of free fatty acid (Cronan, J. E., Jr., Weisberg, L. W., and Allen, R. G. (1975) J. Biol. Chem. 250, 5835-5840). We now report that addition of exogenous oleic acid to these cultures results in no decrease in the synthesis of the unsaturated acids of the free fatty acid fraction although a 40 to 60% decrease of [14C]acetate incorporation into phospholipid unsaturated acyl moieties occurs under these conditions. This result indicates that the decreased synthesis of phospholipid unsaturated acyl moieties observed by others during oleic acid supplementation can be attributed to competition between exogenous and endogenously synthesized unsaturated fatty acids rather than a curtailment of unsaturated fatty acid synthesis per se.     

Effect of exogenous fatty acids on the retention of phospholipid acyl groups by mouse L fibroblasts

Exogenous oleic or linoleic acid, given at a high but nontoxic level (1 mg fatty acid/day for 20 . 10(6) cells in 50 ml medium), caused substantial redistribution of the otherwise permanently retained phospholipid acyls in mouse L fibroblasts. 18--40% of the preformed phospholipid acyls were shifted to triglycerides but most returned to phospholipids when the supply of exogenous fatty acid was removed. The phospholipid acyls could be reshuttled back to triglycerides again whenever an adequate amount of exogenous fatty acid was provided. Daily changes of medium containing oleic acid bound to bovine serum albumin caused a still greater total loss of phospholipid acyls into the medium. The removal of the prelabeled phospholipid acyls also occurred with phospholipid acyls which had been synthesized from [1-(14C)]acetate 3 days earlier. The results demonstrate the fact that the apparent permanently retained phospholipid acyl groups found in L-cells could in fact be displaced through experimental manipulations.     

Metabolism of saturated fatty acids by Paramecium tetraurelia

Paramecium requires oleate for growth. The phospholipids of the ciliate contain high concentrations of palmitate and 18- and 20-carbon unsaturated fatty acids. We previously showed that radiolabeled oleate is desaturated and elongated to provide these 18- and 20-carbon unsaturated acids. We now report on saturated fatty acid (SFA) metabolism in Paramecium. Radiolabeled palmitate and stearate were incorporated directly into cellular phospholipids with little or no desaturation and/or elongation. Radiolabeled acetate, malonate, pyruvate, citrate, or glucose added to cultures were not incorporated into cellular phospholipid fatty acids indicating that these exogenously supplied putative precursors were not utilized for fatty acid synthesis by Paramecium. Radiolabel from octanoate or hexanoate appeared in fatty acyl groups of phospholipids, possibly by partial beta-oxidation and reincorporation of the label. Under oleate-free conditions in which cultures do not grow, radiolabel from these shorter chain SFA were beta-oxidized and preferentially used for the formation of arachidonate, the major end-product of fatty acid synthesis in Paramecium. Cerulenin inhibited culture growth apparently by inhibiting de novo fatty acid synthesis. Cerulenin-treated cells did not incorporate radioactivity from [1-14C]octanoate into esterified palmitate. However, total saponifiable phospholipid fatty acids, including SFA, per cell increased under these conditions.     

Preservation of arachidonoyl phospholipids during tissue processing for electron microscopic autoradiography

To facilitate autoradiographic subcellular localization of arachidonoyl phospholipids, the retention of radioactivity during tissue processing of murine fibrosarcoma cells labeled in vitro with 3H-arachidonate was assessed. Approximately 94% of cell radioactivity was incorporated into phospholipids. During tissue processing, extraction of radioactivity was monitored by liquid scintillation spectrometry. Fixation of cells in glutaraldehyde-tannic acid, postfixation in osmium tetroxide, en bloc staining in uranyl magnesium acetate, dehydration in ethanol, and embedding in Epon resulted in preservation of 93.5% of total tissue radioactivity. Analysis of extracted radioactivity by thin layer chromatography revealed that no specific class of phospholipids was selectively extracted. Fixation with osmium tetroxide alone was nearly as effective as the complete fixation protocol and resulted in retention of 90.0% of radioactivity. However, fixation with glutaraldehyde-tannic acid alone without osmium tetroxide post-fixation led to extraction of 69.8% of total cell radioactivity. Thus, osmium tetroxide is crucial in the preservation of arachidonoyl phospholipids and presumably forms extensive cross-links between polyunsaturated acyl residues. This degree of preservation of arachidonoyl phospholipids is indicative of spatial fixation of the radiolabeled moieties and will permit quantitative studies of subcellular loci of eicosanoid metabolism by electron microscopic autoradiography.     

Release of phospholipid acyl groups from fetal human fibroblasts

Human diploid fibroblasts incorporate [1-¹⁴C]oleate primarily into phospholipids; this radioactivity is gradually lost by cells of adult origin, but retained, after an initial loss, by fetal cells during growth in unlabeled unchanged medium. [1-¹⁴C]oleate is released from fetal cells after each subsequent medium change even when conditioned medium is used. Unlabeled cells readily incorporate medium radioactivity previously released by labeled cells. Raising the serum concentration of the medium increases acyl group release. The apparent retention of acyl groups by fetal cells in the absence of medium changes results from a dynamic equilibrium of fatty acid moieties between cellular phospholipids and the culture medium.     

Preferential incorporation of eicosanoid precursor fatty acids into human umbilical vein endothelial cell phospholipids

We have examined the preferential incorporation of specific fatty acids into phospholipid classes of cultured human umbilical vein endothelial cells. Pulse-labeling of human umbilical vein endothelial cell phospholipids with radiolabeled fatty acids and inhibition of radiolabeled fatty acid incorporation by competition with excess, unlabeled fatty acids in pair-wise combinations revealed two distinct classes of esterification systems into human umbilical vein endothelial cell phospholipids. The eicosanoid precursor fatty acids, including arachidonate, 8,11,14-eicosatrienoate (ETA) and 5,8,11,14,17-eicosapentaenoate (EPA), exhibited high affinity incorporation into total phospholipids, whereas other fatty acids, including docosahexaenoate and monohydroxy eicosatetraenoates, showed low affinity incorporation. The relative degree of incorporation of eicosanoid precursor fatty acids into phospholipid classes was phosphatidylcholine (PC) greater than phosphatidylethanolamine (PE) greater than phosphatidylinositol (PI) greater than phosphatidylserine (PS). The specific activity of [14C]arachidonic acid-labeled PI was two times higher than that of any other radiolabeled phospholipids. When competitive incorporation of eicosanoid precursor fatty acids into phospholipid classes was studied, they were found to be acylated into different phospholipid classes at different rates. Although eicosanoid precursor fatty acids were not preferentially incorporated into PC, arachidonic acid was preferentially incorporated into the other phospholipids and exhibited particular selectivity in comparison with the other eicosanoid precursor fatty acids for incorporation into PI. These results demonstrate that human umbilical vein endothelial cells possess selective incorporation mechanisms for specific fatty acids into various phospholipids via the deacylation-reacylation pathway.     

Phenethyl alcohol inhibition of sn-glycerol 3-phosphate acylation in Escherichia coli.

In vivo and in vitro experiments were performed to determine how phenethyl alcohol (PEA) inhibits phospholipid synthesis in Escherichia coli. This drug drastically reduced the rate of incorporation of sn-glycerol 3-phosphate into the phospholipids of an sn-glycerol 3-phosphate auxotroph. PEA also reduced the rate of fatty acid incorporation into the phospholipids of a fatty acid auxotroph. The kinetics of PEA inhibition of the rate of incorporation of sn-glycerol 3-phosphate were almost identical to those of PEA inhibition of the rate of fatty acid incorporation into phospholipids. The in vivo experiments suggested that the rate-limiting step(s) in phospholipid biosynthesis inhibited by PEA is at the level of the acylation of sn-glycerol 3-phosphate or beyond this step. PEA inhibited the sn-glycerol 3-phosphate acyltransferase with either palmitoyl coenzyme A or palmitoyl-acyl carrier protein as the acyl donor. This drug, however, had no effect on the cytidine 5'-diphosphate-diglyceride:glycerol 3-phosphate phosphatidyl transferase, cytidine 5'-diphosphate-diglyceride:L-serine phosphatidyl transferase, and acyl coenzyme A:lysophatidic acid acyltransferase. The in vitro findings suggested that PEA inhibits phospholipid synthesis primarily at the level of sn-glycerol 3-phosphate acyltransferase.     

Replacement of the aliphatic chains of Clostridium acetobutylicum by exogenous fatty acids: regulation of phospholipid and glycolipid composition.

The membrane lipid aliphatic chains of Clostridium acetobutylicum ATCC 4259 have been extensively modified by growth in biotin-free medium containing vitamin-free casein hydrolysate supplemented with either elaidic acid, oleic acid, or mixtures of palmitic and oleic acids. Growth with elaidic acid resulted in polar lipids containing 88.6% 18:1 acyl chains and 94.5% 18:1 ether-linked chains. Growth with oleic acid resulted in comparable levels of enrichment of the lipids with 18:1 chains and C19 chains containing cyclopropane rings. When cells were grown with mixtures of palmitic and oleic acids, the ether-linked chains of the plasmalogens were greater than or equal to 64% 18:1 plus C19 chains containing cyclopropane rings at all ratios of oleic to palmitic acid in the medium. The acyl chains reflected the palmitic acid content of the medium more closely. Marked changes were observed in both phospholipid and glycosyldiglyceride compositions as the lipid acyl and ether-linked chains became more enriched with unsaturated and cyclopropane chains. The ratio of the glycerol acetal of plasmenylethanolamine to phosphatidylethanolamine increased, the ratio of cardiolipin to phosphatidylglycerol decreased, and the ratio of diglycosyldiglyceride to monoglycosyldiglyceride increased. However, the monoglycosyldiglyceride/diglycosyldiglyceride ratio was lower for cells grown on 100% oleic acid than for cells grown on 60 or 80% oleic acid. In the membranes of cells grown on 100% oleic acid, the ratio of glycolipids to phospholipids was lower than that found in cells grown on 60% oleic acid. These results indicate that C. acetobutylicum regulates its polar lipid composition in a complex manner involving phospholipids and glycosyldiglycerides. These changes can affect the equilibria between those lipids that form bilayers and those lipids that tend to form nonlamellar phases when enriched with unsaturated aliphatic chains. Phosphoglycolipids of unknown structure were also observed in cells grown either with biotin or with fatty acids. The content of the most abundant phosphoglycolipid also varied with the degree of unsaturation of the cellular lipids.     

The elongation of exogenous fatty acids and the control of phospholipid acyl chain length in Micrococcus cryophilus.

The synthesis of fatty acids de novo from acetate and the elongation of exogenous satuated fatty acids (C12-C18) by the psychrophilic bacterium Micrococcus cryophilus (A.T.C.C. 15174) grown at 1 or 20 degrees C was investigated. M. cryophilus normally contains only C16 and C18 acyl chains in its phospholipids, and the C18/C16 ratio is altered by changes in growth temperature. The bacterium was shown to regulate strictly its phospholipid acyl chain length and to be capable of directly elongating myristate and palmitate, and possibly laurate, to a mixture of C16 and C18 acyl chains. Retroconversion of stearate into palmitate also occurred. Fatty acid elongation could be distinguished from fatty acid synthesis de novo by the greater sensitivity of fatty acid elongation to inhibition by NaAsO2 under conditions when the supply of ATP and reduced nicotinamide nucleotides was not limiting. It is suggested that phospholipid acyl chain length may be controlled by a membrane-bound elongase enzyme, which interconverts C16 and C18 fatty acids via a C14 intermediate; the activity of the enzyme could be regulated by membrane lipid fluidity.     

Lipid composition and metabolism in testicular and ejaculated ram spermatozoa

1. Spermatozoa collected directly from the testis of the conscious ram contain 25% more phospholipid than ejaculated spermatozoa. The concentration of lecithin, phosphatidylethanolamine and ethanolamine plasmalogen was greater in testicular spermatozoa; little difference was observed in choline plasmalogen. Both types of spermatozoa had significant amounts of cardiolipin and alkyl ether phospholipid. 2. The fatty acids in the phospholipid extracted from testicular spermatozoa have a very high content of palmitic acid. The phospholipids of ejaculated spermatozoa contained less palmitic acid, but more myristic acid. 3. Ejaculated spermatozoa contained less acyl ester and cholesterol. It is suggested that lipids are a source of substrate for spermatozoa during their passage through the epididymis. 4. Testicular spermatozoa when incubated with [U-¹⁴C]glucose incorporated more radioactivity into the glycerol part of the phospholipid and neutral lipid fractions than did ejaculated cells. The distribution of radioactivity in the individual phospholipids and neutral lipids was similar for both cell types. No radioactivity was detected in choline plasmalogen, which accounted for approx. 40% of the total phospholipid. 5. Testicular spermatozoa incorporated more radioactivity from glucose into formate than into acetate, whereas a higher proportion of radioactivity was found in acetate in ejaculated cells. 6. The implications of these lipid changes in the process of spermatozoal maturation are discussed.     

Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa

Intact human sperm incorporated radiolabelled fatty acids into membrane phospholipids when incubated in medium containing bovine serum albumin as a fatty acid carrier. The polyunsturated fatty acids were preferentially incorporated into the plasmalogen fraction of phospholipid. Uptake was linear with time over 2 hr; at this time sufficient label was available to determine the loss of fatty acids under conditions of spontaneous lipid peroxidation. Loss of the various phospholipid types, the loss of the various fatty acids from these phospholipids, and the overall loss of fatty acids were all first order. The loss of saturated fatty acids was slow with first order rate constant k₁ = 0.003 hr^?1; for the polyunsaturated fatty acids, arachidonic and docosahexaenoic acids, k₁ = 0.145 and 0.162 hr^?1, respectively. The rate of loss of fatty acids from the various phospholipid types was dependent on the type, with loss from phosphatidylethanolamine being the most rapid. Among the phospholipid types, phosphatidylethanolamine was lost at the greatest rate. Analysis of fatty acid loss through oxidation products was determined for radiolabelled arachidonic acid. Under conditions of spontaneous lipid peroxidation at 37°C under air in the absence of albumin, free arachidonic acid was found in the medium, along with minor amounts of hydroxylated derivative. All the hydroperoxy fatty acid remained in the cells. In the presence of albumin, all the hydroperoxy fatty acid was found in the supernatant bound to albumin; none could be detected in the cells. Albumin is known as a very potent inhibitor of lipid peroxidation in sperm; its action may be explained, based on these results, as binding the damaging hydroperoxy fatty acids. These results also indicate that a phospholipase A₂ may act in peroxidative defense by excising a hydroperoxy acyl group from phospholipid and providing the hydroperoxy fatty acid product as substrate to glutathione peroxidase. This formulation targets hydroperoxy fatty acid as a key intermediate in peroxidative degradation. © 1995 wiley-Liss, Inc.     

Phospholipid Alterations During Growth of Escherichia coli

As cultures of Escherichia coli progressed from the exponential growth phase to the stationary growth phase, the phospholipid composition of the cell was altered. Unsaturated fatty acids were converted to cyclopropane fatty acids, and phosphatidyl glycerol appears to have been converted to cardiolipin. With dual isotope label experiments, the kinetics of synthesis of cyclopropane fatty acid for each of the phospholipids was examined in vivo. The amount of cyclopropane fatty acid per phospholipid molecule began to increase in phosphatidyl ethanolamine at a cell density below the density at which this increase was observed in phosphatidyl glycerol or cardiolipin. The rate of this increase in phosphatidyl glycerol or in cardiolipin was faster than the rate of increase in phosphatidyl ethanolamine. After a few hours of stationary-phase growth, all the phospholipids were equally rich in cyclopropane fatty acids. It is suggested that the phospholipid alterations observed are a mechanism to protect against phospholipid degradation during stationary phase growth. Cyclopropane fatty acid synthetase activity was assayed in cultures at various stages of growth. Cultures from all growth stages examined had the same specific activity in crude extracts.     

The interaction of cholesterol with bilayers of phosphatidylethanolamine

The interaction of cholesterol with the glycerol backbone segments of phospholipids was studied in bilayers of phosphatidylethanolamine containing equimolar amounts of cholesterol. Glycerol selectively deuterated at various positions was supplied to the growth medium of Escherichia coli strain 131 GP which is defective in endogeneous glycerol synthesis. The procedure enables the stereospecific labeling of the three glycerol backbone segments of the membrane phospholipids. Phosphatidylethanolamine with wild-type fatty acid composition was purified from E. coli cells and deuterium magnetic resonance spectra were obtained either from dispersions of pure phosphatidylethanolamine or from equimolar mixtures of phosphatidylethanolamine with cholesterol. For comparative purposes 1,2-di[9,10-²H₂]elaidoyl-sn-glycero-3-phosphoethanolamine and [3-α-²H]cholesterol were synthesized in order to monitor the behavior of the fatty acyl chains and of the cholesterol molecule itself. For all deuterated segments the deuterium quadrupole splittings as well as the deuterium spin-lattice (T₁) relaxation times were measured as a function of temperature. The glycerol backbone was found to be a remarkably stable structural element of the phospholipid molecule. The quadrupole splittings of the backbone segments changed only by at most 2 kHz upon incorporation of 50 mol % cholesterol. This was in contrast to the fatty acyl chains where the same amount of cholesterol increased the quadrupole splitting by more than 20 kHz. The glycerol segments exhibited the shortest T₁ relaxation times of all CH₂ segments indicating that the glycerol backbone is the slowest motional moiety of the lipid molecule. Addition of cholesterol has no effect on the backbone motion but the fast reorientation rate of the trans-double bonds in 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine is increased dramatically.