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.     

The structure of a glycerylphosphoryldiglucosyl diglyceride from the lipids of Acholeplasma laidlawii strain B

1. The phosphatidylglucose structure proposed previously (Smith & Henrikson, 1965) for the glucose-containing phospholipid from Acholeplasma laidlawii is incorrect. 2. The structure now proposed is 3-(sn-glycerol-3-phosphoryl-6'-[O-alpha-d-glucopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl])- sn-1,2-diglyceride, a new type of bacterial lipid. 3. Deacylation of the lipid gave a single water-soluble phosphate ester which could be distinguished on chromatography from synthetic samples of glucosylphosphorylglycerols. 4. Hydrolysis of the lipid with alkali gave a mixture of fatty acids, glycerol 2-phosphate, sn-glycerol 3-phosphate and O-alpha-d-glucopyranosyl-(1-->2)-O-alpha- d-glucopyranosyl-(1-->1)-d-glycerol. 5. The lipid was unaffected on incubation with phospholipases A, C and D. 6. Diglucosyl diglyceride was isolated after treatment of the lipid with 60% HF, establishing the location of the fatty acid residues. 7. Periodate oxidation studies showed that the sn-glycerol 3-phosphate was esterified to the 6-hydroxyl group of one of the glucose residues in diglucosyl diglyceride.     

The diglyceride kinase of rat cerebral cortex

1. Formation of phosphatidic acid by diglyceride kinase (EC 2.7.1.-) in the presence of ATP and Mg(2+) was shown in a homogenate and subcellular fractions of rat cerebral cortex. 2. The kinase was activated by Mg(2+). Ca(2+) activated to a smaller extent but was inhibitory in the presence of optimum concentration of Mg(2+). Activity was greatly increased in the presence of added 1,2-diglyceride. 3. Sodium deoxycholate markedly stimulated the reaction, but other detergents (Cutscum and Triton X-100) did not. 4. Diglyceride kinase was concentrated in the supernatant and microsomal fractions from rat cerebral cortex. The distribution of the kinase in the particulate fractions resembled that of acetylcholinesterase and 5'-nucleotidase. 5. The rate of phosphatidic acid synthesis by the diglyceride kinase route was much greater than reported rates for acylation of 3-glycerophosphate and was also very rapid in comparison with the rates of other steps in the synthesis of phosphoinositides. 6. Acetylcholine had no stimulatory effect on diglyceride kinase of isolated intact nerve-ending particles or of nerve-ending membranes obtained after osmotic shock.     

Neutral lipid accumulation in the membranes of Escherichia coli mutants lacking diglyceride kinase.

We have developed a rapid autoradiographic screening assay for detecting diglyceride kinase in colonies of Escherichia coli and have isolated four strains lacking this enzyme. The gene (designated dgk) which is altered in these mutants is cotransduceable with the malB locus, near minute 90 on the chromosome. The membranes of strain RZ60 (which carries the dgk-6 lesion) contain substantial amounts of 1,2-diglyceride, representing approximately 8% of the total lipid. In contrast, wild type cells of E. coli (dgk+) only contain about 0.5% 1,2-diglyceride. The phospholipid composition of these mutants is not dramatically altered, and they are not temperature sensitive for growth. However, strains bearing the dgk-6 mutation do not grow well on nutrient media of low osmolarity. This can be corrected by the inclusion of 1% NaCl or 0.5 M sucrose. These results suggest that 1,2-diglyceride is the true substrate for the kinase in vivo and that the kinase functions as a minor route for phosphatidic acid synthesis. Genetic modification of the diglyceride content of the E. coli membrane has not been reported previously.     

Cyanamide mediated syntheses under plausible primitive earth conditions

Summary A mixture of ammonium palmitate,¹⁴C-sn-glycero-1(3)-phosphate, cyanimide and imidazole when heated for several hours formed significant quantities of phospholipids. These reaction products were shown by chromatographic, chemical and enzymatic procedures to be monopalmitoylglycerophosphate (MPGP), dipalmitoylglycerophosphate (DPGP) and monopalmitoyl cyclic glycerophosphate (cMPGP). A portion of the MPGP and DPGP possessed the same steric configuration as naturally occurring lysophosphatidic acid and phosphatidic acid. The yield of total phospholipid was maximal at temperatures between 60° and 90° after 8 h. When ratios of reactants were varied, up to 45% of radioactive glycerophosphate was converted into phospholipids. The average proportions of individual phosphatidic acids were: 60% MPGP, 27% DPGP and 13% cMPGP. Evidence was obtained for a synergistic relationship between cyanamide and imidazole in promoting the formation of phosphatidic acids. These results suggest that phosphatidic acids, which are essential precursors for the biochemical synthesis of more complex membrane phospholipids, could have been produced on the primitive Earth.The following abbreviations are employed: for phospholipid standards with designated steric configuration LPA lysophosphatidic acid (1-acyl-sn-glycero-3-phosphate) - PA phosphatidic acid (1, 2-di-acyl-sn-glycero-3-phosphate) - MPGP monopalmitoylglycerophosphate - cMPGP monopalmitoyl cyclic glycerophosphate (1(3)-acyl-sn-glycero-2,3 (1,2)-cyclic phosphate) - DPGP dipalmitoylglycerophosphate - GP glycerophosphate - cGP cyclic glycerophosphate (sn-glycero-2,3 (1,2)-cyclic phosphate) - TLC thin layer chromatography To whom reprint requests should be addressed.     

Biosynthesis of Glucosyl Diglycerides by Mycoplasma laidlawii Strain B

Monoglucosyl diglyceride is synthesized from 1,2-diglyceride and uridine-5'-diphosphoglucose (UDP); diglucosyl diglyceride from monoglucosyl diglyceride, and uridine-5'-diphosphoglucose by membranes of Mycoplasma laidlawii strain B. All of these enzymatic activities reside in the membrane. Membranes solubilized by detergent action or succinylation and acetone powders of membranes were inactive. Requirements for Mg(2+), UDP, and appropriate lipid acceptor were demonstrated for biosynthesis of both glycolipids. Glucose-1-phosphate plus uridine triphosphate could replace the UDP requirement. A medium of relatively high ionic strength and a critical concentration of sodium lauryl sulfate stimulated biosynthesis of the monoglucosyl diglyceride. The optimal pH for both reactions was 8.0. A specificity for 1,2-diglyceride from the homologous organism was found for optimal synthesis of the monoglucosyl diglyceride, and a specificity for monoglucosyl diglyceride was found in the case of diglucosyl diglyceride synthesis. Both reactions were specific for UDP.     

Phosphatidylglycerol as biosynthetic precursor for the poly(glycerol phosphate) backbone of bifidobacterial lipoteichoic acid.

Phosphatidylglycerol functions as donor of the sn-glycerol 1-phosphate units in the synthesis in vitro of the 1,2-phosphodiester-linked glycerol phosphate backbone of the lipoteichoic acids of Bifidobacterium bifidum subsp. pennsylvanicum. The incorporation was catalysed by a membrane-bound enzyme system. After addition of chloroform/methanol the product formed coprecipitated with protein. The material was phenol-extractable and was co-eluted with purified lipoteichoic acid on Sepharose 6B. The reaction was stimulated by Triton X-100, UDP-glucose and UDP-galactose, but Mg2+ ions had no effect. The apparent values for Km and Vmax. of the phosphatidylglycerol incorporation were 1.4 mM and 3.1 nmol/h per mg of membrane protein, respectively. Labelled UDP-glucose and UDP-galactose were not incorporated into the lipoteichoic acid fraction by the particulate membrane preparation.     

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli: effect on cell growth and metabolism.

sn-Glycerol 3-phosphorothioate was found to be bacteriocidal to strains of Escherichia coli which have a functional sn-glycerol 3-phosphate transport system. This effect was manifest in strains 7 and 8, which are constitutive mutants for the utilization and transport of sn-glycerol 3-phosphate (glpRc2). Strain E15, which is considered to be wild type for the glycerol phosphate functional units, was affected by the phosphorothioate analog only under conditions that are known to induce the transport system for sn-glycerol 3-phosphate. In addition, another strain of E. coli, strain 6, which is isogenic with strain E15 but has an impaired sn-glycerol 3-phosphate transport system (glpT13), was not affected by similar concentrations of sn-glycerol 3-phosphorothioate. Transport studies in which [3H]glycerol phosphate and its phosphorothioate analog were used demonstrated that the latter compound was taken up via the specific active transport system for sn-glycerol 3-phosphate; the Km values were 9 and 11 microM, respectively. The rates of macromolecular synthesis were found to be inhibited severely by sn-glycerol 3-phosphorothioate at a concentration at which sn-glycerol 3-phosphate had no effect (5 microM). At a lower concentration of the analog (0.5 microM), the rates of protein synthesis and RNA synthesis (52 and 58% below control values after 90 min, respectively) were more sensitive than the rates of DNA synthesis and cell wall synthesis (18% below control values after 3 h for DNA; transient decrease in the cell wall values after 90 min). The levels of the nucleoside triphosphates were not affected by the presence of the phospholipid precursor or its analog at a concentration of 5 microM. The phospholipid composition was significantly altered in the presence of bacteriocidal concentrations (5 microM) of sn-glycerol 3-phosphorothioate. The amount of phosphatidylglycerol in the membranes decreased from 13.5 to 3.5%. Concomitant with this decrease in phosphatidylglycerol content was a fourfold increase in the 32P content of cardiolipin (from 6.8 to 24.2%), whereas the phosphatidylethanolamine content showed only a minor reduction (8%) after 3 h. The rates of synthesis of all of the phospholipids decreased in the presence of 5 microM sn-glycerol 3-phosphorothioate, with the most significant effects observed for phosphatidylglycerol (63% after 3 h). Phosphatidylglycerol showed increased rates of turnover after 90 min (21%) and 3 h (11%), with concomitant increases in the levels of cardiolipin of more than twofold. Our data suggest that a considerably greater proportion of phosphatidylglycerol turnover may be recover in cardiolipin than is metabolized via other pathways (e.g., the membrane-derived oligosaccharide pathway).     

Synthesis of biologically active spin-labelled radioactive cytidine diphosphodiglyceride, a novel probe for biological membranes.

A versatile synthesis of spin-labelled radioactive cytidine diphospho-sn-1,2-diacylglycerol (CDP-diglyceride) has been developed based on the combination of the enzymatic acylation of radioactive sn-glycero-3-phosphate with 12-doxyl stearic acid and the chemical conversion of the thus obtained spin-labelled radioactive phosphatidic acid with cytidine monophosphomorpholi-date into spin-labelled radioactive CDP-diglyceride. The method for the isolation and purification of the latter compound was described. This obtained CDP-[2-3H]diglyceride contained 10% of fatty acids of paramagnetic nature, presumably present as a covalently bound 12-doxyl stearic acid esters. The biological activity was tested by using the synthesized compound as a substrate in the mitochondrial biosynthesis of phosphatidylglycerol. It was found that spin-labelled CDP-[2-3H]diglyceride prepared as described can be converted in the presence of sn-[2-14C]-glycero-3-phosphate into a spin-labelled [2-3H, 2'-14C]phosphatidylglycerol with isolated rat liver mitochondria, establishing therefore that the site of its utilization is identical with the site of phosphatidylglycerol synthesis in isolated mitochondria, i.e. inner mitochondrial membrane. Results described demonstrate that the synthesized spin-labelled CDP-diglyceride can be used as a specific probe for the spin- and radioactive covalent labelling of polyglycerophosphatides of mitochondrial membranes. Some implications and further possibilities in the study of biological membranes using the spin-labelled radioactive CDP-diglyceride are discussed.     

Diglyceride Kinase Mutants of Escherichia coli: Inner Membrane Association of 1,2-Diglyceride and Its Relation to Synthesis of Membrane-Derived Oligosaccharides

Mutants of Escherichia coli defective in diglyceride kinase contain 10 to 20 times more sn-1,2-diglyceride than normal cells. This material constitutes about 8% of the total lipid in such strains. We now report that this excess diglyceride is recovered in the particulate fraction, primarily in association with the inner, cytoplasmic membrane. The diglyceride kinase of wild-type cells was recovered in the same inner membrane fractions. The conditions employed for the preparation of the membranes did not appear to cause significant redistribution of lipids and proteins. The biochemical reactions leading to the formation of diglyceride in E. coli are not known. To determine whether diglyceride formation requires concurrent synthesis of the membrane-derived oligosaccharides (H. Schulman and E. P. Kennedy, J. Biol. Chem. 252:4250-4255, 1977), we have constructed a double mutant defective in both the kinase (dgk) and phosphoglucose isomerase (pgi). When oligosaccharide synthesis was inhibited in this organism by growing the cells on amino acids as the sole carbon source, the diglyceride was no longer present in large amounts. When glucose was also added to the medium, the pgi mutation was bypassed, oligosaccharide synthesis resumed, and diglyceride again accumulated. These findings suggest that diglyceride may arise during the transfer of the sn-glycero-1-P moiety from phosphatidylglycerol (and possibly cardiolipin) to the oligosaccharides. In wild-type cells the kinase permits the cyclical reutilization of diglyceride molecules for phospholipid biosynthesis.     

A novel cytokine-inducing glycolipid isolated from the lipoteichoic acid fraction of Enterococcus hirae ATCC 9790: A fundamental structure of the hydrophilic part

Previously, we showed that quantitatively minor several glycolipids only less than 5% of the lipoteichoic acid (LTA) fraction from Enterococcus hirae ATCC 9790 possessed cytokine-inducing activity, whereas the major component (over 90%) did not [Suda et al. (1995) FEMS Immun Med Microbiol 12:97–112]. The major inactive component was shown to have the chemical structure as was proposed for the LTA by Fischer [Hashimoto et al. (1997) J Biochem 121:779–86], suggesting that so-called LTA is not a cytokine-inducing component in the Gram-positive bacteria. In the present paper, the structure of the hydrophilic part of one of the cytokine-inducing glycolipid tentatively named GL4 is elucidated. GL4 was first subjected to hydrolysis with aqueous HF to give a polysaccharide and a mixture of low molecular weight products. The polysaccharide was composed mainly of highly branching mannan as concluded from NMR and MS analyses of its acetolysis products. The low molecular weight products consisted of phosphate and glycerol, suggesting the presence of a poly(glycerophosphate) structure in the original GL4. From these observations, the hydrophilic part of GL4 was shown to consist of mannose-rich polysaccharide and poly(glycerophosphate), the latter being bound to the former by a phosphodiester linkage.     

Biosynthesis of murein lipoprotein in Escherichia coli: effects of 3,4-dihydroxybutyl-1-phosphonate.

The effects of 3,4-dihydroxybutyl-1-phosphonate, a four-carbon analog of sn-glycerol 3-phosphate, on the biosynthesis of the glyceryl moiety in murein lipoprotein of Escherichia coli were studied. The compound at a concentration of 55 microM strong inhibits in the incorporation of [2-3H]glycerol radioactivity into lipoprotein by virtue of its inhibition of the synthesis of phosphatidylglycerol. On the other hand, the incorporation of prelabeled [2-3H]glycerol radioactivity into lipoprotein was only partially inhbited by 3,4-dihydroxybutyl-1-phosphonate even at a much higher concentration (1 mM). These data were consistent with the postulated pathway for the biosynthesis of the lipid moiety in lipoportein: cysteine-lipoprotein + phosphatidylglycerol leads to glycerylcystein-lipoprotein + phosphatidic acid.     

Epidermal growth factor-induced hydrolysis of phosphatidylcholine by phospholipase D and phospholipase C in human dermal fibroblasts

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining 20% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phosphatidylcholine.     

Phosphatidylglycerol synthesis in pea chloroplasts: pathway and localization

Isolated intact pea chloroplasts synthesized phosphatidylglycerol from either [¹⁴C]acetate or [¹⁴C]glycerol 3-phosphate. Both time-course and pulse-chase labeling studies demonstrated a precursor-product relationship between newly synthesized phosphatidic acid and newly synthesized phosphatidylglycerol.

The synthesis both of CDP-diacylglycerol from exogenous phosphatidic acid and CTP, and of phosphatidylglycerol from exogenous CDP-diacylglycerol and glycerol 3-phosphate, could be assayed in fractions obtained from disrupted chloroplasts. Moreover, the enzymes catalyzing these reactions were localized in the inner envelope membrane. Exogenous phosphatidic acid was incorporated into phosphatidylglycerol, but only following its incorporation into CDP-diacylglycerol. Finally, radio-active phosphatidic acid synthesized in the envelope membranes from [¹⁴C]palmitoyl-ACP and 1-oleoyl-glycerol 3-phosphate was sequentially incorporated into labeled CDP-diacylglycerol and phosphatidylglycerol upon the addition of appropriate substrates and cofactors. Thus, we have demonstrated that (a) the synthesis of phosphatidylglycerol in chloroplasts occurs by the pathway: phosphatidic acid → CDP-diacylglycerol →→ phosphatidylglycerol, and (b) phosphatidylglycerol synthesis is located in the inner envelope membrane.

     

Phosphatidic acid metabolism in rat liver microsomes.

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

Function of phospholipids on the regulatory properties of solubilized and membrane-bound sn-glycerol-3-phosphate acyltransferase of Escherichia coli.

Glycerophosphate acyltransferase (acyl-CoA:sn-glycerol-3-phosphate O-acyltransferase, EC 2.3.1.15) solubilized from Escherichia coli membranes was highly activated by phosphatidylglycerol. Phosphatidylethanolamine, cardiolipin and 1,2-diacyl-sn-glycerol 3-phosphate showed no effect. The Km of the enzyme for sn-glycerol 3-phosphate was increased 20-fold by solubilization. The value could not be restored by the addition of phospholipids. Temperature-sensitive regulation of the synthesis of either 1-palmitoyl- or cis-vaccenoyl-sn-glycerol 3-phosphate by the solubilized enzyme was identical with that by the membrane-bound enzyme in vivo and in vitro. The proportion of the molecular species of 1-acyl-sn-glycerol 3-phosphate varied when the ratios of palmitoyl-CoA and cis-vaccenoyl-CoA were changed, but changes in the sn-glycerol 3-phosphate concentration had no effect on selective acylation by both the solubilized and membrane-bound enzymes.     

The formation and continuous turnover of a fraction of phosphatidic acid on stimulation of NaC1 secretion by acetylcholine in the salt gland

Acetylcholine, which stimulates NaCl secretion in the avian salt gland, causes the rapid formation of a fraction of phosphatidic acid, as measured by ³²P incorporation, which amounts maximally to about 0.18 µmoles per g of fresh tissue. This does not appear to involve synthesis of the diglyceride moiety of phosphatidic acid, as measured by glycerol-1-¹⁴C incorporation. It presumably involves formation of phosphatidic acid by the diglyceride kinase pathway from preformed diglyceride and ATP. The specific activity of the AT³²P of the tissue is not increased in the presence of acetylcholine. At time intervals after addition of acetylcholine during which a full response, measured as increased O₂ uptake, may be observed, phosphatidic acid appears to be the only phosphatide which shows any increase either in total ³²P radioactivity or in net specific acitvity. This responsive fraction of phosphatidic acid undergoes continuous turnover of its phosphate moiety. There is no evidence that this turnover is due to the phosphatidic acid acting as a pool of intermediate for the synthesis of other phospholipids or glycerides. The responsive fraction amounts to not more than 20% of the total phosphatidic acid of the tissue; it does not mix with the other (non-responsive) phosphatidic acid of the tissue. The observations suggest that this phosphatidic acid plays some role in the over-all secretory process.     

Structure and properties of lipoteichoic acid of group A Streptococcus type M 29

Lipoteichoic acid (LTA) of group A streptococci, type M 29 was studied. Chemical and 13C NMR spectroscopic analysis showed that the polymer contained poly(glycerophosphate) chain consisting of 12-14 glycerophosphate elements united by the 1----3 type phosphodiether bond and diglucosylglyceride. Oleic, stearic, palmitic and palmitoleic fatty acids predominated in the polymer composition. The content of the fatty acids amounted approximately to 2 per cent of LTA dry weight. The poly(glycerophosphate) chain contained 6-7 ether linked alanyl moieties. The results of the LTA biological study were analyzed in comparison to the data on a previous study of antitumor and cardiotoxic properties of teichoic acid from Streptomyces levoris K-3053 which is structurally close to the LTA hydrophilic moiety. It was assumed that the molecule negative charge had an effect on the cardiotoxic and antitumor activity.     

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

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

Multiple controls exerted on in vivo expression of the pepN gene in Escherichia coli: studies with pepN-lacZ operon and protein fusion strains.

Lipoteichoic acid (LTA) from Lactobacillus casei contains poly(glycerophosphate) substituted with D-alanyl ester residues. The distribution of these residues in the in vitro-synthesized polymer is uniform. Esterification of LTA with D-alanine may occur in one of two modes: (i) addition at random or (ii) addition at a defined locus in the poly(glycerophosphate) chain followed by redistribution of the ester residues. A time-dependent transacylation of these residues from D-[14C]alanyl-lipophilic LTA to hydrophilic acceptor was observed. The hydrophilic acceptor was characterized as D-alanyl-hydrophilic LTA. This transacylation requires neither ATP nor the D-alanine incorporation system, i.e., the D-alanine activating enzyme and D-alanine:membrane acceptor ligase. No evidence for an enzyme-catalyzed transacylation reaction was observed. We propose that this process of transacylation may be responsible for the redistribution of D-alanyl residues after esterification to the poly(glycerophosphate). As a result, it is difficult to distinguish between these proposed modes of addition.