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PubMed1979

Glycerol 3-phosphate analogues as metabolic inhibitors in Escherichia coli, 3-hydroxy-4-oxobutyl-1-phosphonate, a drug that interferes with normal phosphoglyceride metabolism.

3-Hydroxy-4-oxobutyl-1-phosphonate, the phoshonic acid analogue of glyceraldehyde 3-phosphate, enters Escherichia coli via the glycerol 3-phosphate transport system. There is no differential effect upon the accumulation of deoxyribonucleic acid, ribonucleic acid, or phosphoglycerides, although the accumulation of proteins was less effected. Examination of the phospholipids revealed that phosphatidylglycerol accumulation was most severely inhibited and cardiolipin accumulation was least affected. Concentrations of glyceraldehyde 3-phosphate and its phosphonic acid analogue that markedly inhibit macromolecular and phosphoglyceride biosynthesis have no effect upon the intracellular nucleoside triphosphate pool size. The phosphonate is a competitive inhibitor of sn-glycerol 3-phosphate in reactions catalyzed by acyl coenzyme A:sn-glycerol-3-phosphate acyltransferase and CDP-diacylglycerol:sn-glycerol-3-phosphate phosphatidyltransferase. A Km mutant for the former enzyme was susceptible to the phosphansferase activity. Studies with mutant strains ruled out the aerobic glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate synthase, and fructose-1,6-biphosphate aldolase as the primary sites of action.

Bacterial Proteins↗
PubMed1980

Biosynthesis of phosphatidylinositol in Crithidia fasciculata.

Microsomal preparations from the protozoan (Crithidia fasciculata were shown to incorporate myo-[2-3H]inositol into phosphatidylinositol by both the CDPdiacylglycerol:myo-inositol phosphatidyltransferase reaction and by a myo-inositol exchange reaction. Non-ionic detergent and Mg2+ were necessary for the measurement of transferase activity. Untreated preparations could not be saturated with Mg2+, even at very high concentrations (50-75 mM). However, low concentrations of EGTA (75 micro M) both stimulated the activity 3-fold and reduced the Mg2+ required for saturation to 15-20 mM. EGTA also increased the apparent Km for CDPdiacylglycerol while increasing the sensitivity to substrate inhibition above 1 mM. The transferase activity was inhibited by relatively low concentrations of Ca2+ (50 micro M). This and the EGTA effect suggest a possible role for Ca2+ in the modulation of phosphatidylinositol synthesis. The myo-inositol exchange activity required Mn2+, was insensitive to Ca2+ inhibition and was only slightly stimulated by detergents and EGTA. This activity was preferentially inactivated by heating at 50 degrees C in the presence of Triton X-100. In a detergent solubilized preparation the exchange activity but not the transferase exhibited a non-specific requirement for phospholipid. The differences in properties of the two activities suggest the presence of a separate exchange enzyme.

Animals↗
PubMed1980

Cytotoxic liponucleotide analogs. I. Chemical synthesis of CDP-diacylglycerol analogs containing the cytosine arabinoside moiety.

Cytidine and deoxycytidine diphosphate diacylglycerol are metabolic liponucleotides which are substrates for the biosynthesis of several classes of cellular phosphoglycerides. In addition to their essential biochemical function, liponucleotides can be considered unique from the point of view of molecular structure (lipid, phosphorus, sugar, heterocyclic moieties) and biophysical properties. Liponucleotides, therefore, have been investigated as possible models for anticancer drug design and development. The chemical synthesis of several liponucleotide analogs of cytidine diphosphate diacylglycerol (CDPdiacylglycerol/dCDPdiacylglycerol) containing the 1-beta-D-arabinofuranosyl moiety was undertaken for the purpose of evaluation of the antitumor activity of these compounds. The analogs were synthesized by reaction of 1-beta-D-arabinofuranosylcytosine-5'-(hydrogen morpholinophosphonate):N,N'-dicyclohexyl-4-morpholine carboxamide (1 : 1) in pyridine with either egg lecithin-derived phosphatidic acid, synthetic phosphatidic acid, or synthetic analogs of phosphatidic acid. The yields of liponucleotide analogs after purification were approx. 25-40%. Although reaction yields were not optimized, the condensation of phosphatidic acids and nucleotides represents an expedient laboratory-scale synthetic approach to liponucleotides, especially when phosphatidic acids are obtained from natural sources or by semisynthetic methods, and when 5'-nucleotides can be synthesized directly (i.e., without use of protecting groups) from precursor nucleosides.

Antineoplastic Agents↗
PubMed1980

Cytotoxic liponucleotide analogs. II. Antitumor activity of CDP-diacylglycerol analogs containing the cytosine arabinoside moiety.

Among events limiting the effectiveness of cancer chemotherapy are the general lack of preferential uptake of anticancer drugs by tumor cells and the occurrence of drug resistance. An approach has been undertaken to explore whether or not such events can be favorably altered or circumvented therapeutically by development of a new class of anticancer molecules, cytotoxic liponucleotide analogs. The design of cytotoxic liponucleotide analogs encompasses both biochemical and biophysical aspects of liponucleotide and glycerophospholipid structure and metabolism. Several cytotoxic liponucleotide analogs of cytidine diphosphate diacylglycerol (CDPdiacylglycerol/dCDPdiacylglycerol), containing the 1-beta-D-arabinofuranosyl moiety, were tested for antitumor activity. Multispecies ara-CDPdiacylglycerol (1-beta-D-arabinofuranosylcytosine 5'-diphosphate diacylglycerol), which contains egg lecithin-derived mixed fatty acyl chains, was more active than 1-beta-D-arabinofuranosylcytosine (ara-C), a clinically used anticancer drug, against leukemia L5178Y and P388 ascites cells in mice. At identical single doses (50 mg/kg per day times 4) administered intraperitoneally, ara-CDPdiacylglycerol prolonged the life spans of L5178Y tumor-bearing mice 93%, while ara-C prolonged life by 18%. Ara-CDPdiacylglycerol increased life spans of P388 tumor-bearing mice by 357% at doses of 50 mg/kg per day times 4; the maximum increase with ara-C was 159% (85 mg/kg per day times 4). Against a P388 ara-C-resistant cell line (P/Ara-C, kinase deficient) in mice, ara-CDPdiacylglycerol prolonged survival times by 34% at a dose of 50 mg/kg per day times 4 and by 55% at 75 mg/kg per day times 4; the drug was not active against two other ara-C-resistant murine leukemia mutants (CA 55, CA5b). With cell line-derived human colon carcinoma HCT-15 grown in mice immunosuppressed with anti-thymocyte serum, ara-CDPdiacylglycerol at a single daily dose of 50 mg/kg per day times 4 significantly reduced tumor weights to 21% of the controls; the same dose schedule of ara-C caused no observable reduction of tumor weights. Results of these preliminary antitumor evaluations indicate that cytotoxic liponucleotide analogs should be investigated further to determine their potential as antineoplastic molecules.

Animals↗
PubMed1980

The stimulation of rat liver microsomal CTP: phosphatidate cytidylyltransferase activity by guanosine triphosphate.

GTP has been found to markedly enhance the formation of CDPdiacylglycerol in rat liver microsomes. The magnitude of the GTP stimulatory effect was influenced by the fatty acid composition of the phosphatidic acid substrate. Heat or proteolytic treatment of microsomal membranes prevented GTP from stimulating liponucleotide formation. Treatment of microsomes with low levels of detergent reduced the stimulatory action of the GTP. The increased synthesis of CDPdiacylglycerol obtained by the addition of GTP to the incubation medium is due to an increase in CTP:phosphatidate cytidylyltransferase activity, rather than to a reduction in the breakdown of either the substrates or product of the reaction. The results suggest that some other protein or enzyme mediates the effect of GTP upon CTP:phosphatidate cytidylyltransferase activity.

Animals↗
PubMed1981

Optimal assay and subcellular location of phosphatidylglycerol synthesis in lung.

Synthesis of phosphatidylglycerol from CDPdiacylglycerol and glycerol 3-phosphate by membranous subcellular fractions of rat lung and liver was optimal when assayed in the presence of bovine serum albumin and Triton X-100. Specific activities of glycerolphosphate phosphatidyltransferase in all membranous subcellular fractions of lung were several times higher than the corresponding fractions from liver. Distribution of this enzyme in subcellular fractions of lung or liver closely parallel the activity of the mitochondrial enzymes monoamine oxidase and succinate cytochrome c reductase. The phosphatidylglycerol-synthesizing activity in microsomes of both lung and liver was a minor fraction of total tissue activity and could be interpreted as due either to contamination with outer mitochondrial membrane or to a small amount of activity innate to microsomes. These results suggest that phosphatidylglycerol, which is believed to be a component of pulmonary surfactant, is synthesized by lung at a rapid rate relative to liver and that the subcellular distribution of its synthesis is similar in both tissues, with mitochondria as the major site.

Animals↗
PubMed1981

Topography of glycerolipid synthetic enzymes. Synthesis of phosphatidylserine, phosphatidylinositol and glycerolipid intermediates occurs on the cytoplasmic surface of rat liver microsomal vesicles.

The topography of glycerolipid biosynthetic enzymes within the transverse plane of rat liver microsomal vesicles was investigated: (1) by use of the impermeant inhibitor, mercury-dextran; (2) by use of proteases; and (3) by determining whether the enzyme activities are latent. The seven enzyme activities investigated (dihydroxyacetone-phosphate acyltransferase, acyldihydroxyacetone-phosphate oxidoreductase, phosphatidic acid : CTPcytidyltransferase, CDPdiacylglycerol : inositol phosphatidyltransferase, 2-monoacylglycerol acyltransferase, diacylglycerol kinase, and the serine base exchange enzyme) function in phosphatidylinositol and phosphatidylserine synthesis and at intermediate levels in glycerolipid synthesis including steps of ether lipid synthesis. Mercury-dextran inhibited four of these enzymes greater than 60% in intact microsomal vesicles. One or more of the proteases employed (chymotrypsin, trypsin and pronase) inactivated each of the seven enzyme activities in intact microsomal vesicles. These two approaches indicate that each of these enzymes has important domains located on the cytoplasmic surface of microsomal vesicles. These enzyme activities could be assayed in intact microsomal vesicles. None appeared to be highly latent, indicating that substrates have free access to active sites. One substrate for each of these enzymes had been shown previously to be unable to cross the microsomal membrane. These data indicate that the active sites of these enzymes are located on the cytoplasmic surface of microsomal vesicles. It is concluded that the synthesis of phosphatidylserine and phosphatidylinositol, intermediates of ether lipid formation and other intermediates of glycerolipid synthesis occur asymmetrically on the cytoplasmic surface of the endoplasmic reticulum. These findings and our previous investigations on the topography of seven enzymes of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine biosynthesis (Ballas, L.M. and Bell, R.M., Biochim. Biophys. Acta 602, (1980) 578-590) indicate that the synthesis of the major cellular glycerolipids occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.

Acyltransferases↗
PubMed1982

Stereospecific synthesis and enzyme studies of CDP-diacylglycerols.

The fatty acid specificity of two enzymes that metabolize CDPdiacylglycerol, CDPdiacylglycerol hydrolase (EC 3.6.1.26) and CDPdiacylglycerol: inositol phosphatidyltransferase (EC 2.7.8.11), has been examined in guinea pig brain. Mixed CDPdiacylglycerols were stereospecifically synthesized by the following sequence: (i) hydrolysis of a homodiacyl lecithin to 1-acyl lysoPC by action of snake venom phospholipase A2, (ii) reacylation with the anhydride of the desired second fatty acid and dimethylaminopyridine, (iii) hydrolysis of the resultant heterodiacyl lecithin to phosphatidate with cabbage phospholipase D, and (iv) reaction of phosphatidate with CMPmorpholidate to give CDPdiacylglycerol. CDPdiacylglycerol: inositol phosphatidyltransferase showed the following rates of conversion of 40-microM suspensions of CDPdiacylglycerol in 0.15% Triton X-100 to phosphatidylinositol relative to the 1-stearoyl-2-oleoyl derivative (100%): dipalmitoyl, 70%; distearoyl, 38%; diarachidonoyl, 9%; 1-arachidonoyl-2-stearoyl, 6%; 1-stearoyl-2-arachidonoyl, 4%. These results indicate that the composition of isolated phosphatidylinositol and related lipids is not explained by the fatty acid specificity of the biosynthetic enzymes and supports the intervention of a deacylation-reacylation sequence. The rates of hydrolysis of the synthetic CDPdiacylglycerols at 76 microM, in 0.3% Triton X-100, by the CDPdiacylglycerol hydrolase relative to the 1-stearoyl-2-oleoyl derivative (100%) were: dipalmitoyl, 70%; distearoyl, 32%; 1-arachidonoyl-2-stearoyl, 30%; 1-stearoyl-2-arachidonoyl, 28%; diarachidonoyl, 22%. Inhibition of this enzyme by AMP was shown to be non-competitive, with a Ki of 40 microM. The lysosomal localization of the mammalian hydrolase was confirmed.

Animals↗
PubMed1983

The metabolism of CDP-diacylglycerol and phosphatidylinositol in the microsomal fraction of rat liver. Effects of chlorpromazine, magnesium and manganese.

1. The metabolism of CDPdiacylglycerol and phosphatidylinositol was measured using substrates bound to the microsomal membranes of rat liver. 2. Chlorpromazine inhibited the degradation of [14C]CDPdiacylglycerol and the concomitant inositol-independent release of 14C in water-soluble products in the presence of various concentrations of Mg2+ and Mn2+. 3. The activity of CDPdiacylglycerol inositol phosphatidyltransferase was measured by determining the rate of incorporation of [3H]inositol into phosphatidylinositol, and by the inositol-dependent release of water-soluble 14C from [14C]CDPdiacylglycerol. Both of these parameters were inhibited by chlorpromazine in incubations that contained rate-limiting concentrations of Mg2+. However, chlorpromazine stimulated the reaction when 20 mM Mg2+, 0.5 mM Mn2+, 2 mM Mn2+ or 20 mM Mn2+ was added to the incubations. 4. Low concentrations of chlorpromazine increased an inositol-exchange reaction in the presence of 0.5 mM Mn2+ whereas higher concentrations of chlorpromazine inhibited. Chlorpromazine had relatively less effect on the inositol-exchange reaction at higher concentrations of Mn2+. 5. The action of chlorpromazine in decreasing the breakdown of CDPdiacylglycerol and in stimulating its conversion to phosphatidylinositol could explain part of the mechanism by which this compound and other amphiphilic cations increase the synthesis of acidic phospholipids.

Animals↗
PubMed1984

Inhibition of platelet phosphatidylinositol synthetase by an analog of CDP-diacylglycerol.

Unpublished portions of the synthesis of a phosphinate-phosphonate diether analog of CDPdiacylglycerol are reported. The liponucleotide analog was found to be a very powerful inhibitor of platelet PI synthetase; kinetic data suggest a competitive inhibition mechanism. The structural specificity of CDPdiacylglycerol for liponucleotide-mediated biosynthetic reactions is discussed.

Blood Platelets↗
PubMed1985

Biophysical properties of cytidine diphosphate diacylglycerol in solution.

The physical properties of CDP diacylglycerol derived from egg phosphatidylcholine are very different from those of the common glycerophospholipids, such as phosphatidylcholine. Gently dispersed in buffer (5 mM phosphate, 0.15 M NaCl, pH 7.4), the liponucleotide initially forms an opalescent suspension of spherical vesicles, up to 50 micron in diameter, which appear to be unilamellar. These large vesicles are unstable and, independently of initial concentration, unstirred suspensions are no longer turbid after being incubated for about 1 h at room temperature. The passage of samples through Sepharose and Sephadex at increasing time intervals after the first hour reveals a continuing but slow diminution in size until, at about two days, a final peak is obtained which remains invariant for longer times. Chromatography of these ultimate stable micelles on Sephadex G-200 gives a Stokes radius of 4.2 nm. Their sedimentation coefficient extrapolated to zero concentration is 6.1 S. These numbers, combined with a partial specific volume of 0.835 ml X g-1, give an anhydrous mass of 155 000 Da and an aggregation number of 158. Although the data suggest the particles to be spherical, other compact forms cannot be excluded. Proton NMR at 220 MHz shows time-dependent spectral changes which are consistent with the slow structural transformation observed by gel-filtration chromatography, and indicate that the sugar and cytosine groups in the ultimate micelles apparently are motionally restricted. The critical micelle concentration is near 6 microM, but micelle-free molecule equilibration requires at least 7 days at a total concentration of 89 microM. Sonication considerably decreases the time required for the vesicle-micelle transformation and the micelle-free molecule equilibration. Some implications for enzymology are discussed.

Biophysical Phenomena↗
PubMed1988

Comparison of the HPLC-separated species patterns of phosphatidic acid, CDP-diacylglycerol and diacylglycerol synthesized de novo in rat liver microsomes (a new method).

The species pattern of phosphatidic acid was compared with that of CDP-diacylglycerol and diacylglycerol synthesized de novo by glycerol 3-phosphate acylation in a CoA ester-generating system in liver microsomes. The similarity of the species patterns of phosphatidic acid and CDP-diacylglycerol indicated that the CTP-phosphatidyl cytidylyltransferase showed no selectivity for individual species of its phosphatidic acid substrate. Since the species pattern of diacylglycerol deviated from that of phosphatidic acid, a slight acyl selectivity of the phosphatidic acid phosphohydrolase or a slight inhomogeneity of its substrate pool might be assumed. For the determination of the molecular species of CDP-diacylglycerol, a new method was developed. By incubation of CDP-diacylglycerol with oligonucleate 5'-nucleotidohydrolase (phosphodiesterase), phosphatidic acid was produced. The CDP-diacylglycerol-derived phosphatidic acid was methylated with diazomethane and then separated by reverse-phase HPLC in 15 molecular species.

Animals↗
PubMed1989

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

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

Animals↗
PubMed1990

Biochemical characterization and regulation of cardiolipin synthase in Saccharomyces cerevisiae.

Cardiolipin (CL) synthase activity was characterized in mitochondrial extracts of the yeast Saccharomyces cerevisiae and was shown for the first time to utilize CDP-diacylglycerol as a substrate. CL synthase exhibited a pH optimum of 9.0. Maximal activity was obtained in the presence of 20 mM magnesium with a Triton X-100: phospholipid ratio of 1:1. The apparent Km values for phosphatidylglycerol and CDP-diacylglycerol were 1 mM and 36 microM, respectively. CL synthase activity was maximal at 45 degrees C and heat inactivation studies showed that the enzyme retained greater than 75% of its activity at temperatures up to 55 degrees C. To study the regulation of CL synthase, the enzyme was assayed in cells grown under conditions known to affect general phospholipid synthesis. Unlike many phospholipid biosynthetic enzymes including PGP synthase, which catalyzes the initial step in CL biosynthesis, CL synthase was not repressed in cells grown in the presence of the phospholipid precursor inositol. Detailed procedures for the enzymatic synthesis of 32P-labelled substrates are described.

Cytidine Diphosphate Diglycerides↗
PubMed1991

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes.

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodelling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the I-positive of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.

Acyl Coenzyme A↗
PubMed1992

Comparison of the molecular species patterns of phosphatidic acid, CDP-diacylglycerols and phosphatidylinositol in potato tuber, pea leaf and soya-bean microsomes: consequences for the selectivity of the enzymes catalyzing phosphatidylinositol biosynthesis.

Microsomes prepared from pea leaf, potato tuber or germinated soya-beans, were incubated for 30 min with [14C]glycerol 3-phosphate. In the three tissues, phosphatidic acid (PA), CDP-diacylglycerols (CMP-PA) and phosphatidylinositol (PI) were labelled and could be separated by TLC. After methylation of phosphatidic acid, or treatment of CMP-PA by a nucleotidase, the molecular species composition of the three lipid classes could be determined by radio-HPLC. The similarity observed between the distributions of radioactivity among CMP-PA and PA molecular species, in the three tissues, indicates that the enzyme CTP:PA cytidylyltransferase did not present any selectivity towards any molecular species of PA. In contrast, only two molecular species containing palmitic acid (16:0/18:2 and 16:0/18:3) were labelled in PI whereas labelled PA and CMP-PA contained molecular species possessing stearic acid (18:0/18:2, 18:0/18:3 and 18:0/18:1). This indicates that the enzyme PI-synthase utilizes preferentially those molecular species of CMP-PA containing palmitic acid as substrates. However, mass analyses of PI prepared from the microsomes of the three tissues used in this study, indicated the presence of molecular species containing stearic acid (18:0/18:2 and 18:2/18:2). Except in soya-bean microsomes (where 18:0/18:2-PI represented 16% of total PI), those last molecular species were always present in small amounts.

CDP-Diacylglycerol-Inositol 3-Phosphatidyltransfer↗
PubMed1993

Biosynthesis and characterization of phosphatidylglycerophosphoglycerol, a possible intermediate in lipoteichoic acid biosynthesis in Streptococcus sanguis.

A membrane enzyme preparation from Streptococcus sanguis was shown to convert sn-[14C]glycerol 3-phosphate and CDP-diacylglycerol (or deoxyCDP-diacylglycerol) into a series of progressively higher-molecular-weight [14C]oligophosphoglycerophospholipids in vitro. The first oligophosphoglycerophospholipid to accumulate (termed lipid-1) was purified to homogeneity; chemical analysis, gas-liquid chromatography and chemical degradation studies indicated the most likely structure to be phosphatidylglycerophosphoglycerol (PGpG). PGpG is formed directly from two molecules of phosphatidylglycerol (PG), one molecule of PG serving as a sn-glycerol 1-phosphate (pG) donor and the second serving as the pG acceptor, with co-production of diacylglycerol. These oligophosphoglycerophospholipids may be intermediates in the biosynthesis of lipoteichoic acids.

Carbon Radioisotopes↗