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PubMed1976

Partial purification and characterization of cytidine 5'-diphosphate-diglyceride hydrolase from membranes of Escherichia coli.

Cytidine 5'-diphosphate (CDP)-diglyceride is hydrolyzed to phosphatidic acid and cytidine 5'-monophosphate by a specific membrane-bound enzyme in cell-free extracts of Escherichia coli. The hydrolase can be extracted from the particulate fraction with Triton X-100 and purified 1,000-fold in the presence of this detergent. Several nucleoside disphosphate diglycerides were synthesized to determine the substrate specificity of the hydrolase. CDP-diglyceride was hydrolyzed preferentially, although uridine 5'-diphosphate-diglyceride, guanosine 5'-diphosphate-diglyceride, and adenosine 5'-diphosphate (ADP)-diglyceride were also slowly hydrolyzed. Surprisingly, the purified enzyme did not catalyze detectable cleavage of deoxy-CDP (dCDP)-diglyceride. The liponucleotide pool of E. coli contains dCDP-diglyceride and CDP-diglyceride in approximately equal amounts (Raetz and Kennedy, 1973). Water-soluble nucleoside pyrophosphates, such as CDP-choline, nicotinamide adenine dinucleotide, or adenosine 5'-triphosphate are not attacked by this specific hydrolase. Hydrolysis of CDP-diglyceride is strongly inhibited by adenosine 5'-monophosphate and by ADP-diglyceride.

Cell Membrane↗
PubMed1992

Formyl peptide stimulates and ATP gamma S potentiates [3H]cytidine 5'-diphosphate diglyceride accumulation in human neutrophils.

Although it is evident that the chemotactic peptide FMLP activates O2-formation in neutrophils via the phosphoinositidase-mediated second messenger system, it is less clear how endogenous priming agents such as ATP and platelet activating factor potentiate FMLP action. In our study, we have examined the possible effects of the stable ATP analog adenosine 5'-O-[3-thiotriphosphate] (ATP gamma S) on cellular levels of inositol 1,4,5-trisphosphate, [Ca2+]i and diglyceride (DG), in resting and in FMLP-stimulated neutrophils. Although all three measures were increased in the presence of FMLP, only the increase in DG was enhanced by pretreatment (priming) with ATP gamma S. We also measured the accumulation of the phosphoinositide cycle intermediate cytidine 5'-diphosphate (CDP)-DG to assess possible effects of priming on phosphoinositide resynthesis. The addition of FMLP to [3H]cytidine-prelabeled neutrophils elicited an increase in the accumulation of [3H]CDP-DG that was maximally enhanced in cells that were pretreated with cytochalasin B. The stimulated accumulation of [3H]CDP-DG was completely reversed by the addition of myo-inositol. Treatment of [3H]cytidine-prelabeled neutrophils with ATP gamma S (10-100 microM) resulted in a dose-dependent synergistic increase in FMLP-stimulated [3H]CDP-DG accumulation, whereas ATP gamma S alone had no effect. The observed increases in DG and in [3H]CDP-DG, in contrast to inositol 1,4,5-trisphosphate and [Ca2+]i responses, correlates well with the ATP gamma S-priming of FMLP-induced O2-formation. A similar potentiation of FMLP-induced stimulation of CDP-DG formation was also observed with platelet-activating factor. It is proposed that the priming of FMLP responses in neutrophils proceeds via a mechanism that selectively enhances DG production through a mechanism that is independent of FMLP-induced breakdown of phosphatidylinositol bisphosphate.

Adenosine Triphosphate↗
PubMed1977

A phospholipid derivative of cytosine arabinoside and its conversion to phosphatidylinositol by animal tissue.

We have synthesized an analog (ara-CDP-DL-dipalmitin) of cytidine diphosphate diglyceride (CDP-diglyceride) in which the antitumor drug, cytosine arabinoside, is substituted for the cytidine moiety. Enzymes in rat and human liver convert this analog to phosphatidylinositol, thereby releasing cytosine arabinoside-5'-monophosphate, an obligatory intermediate in the activation of cytosine arabinoside. Unlike cytidine diphosphate diglyceride, however, ara-CDP-DL-diapalmitin is not an efficient substrate for phosphatidylglycerophosphate synthesis in liver or phosphatidylserine in Escherichia coli. The antitumor activity of ara-CDP-DL-dipalmitin in mice bearing L5178Y leukemia is described.

Animals↗
PubMed1970

The structure and biosynthesis of phosphatidyl inositol in cauliflower inflorescence.

Phosphatidyl inositol has been isolated from cauliflower inflorescence and soybean. Analysis of the fatty acid distribution showed that unsaturated fatty acids were preferred at position 2, and that saturated fatty acids were preferred at position 1.Mitochondrial preparations from cauliflower inflorescence were capable of synthesizing phosphatidyl inositol from myo-inositol and cytidine diphosphate diglyceride. The optimal pH for the reaction was 9.0. Metal ions were required for the reaction: Mn(2+) ions were optimally effective at 1.5 mm, and Mg(2+) ions were maximally effective at 30 mm. The maximal rate in the presence of Mn(2+) was 9 times as great as that in the presence of Mg(2+). Phosphatidyl inositol could also be synthesized when cytidine diphosphate diglyceride was replaced by cytidine triphosphate, but the pH optimum then shifted to 8.1.

Journal Article↗
PubMed1975

Identification of cytidine diphosphate-diglyceride in the pineal gland of the rat and its accumulation in the presence of DL-propranolol.

CDP-diglyceride, an important metabolic intermediate in the biosynthesis of phospholipids, has been isolated for the first time from a mammalian tissue. The isolated material, labeled in incubations of intact rat pineal glands with 32P, [3H]cytidine, or [3H]CTP in the presence of DL-propranolol, was chromatographically identical with authentic CDP-diglyceride and was able to serve as phosphatidyl donor in the enzymatic synthesis of phosphatidylinositol and phosphatidyglycerol. It yielded the expected products upon enzymatic and chemical degradation. No dCDP-diglyceride was detected No radioactive CDP-diglyceride was detected following incubations in the absence of propranolol. Stimulation of CDP-diglyceride labeling from 32P1 occurred at propranolol concentrations between 0.03 and 1.0 mM. Net synthesis of the liponucleotide was shown. At 0.1 mM, propranolol incrased the incorporation of radioactivity into phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. When inositol (10 mM) and propranolol (0.1 mM) were both present, phosphatidylinositol labeling was further increased, wheas stimulation of phosphatidylglycerol and CPD-diglyceride labeling was abolished. Since CDP-diglyceride did not accumulate in the absence of the drug, its availability may normally be the limiting factor in phosphatidylinositol and phosphatidylglycerol biosynthesis. When propranol is present, inositol may become limiting and thus may lead to the observed labeling pattern.

Animals↗
PubMed1976

Studies on cardiolipin biosynthesis in Mycobacterium smegmatis.

Supplementation of a growth medium with 5% glucose has been found to stimulate the formation of cardiolipin and phosphatidylethanolamine five- and threefold, respectively, in Mycobacterium smegmatis. The presence of both cytidine diphosphate diglyceride and phosphatidylglycerol pathways of biosynthesis of cardiolipin in cell-free extracts has been demonstrated. The enzymes were localized in the fractions which contained membranes. Isonicotinic acid hydrazide and streptomycin sulfate inhibited the formation of cardiolipin.

Cardiolipins↗
PubMed1975

Biosynthesis of bis(monoacylglyceryl)phosphate and acylphosphatidylglycerol in rat liver mitochondrial.

Bis(monoacylglyceryl)phosphate is present in trace quantities in normal liver where it represents smaller than 1 percent of total liver phospholipids. This compound is of considerable interest since its level can be greatly increased in certain lipidoses, either of a genetic nature or caused by drugs. Biosynthesis of bis(monoacylglyceryl)P in vitro has not previously been demonstrated. This paper reports the enzymatic formation of bis(monoacylglyceryl)P and acylphosphatidylglycerol as minor products from cytidine diphosphate diglyceride and radioactive sn-glycerol-3-P using a crude mitochondrial fraction from normal rat liver; phosphatidylglycerol was the principal product. Evidence is also presented which shows the enzymatic formation of acylphosphatidylglycerol and bis(monoacylglyceryl)P from [1',3'-14C]phosphatidylglycerol by a crude rat liver mitochondrial preparation in vitro. The structures of the radioactive bis(monoacylglyceryl)P and acylphosphatidylglycerol were tentatively established by thin layer chromatography, and chromatography of the products of mild alkaline hydrolysis and acetolysis. Radioactive acylphosphatidylglycerol was stoichiometrically converted to bis(monoacylglyceryl)P by treatment with bee venom phospholipase A, providing additional evidence for the structure of acylphosphatidylglycerol and bis(monoacylglyceryl)P.

Animals↗
PubMed1979

The mechanism of modification by propranolol of the metabolism of phosphatidyl-CMP (CDP-diacylglycerol) and other lipids in the rat pineal gland.

The mechanism underlying the alteration of phospholipid metabolism in rat pineal gland in vitro produced by propranolol and tertiary amine local anesthetics was investigated. 0.1 mM propranolol did not affect either the levels or specific activity of [32P]ATP in glands. In the presence of the drug, the incorporation of cytidine, but not of inorganic phosphate, into phosphatidyl-CMP (CDP-diacylglycerol) was dependent on the cytidine concentration. The incorporation of glycerol into phosphatidyl-CMP, phosphatidylinositol and phosphatidylglycerol was enhanced by propranolol, whereas labeling of phosphatidylcholine was decreased. When both 1 mM propranolol and 1 mM inositol were present, labeling of phosphatidylinositol was further increased, stimulation of phosphatidyl-CMP and phosphatidylglycerol labeling was reduced and incorporation into phosphatidylcholine and triacylglycerol was depressed. The incorporation of [3H]inositol into pineal lipids was also enhanced by propranolol. 10 microM propranolol inhibited rat liver phosphatidic acid phosphohydrolase by 50%, while local anesthetics were less potent in the decreasing order: dibucaine greater than tetracaine greater than lidocaine greater than procaine. The propranolol-induced accumulation of phosphatidyl-CMP was prevented by supplying adequate freely diffusible inositol in the medium. The phosphatidyl-CMP which accumulated was not utilized for the enhanced formation of phosphatidylinositol brought about by norepinephrine. The results indicate that propranolol and local anesthetics redirect pineal phospholipid metabolism in part by inhibition of phosphatidic acid phosphohydrolase.

Adenosine Triphosphate↗
PubMed1985

The CMP-stimulated production of diacylglycerol and CDPdiacylglycerol in neuronal nuclei labelled with radioactive arachidonate.

A neuronal nuclear fraction (N1), isolated from immature rabbit cerebral cortex, was preincubated with [3H]arachidonate, ATP, CoA, Mg2+ and 1-acyl-sn-glycero-3-phosphocholine or 1-acyl-sn-glycero-3-phosphoinositol. Using the former lysophospholipid, a sizeable incorporation of radioactivity was seen in N1 phosphatidylcholine. In subsequent incubations in the presence of CMP and EGTA, there was a generation of radioactive diacylglycerol in N1 and a corresponding decline in phosphatidylcholine radioactivity. Both these changes could be blocked by the addition of CDPcholine. In incubations using N1 phosphatidylinositol or phosphatidylethanolamine prelabelled with [3H]arachidonate, no evidence was found to support a direct generation of diacylglycerol from these phospholipids. The back reaction of cholinephosphotransferase in N1 is likely the principal source of diacylglycerols bearing arachidonate. Using either lysophospholipid in the preincubations described in the opening sentence, more than half of the incorporated radioactivity derived from [3H]arachidonate was found in N1 phosphatidylinositol. In subsequent incubations with EGTA and CMP there was a production of radioactive CDPdiacylglycerol and a decline in radioactive phosphatidylinositol. Both events could be blocked by the presence of myo-inositol. Radioactive CDPdiacylglycerol, produced in N1 in the presence of CMP and EGTA, was converted back into phosphatidylinositol by the addition of myo-inositol. The production of CDPdiacylglycerol is likely the result of the back reaction of CDPdiacylglycerol:inositol phosphatidate transferase in N1.

Animals↗
PubMed1983

Chloroform-soluble nucleotides in Escherichia coli. Role of CDP-diglyceride in the enzymatic cytidylylation of phosphomonoester acceptors.

CDP-diglyceride, the precursor of all the phospholipids in Escherichia coli, is cleaved in vitro to phosphatidic acid and CMP by a membrane-bound hydrolase. Since the physiological function of CDP-diglyceride hydrolase is unknown, we have explored the possibility that this enzyme acts in vivo as either a phosphatidyl- or cytidylyltransferase. To distinguish between these two alternatives, partially purified hydrolase was incubated with CDP-diglyceride in the presence of 50% H218O. Analysis of the reaction products by 31P NMR showed that 18O is incorporated exclusively into CMP, suggesting that the enzyme is a cytidylyltransferase. This conclusion is further supported by the following experimental results: (i) the hydrolase catalyzes the transfer of CMP from CDP-diglyceride to Pi; (ii) numerous phosphomonoesters, such as glycerol 3-phosphate, phosphoserine, and glucose 1-phosphate also function as CMP acceptors, but the corresponding compounds lacking the phosphate residues are not substrates for the enzyme; and (iii) CDP-diglyceride hydrolase exchanges [32P]phosphatidic acid for the phosphatidyl moiety of CDP-diglyceride and 32Pi for the beta-phosphate residue of CDP, indicating the involvement of a novel CMP-enzyme complex. These data suggest a biosynthetic role for CDP-diglyceride hydrolase, and extend the possible functions of CDP-diglyceride in the E. coli envelope.

Chloroform↗
PubMed1978

Partial purification and properties of CTP:phosphatidic acid cytidylyltransferase from membranes of Escherichia coli.

The cytosine liponucleotides CDP-diglyceride and dCDP-diglyceride are key intermediates in phospholipid biosynthesis in Escherichia coli (C. R. H. Raetz and E. P. Kennedy, J. Biol. Chem. 248:1098--1105, 1973). The enzyme responsible for their synthesis, CTP:phosphatidic acid cytidylytransferase, was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin and was purified about 70-fold (relative to cell-free extracts) in the presence of detergent. In studies of the heat stability of the enzyme, activity decayed slowly at 63 degrees C. Initial velocity kinetic experiments suggested a sequential, rather than ping-pong, reaction mechanism; isotopic exchange reaction studies supported this conclusion and indicated that inorganic pyrophosphate is released before CDP-diglyceride in the reaction sequence. The enzyme utilized both CTP and dCTP as nucleotide substrate for the synthesis of CDP-diglyceride and dCDP-diglyceride, respectively. No distinction was observed between CTP and dCTP utilization in any of the purification, heat stability, and reaction mechanism studies. In addition, CTP and dCTP were competitive substrates for the partially purified enzyme. It therefore appears that a single enzyme catalyzes synthesis of both CDP-diglyceride and dCDP-diglyceride in E. coli. The enzyme also catalyzes a pyrophosphorolysis of CDP-diglyceride, i.e., the reverse of its physiologically important catalysis.

Cell Membrane↗
PubMed1981

The influence of divalent cations and substrate concentration on the incorporation of myo-inositol into phospholipids of isolated bovine oligodendrocytes.

The incorporation of myo-inositol into phosphatidylinositol by two routes (CTP-independent and CTP-independent) has been investigated in homogenates prepared from isolated bovine oligodendrocyte perikarya. The CTP-dependent route has the higher maximum velocity of inositol incorporation and can utilise either Mn2+ or Mg2+ as a divalent ion cofactor. This route of inositol incorporation is also strongly inhibited by Ca2+ ions at concentrations less than 1 mM. The primary site of the inhibitory action appears to be the enzyme CDP-diglyceride inositol phosphatidyl transferase (EC 2.7.8.11) though synthesis of CDP-diacylglycerol is also inhibited by endogenous Ca2+ present in the oligodendrocyte homogenate. In contrast, CTP-independent inositol incorporation into phosphatidylinositol is only stimulated by Mn2+ (Zn2+,Cu2+, Mg2+, Ca2+ and Co2+ are ineffective) and is not inhibited by Ca2+, at least up to a concentration of 1 mM.

Animals↗
PubMed1976

Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol.

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDPdiglyceride):L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase) is bound tightly to the ribosomes in crude extracts of Escherichia coli. After separation of the enzyme from the ribosomes by the method of Raetz and Kennedy (Raetz, C.R.H., and Kennedy, E.P. (1974), J. Biol. Chem. 249, 5038), we have purified the enzyme to 97% of homogenekty. The major portion of the overall 5500-fold purification was attained by substrate-specific elution from phosphocellulose using CDP-diglyceride in the presence of detergent. The purified enzyme migrated as a single band with an apparent minimum molecular weight of 54 000 when subjected to electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. The purified enzyme catalyzed exchange reactions between cytidine 5'- monophosphate (CMP) and CDP-diglyceride and between serine and phosphatidylserine. The enzyme also catalyzed the hydrolysis of CDP-diglyceride to form CMP and phosphatidic acid. dCDP-diglyceride was equivalent to CDP-diglyceride in all reactions catalyzed by the enzyme. In addition, the purified enzyme catalyzed the formation of phosphatidylglycerol or phosphatidylglycerophosphate at a very slow rate when serine was replaced as substrate by glycerol or sn-glycero-3-phosphate, respectively. These results suggest catalysis occurs via a ping-pong mechanism through the formation of a phosphatidyl-enzyme intermediate.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗
PubMed1998

Enzymatic synthesis of [3H]Cytidine 5'-diphospho-1, 2-diacyl-sn-glycerol.

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol; CDP-DG) is an important intermediate in the biosynthesis of the major glycerophosphate-based phospholipids of prokaryotes and eukaryotes. This compound is expensive to purchase and inefficient to prepare chemically. Radiolabeled CDP-diacylglycerol is unavailable commercially. We describe a simple and inexpensive method to synthesize [3H]CDP-DG enzymatically. The three-step enzymatic procedure includes phosphorylation of [3H]glycerol to sn-[3H]glycerol 3-phosphate (G3P) by glycerokinase,acylation of [3H]G3P to [3H]phosphatidic acid (PA) by G3P acyltransferase, and conversion of [3H]PA and CTP to [3H]CDP-DG by CDP-DG synthase. This procedure is considerably less labor intensive and less expensive than is chemical synthesis, and the yield is at least 30%.

Acylation↗