Differential selectivity of cholinephosphotransferase and ethanolaminephosphotransferase of Tetrahymena for diacylglycerol and alkylacylglycerol

The glycerophospholipids of the ciliate protozoan Tetrahymena thermophila differ greatly in their content of alkylacylglycerol with phosphatidylcholine, phosphatidylethanolamine, and 2-aminoethylphosphonolipid containing 60, 4, and 53% glyceryl ether, respectively. This difference is achieved by differences in the selectivities of cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) for alkylacylglycerol and diacylglycerol. When the two enzymes are assayed in vitro using only endogenous diglyceride as substrate, the newly formed phosphatidylcholine contains 37% glyceryl ether, while the newly formed phosphatidylethanolamine contains 5% glyceryl ether. The ethanolaminephosphotransferase is stimulated equally well by addition of diolein and dipalmitin, but the diacylglycerols have no effect on the glyceryl ether content of phosphatidylethanolamine. In contrast, the glyceryl ether content of newly formed phosphatidylcholine decreases to 16% when the cholinephosphotransferase is exposed to diolein or dipalmitin. The ethanolaminephosphotransferase is not stimulated by addition of a 60:40 mixture of alkylacylglycerol/diacylglycerol. The cholinephosphotransferase is stimulated by the mixture to the same extent as it is by the diacylglycerols, with the glyceryl ether content of the newly formed phosphatidylcholine increasing to 52%. With the addition of alkylacylglycerol alone, the glyceryl ether content of the newly formed phosphatidylethanolamine increases to 10%, while that of the newly formed phosphatidylcholine increases almost to 60%.     

Regulation of triacylglycerol synthesis in the liver: a decrease in diacylglycerol acyltransferase activity after treatment of isolated rat hepatocytes with glucagon

Isolated rat hepatocytes were used to investigate the possibility of a short-term effect of glucagon on the synthesis of triacylglycerols in the liver. Incubation of hepatocytes in the presence of glucagon, followed by homogenization in a buffer containing F- (50 mM) and EDTA (2.5 mM), resulted in a 53% decrease in activity of microsomal diacylglycerol acyltransferase (EC 2.3.1.20), the only enzyme that is exclusively involved in the synthesis of triacylglycerols. The activity of cholinephosphotransferase (EC 2.7.8.2), which also uses diacylglycerols as substrate, was not decreased after exposure of the hepatocytes to glucagon. This may imply that triacylglycerol synthesis can be regulated independently of phosphatidylcholine synthesis. The activity of diacylglycerol acyltransferase in microsomes isolated from a homogenate of whole liver could be reduced by preincubating the microsomes with Mg2+ (5 mM), ATP (1 mM) and 105 000 X g supernatant. The enzyme could be reactivated by incubation of the washed microsomes with a 105 000 X g supernatant in the presence of dithiothreitol (5 mM). Fluoride (50 mM) inhibited this reactivation. It is concluded that the activity of diacylglycerol acyltransferase is subject to hormonal short-term control, possibly via a phosphorylation-dephosphorylation mechanism.     

Topography of phosphatidylcholine, phosphatidylethanolamine and triacylgycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the sn-glycerol-3-phosphate acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of thease enzymes are located on the external surface of microsomal vesicles. It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

Phosphoglyceride biosynthesis by brain microsomes: centrophenoxine, SaH-42-348, and DH-990 inhibit phospholipid N-methylation

The effects of centrophenoxine, SaH-42-348, and DH-990 on several enzymes involved in aminophospholipid biosynthesis in brain have been examined in vitro. Relatively high concentrations of centrophenoxine were required to achieve 50% inhibition of the microsomal enzymes CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (EPT), CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT), phosphatidyl-N-methylethanolamine N-methyltransferase (PME-NMT), and phosphatidyl-N,N-dimethylethanolamine N-methyltransferase (PDE-NMT). Intermediate concentrations of SaH-42-348 inhibited CPT (IC50 = 2.0 mM), EPT (IC50 = 1.9 mM), PME-NMT (IC50 = 0.19 mM), and PDE-NMT (IC50 = 0.17 mM). Of the three drugs tested, DH-990 was the most potent inhibitor of the phospholipid-synthesizing enzymes. Phosphatidylserine decarboxylase, a mitochondrial inner-membrane enzyme [A. K. Percy, J. F. Moore, M. A. Carson, and C. J. Waechter (1983) Arch. Biochem. Biophys. 223, 484-494], was virtually unaffected by the three drugs added at millimolar concentrations. Kinetic analyses indicated that the inhibitory action of DH-990 on the brain enzymes was noncompetitive with respect to all substrates. The relatively high sensitivity of CPT (IC50 = 0.6 mM), EPT (IC50 = 2.2 mM), PME-NMT (IC50 = 2.5 microM), and PDE-NMT (IC50 = 2.5 microM) to inhibition by DH-990 in brain microsomes suggests that this compound may be useful for cellular studies on the possible relationships between phospholipid metabolism and neurobiological functions.     

Aromatic boronic acids as probes of the catalytic site of human plasma lecithin-cholesterol acyltransferase

We have recently proposed a catalytic mechanism for human plasma lecithin-cholesterol acyltransferase (EC 2.3.1.43) (J. Biol. Chem. (1986) 261, 7032-7043), implicating single serine and histidine residues in phosphatidylcholine cleavage and two cysteine residues in cholesterol esterification. We now confirm the involvement of serine and histidine in catalysing the phospholipase A2 action of lecithin-cholesterol acyltransferase by demonstrating the inhibition of this activity by phenylboronic acid (Ki = 1.23 mM) and m-aminophenylboronic acid (Ki = 2.32 mM), inhibitors of known serine/histidine hydrolases. The specificity of the interaction of aromatic boronic acids with catalytic serine and histidine residues and the putative formation of a tetrahedral adduct between boron and the lecithin-cholesterol acyltransferase serine hydroxyl group which is similar to the transition-state intermediate formed between phosphatidylcholine and the catalytic serine residue was suggested by: substrate protection against inhibition by phenylboronic acids; a much reduced incorporation of phenylmethane[35S]sulphonyl fluoride into the enzyme in the presence of phenylboronic acid; the lack of interaction of histidine- or serine-modified enzyme with immobilized phenylboronic acid in the presence of glycerol (Ve/Vo = 2.7 and 2.3 respectively) when compared to the native enzyme (Ve/Vo = 5.25). Fatty acyl-lecithin-cholesterol acyltransferase, produced by incubation of the enzyme with a lecithin-apolipoprotein A-I proteoliposome substrate, was not retarded upon the sorbent column (Ve/Vo = 1.5). Modification of the enzyme's two free cysteine residues with 5,5'-dithiobis(2-nitrobenzoic acid) or potassium ferricyanide reduced (Ve/Vo = 3.5) but did not abolish retardation on the sorbent column, indicating that these modifications resulted in steric hinderance of the interaction of the boron atom with the lecithin-cholesterol acyltransferase serine hydroxyl group. These data suggest that the serine and histidine residues are proximal within the enzyme catalytic site and that both cysteine thiol groups are close to the serine hydroxyl group. The presence of significant amino-acid sequence homologies between lecithin-cholesterol acyltransferase, triacylglycerol lipases and the transacylases of fatty acid synthase is also reported.     

Topography of phosphatidylcholine,phosphatidylethanolamine and triacylglycerol biosynthetic enzymes in rat liver microsomes

The topography of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol biosynthetic enzymes within the transverse plane of rat liver microsomes was investigated using two impermeant inhibitors, mercury-dextran and dextran-maleimide. Between 70 and 98% of the activities of fatty acid : CoA ligase (EC 6.2.1.3), $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase (EC 2.3.1.15), phosphatidic acid phosphatase (EC 3.1.3.4), diacylglycerol acyltransferase (EC 2.3.1.20), diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) were inactivated by mercury-dextran. Dextran-maleimide caused 52% inactivation of the $\text{sn-}\text{glycerol}\text{-3-}\text{phosphate}$ acyltransferase. Inactivation of each of these activities except fatty acid : CoA ligase occurred in microsomal vesicles which remained intact as evidenced by the maintenance of highly latent mannose-6-phosphatase activity (EC 3.1.3.9). These glycerolipid biosynthetic activities were not latent, indicating that substrates have free access to the active sites. Moreover, ATP, CDP-choline and CMP appeared unable to penetrate the microsome membrane. These data indicate that the active sites of these enzymes are located on the external surface of microsomal vesicles.It is concluded that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.     

Cholinephosphotransferase and ethanolaminephosphotransferase activities in Plasmodium knowlesi-infected erythrocytes. Their use as parasite-specific markers

CDPcholine: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) activities were investigated in Plasmodium knowlesi-infected erythrocytes obtained from Macaca fascicularis monkeys. Disrupted infected erythrocytes possess a cholinephosphotransferase activity (1.3 +/- 0.2 nmol phosphatidylcholine/10(7) infected cells per h) 1.5-times higher than the ethanolaminephosphotransferase activity. Optimal activities of both enzymes were observed in the presence of 12 mM MnCl2, which was about 3-times as effective as 40 mM MgCl2 as a cofactor. The two activities had similar dependences on pH and thermal inactivation. Their Arrhenius plots show an identical break at 17 degrees C and the corresponding activation energies below and above the critical temperature were similar for the two activities. Sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, beta-D-octylglucoside and lysophosphatidylcholine strongly inhibited the two activities above their critical micellar concentration, but the first three detergents stimulated the activities at lower concentrations. Saponin (0.004-0.5%) either did not affect the two activities or else increased them. Cholinephosphotransferase and ethanolaminephosphotransferase activities had apparent Km values for the CDP ester of 23.4 and 18.6 microM, respectively. CDPcholine and CDPethanolamine competitively inhibited the ethanolaminephosphotransferase and cholinephosphotransferase activities, respectively. The high selectivity of these activities for individual molecular species of diradylglycerol suggests that substrate specificity is responsible for the various molecular species of Plasmodium-infected erythrocyte phospholipids. However, cholinephosphotransferase and ethanolaminephosphotransferase had different dependences on 1,2-dilauroylglycerol and 1-oleylglycerol, which were substrates for cholinephosphotransferase but not for ethanolaminephosphotransferase under our conditions. These data provide the first characterization of an enzyme involved in the intense lipid metabolism in Plasmodium-infected erythrocytes, and the presence of cholinephosphotransferase demonstrates a biosynthesis of phosphatidylcholine by the Kennedy pathway after infection. Our data suggest that cholinephosphotransferase and ethanolaminephosphotransferase activities could be catalyzed by the same enzyme. Furthermore, since host erythrocytes are devoid of these enzymatic activities, cholinephosphotransferase is a parasite-specific membrane-associated enzyme which can be used as a probe or marker.     

The capacity of the sarcoplasmic reticulum for phospholipid synthesis: a developmental study

The activities of three enzymes involved in phospholipid synthesis, sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15), cholinephosphate cytidylyltransferase (EC 2.7.7.15), and cholinephosphotransferase (EC 2.7.8.2), were assayed in adult skeletal muscle. The acyltransferase and cholinephosphotransferase were concentrated in the sarcoplasmic reticulum, where their specific activities were 80 and 33%, respectively, of the specific activity in liver microsomes. Cytidylyltransferase activity was distributed throughout the cell with most of the activity in the cytosol. Its activity in muscle was only 10% of liver activity. Functional sarcoplasmic reticulum was isolated by density gradient centrifugation after calcium loading in the presence of phosphate. The specific activities of these enzymes wee undiminished in the calcium-loaded fraction, suggesting that these enzymes are intrinsic components of the sarcoplasmic reticulum. In developing muscle (2 and 6 days postnatal) acyltransferase and cholinephosphotransferase activities were also present in a calcium-loaded microsomal subfraction at the same level as in the adult. Cytidylyltransferase activity, on the other hand, was 8-fold higher in developing muscle. In addition, developing muscle had a 3-fold increase in the proportion of cytidylyltransferase associated with the microsomal fraction. These data suggest that sarcoplasmic reticulum has the capacity for phospholipid synthesis in mature and developing muscle, and that the rate of phosphatidylcholine synthesis may be regulated by the levels of cytidylyltransferase and by translocation of this enzyme between the sarcoplasmic reticulum and the cytosol.     

Regulation of phosphatidylcholine synthesis in rat liver endoplasmic reticulum.

The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5'-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5'-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation.     

Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities.

Yeast acyl-coenzyme A:dihydroxyacetone-phosphate O-acyltransferase (DHAP acyltransferase; EC 2.3.1.42) was investigated to (i) determine whether its activity and that of acyl-coenzyme A:sn-glycerol-3-phosphate O-acyltransferase (glycerol-P acyltransferase; EC 2.3.1.15) represent dual catalytic functions of a single membranous enzyme, (ii) estimate the relative contributions of the glycerol-P and DHAP pathways for yeast glycerolipid synthesis, and (iii) evaluate the suitability of yeast for future genetic investigations of the eucaryotic glycerol-P and DHAP acyltransferase activities. The membranous DHAP acyltransferase activity showed an apparent Km of 0.79 mM for DHAP, with a Vmax of 5.3 nmol/min per mg, whereas the glycerol-P acyltransferase activity showed an apparent Km of 0.05 mM for glycerol-P, with a Vmax of 3.4 nmol/min per mg. Glycerol-P was a competitive inhibitor (Ki, 0.07 mM) of the DHAP acyltransferase activity, and DHAP was a competitive inhibitor (Ki, 0.91 mM) of the glycerol-P acyltransferase activity. The two acyltransferase activities exhibited marked similarities in their pH dependence, acyl-coenzyme A chain length preference and substrate concentration dependencies, thermolability, and patterns of inactivation by N-ethylmaleimide, trypsin, and detergents. Thus, the data strongly suggest that yeast glycerol-P and DHAP acyltransferase activities represent dual catalytic functions of a single membrane-bound enzyme. Furthermore, since no acyl-DHAP oxidoreductase activity could be detected in yeast membranes, the DHAP pathway for glycerolipid synthesis may not operate in yeast.     

Regulation of selectivity of CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase in rat liver microsomes towards different molecular species of 1,2-diacyl-sn-glycerols.

The suitability of monoenoic, dienoic, tetraenoic, and hexaenoic molecular species of 1,2-diacyl-sn-glycerols as substrates for the CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied in rat liver microsomes. No statistically significant difference in the rates of phosphatidylcholine synthesis with the various diacylglycerols was found at 0.40 mM, although a moderate discrimination against hexaenoic species relative to monoenoic and dienoic species was observed at 0.25 mM. The addition of palmitoyl-CoA (7.5 micron) significantly enhanced cholinephosphotransferase activity when tetraenoic diacylglycerols were added at 0.25 or 0.40 mM. CDPethanolamine at 24.4 micron was found to inhibit the rates of phophatidylcholine biosynthesis by 54 and 39% with hexaenoic and monoenoic 1,2-diacyl-sn-glycerols, respectively, whereas no significant effects were observed in the case of dienoic and tetraenoic species. These latter findings may partially explain why 1-saturated 2-docosahexaenoyl diacylglycerols are used to a greater extent for phosphatidylethanolamine than for phosphatidylcholine synthesis in rat liver in vivo. The present results also suggest that the selectivity of the cholinephosphotransferase for certain molecular species of 1,2-diacyl-sn-glycerols is a function of diacylglycerol concentration and may be mediated under physiological conditions by substrates for enzymes which compete for common diacylglycerol precursors.     

Effects of betamethasone on phospholipid content,composition and biosynthesis in the fetal rabbit lung

Administration of betamethasone (0.2 mg/kg, intramuscularly) to pregnant rabbits had the following effects on the fetal lung at 26–27 days gestation. It increased the amount of phosphatidylcholine in lung lavage by 70% and almost doubled the phosphatidylcholine/sphingomyelin ratio, it increased the rate of incorporation of choline into phosphatidylcholine in fetal lung slices by up to 90%, it increased the activities of pulmonary cholinephosphate cytidylyltransferase and phosphatidate phosphatase by 50% and it reduced the amount of lung glycogen to 60% of the amount in the controls. Betamethasone had no effect on the activities of pulmonary cholinephosphotransferase or lysolecithin: lysolecithin acyltransferase but it sligthly decreased the activity of choline kinase.Betamethasone administration to the doe did not increase the amount of surfactant phospholipid in fetal lung lavage to as great an extent as did direct administration of cortisol to the fetuses. Neither did betamethasone stimulate the activity of pulmonary cholinephosphotransferase. These data suggest that agents other than glucocorticoids mediate the stress-induced acceleration of fetal lung maturation and surfactant production.     

The reverse reaction of cholinephosphotransferase in rat brain microsomes. A new pathway for degradation of phosphatidylcholine

The synthesis of phosphatidylcholine is catalyzed by cholinephosphotransferase (EC 2.7.8.2) which is known to be reversible in liver. The reversibility of cholinephosphotransferase in rat brain in demonstrated in this paper. Labeled microsomes were prepared from young rats which had been given an intracerebral injection of labeled choline or oleate 2 h before killing. During incubation of choline-labeled microsomes with CMP, label was lost from ;choline glycerophospholipids and labeled CDPcholine was produced. The Km for CMP was 0.35 mM and V was 3.3 nmol/min per mg protein. Neither AMP nor UMP could substitute for CMP. Oleate-labeled microsomes were pretreated with e mM diisopropylfluorophosphate (lipase inhibitor). During incubation with CMP, label was lost from choline, and ethanolamine glycerophospholipid and labeled diacylglycerols were produced. When the lipase was not inhibited, labeled oleate was produced. We propose that a principal pathway for degradation of phosphatidylcholine, particularly during brain ischemia, is by reversal of cholinephosphotransferase, followed by hydrolysis of diacylglycerols by the lipase.     

Lysolecithin as regulator of de novo lecithin synthesis in rat liver microsomes.

1-Acyl-sn-glycero-3-phosphocholine (lysolecithin) was found to affect 1,2-diacyl-sn-glycerol:CDPcholine cholinephosphotransferase (CPT; EC 2.7.8.2) activity of rat liver microsomes in a concentration dependent, characteristic manner. Cholinephosphate transfer was activated at lysolecithin concentrations below 0.5 mM with a maximum stimulation occurring at 75–100 μM lysolecithin levels. At concentrations above 0.5 mM, CPT activity was inhibited by lysolecithin. It was shown that CPT inhibition by lysolecithin is competitive (Ki ≈ 0.6 mM) with respect to CDPcholine. The possible role of lysolecithin as regulator of de novo lecithin synthesis in vivo is outlined.     

Role of lamellar bodies in the biosynthesis of phosphatidylcholine in mouse lung.

1. A lamellar body-enriched fraction was isolated from whole lung homogenates of mouse lung and its contamination with microsomes, mitochondria, and cytosol protein assessed by marker enzyme analyses. 2. By measuring the activity of cholinephosphotransferase (EC 2.7.8.2) in varying amounts of microsomes in the presence and absence of a fixed quantity of lamellar bodies, it could be demonstrated unequivocally that lamellar bodies of mouse lung lack the capacity to synthesize phosphatidylcholine de novo. 3. A similar approach allowed the conclusion that lamellar bodies of mouse lung do not contain lysophosphatidylcholine acyltransferase (EC 2.3.1.23) and lysophosphatidylcholine:lysophosphatidylcholine acyltransferase (EC 2.3.1.--), enzymes which play a putative role in the formation of pulmonary 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine. The activities of these enzymes observed in lamellar body fractions could be attributed completely to contaminating microsomes and cytosol respectively. 4. Lamellar bodies contributed to the activity of microsomal lysophosphatidylcholine acyltransferase by a cooperative effect. The possible role of this cooperation in the biosynthesis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine is discussed.     

Phosphatidylcholine biosynthesis and choline transport in the anaerobic protozoon Entodinium caudatum.

Choline accumulation and phosphatidylcholine biosynthesis were investigated in the choline-requiring anaerobic protozoon Entodinium caudatum by incubating whole cells or subcellular fractions with [14C] choline, phosphoryl [14C] choline and CDP-[14C] choline. 2. All membrane fractions contained choline kinase (EC 2.7.1.32) and CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2), although the specific activities were less in the cell-envelope fraction. Choline phosphate cytidylyltransferase (EC 2.7.7.15) was limited to the supernatant, and this enzyme was rate-limiting for phosphatidylcholine synthesis in the whole cell. 3. Synthesis of phosphatidylcholine from free choline by membranes was only possible in the presence of supernatant. Such reconstituted systems required ATP (2.5 mM), CTP (1 mM) and Mg2+ (5 mM) for maximum synthesis of the phospholipid. CTP and Mg2+ were absolute requirements. 4. Hemicholinium-3 prevented choline uptake by the cells and was strongly inhibitory towards choline kinase; the other enzymes involved in phosphatidylcholine synthesis were minimally affected. 5. Ca2+ ions (0.5 mM) substantially inhibited CDP-choline-1,2-diacylglycerol cholinephosphotransferase in the presence of 15 mM-Mg2+, but choline phosphate cytidylyltransferase and choline kinase were less affected. 6. No free choline could be detected intact cells even after short (10-180s) incubations or at temperatures down to 10 degrees C. The [14C] choline entering was mainly present as phosphorylcholine and to a lesser extent as phosphatidylcholine. 7. It is suggested that choline kinase effectively traps any choline within the cell, thus ensuring a supply of the base for future growth. At low choline concentrations the activity of choline kinase is rate-limiting for choline uptake, and the enzyme might possibly play an active role in the transport phenomenon. Thus the choline uptake by intact cells and choline kinase have similar Km values and show similar responses to temperature and hemicholinium-3.     

Phospholipid synthesis in isolated fat cells. Studies of microsomal diacylglycerol cholinephosphotransferase and diacylglycerol ethanolaminephosphotransferase activities.

Diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) activities were investigated in microsomes from isolated rat fat cells. Assays based on the conversion of CDP-[14C]choline of CDP-[14C]ethanolamine to phosphatidylcholine or phosphatidylethanolamine utilized ethanol-dispersed diacylglycerols and 1 to 5 microng of protein. Cholinephosphotransferase and ethanolaminephosphotransferase activities had similar dependences on MgCl2 and pH, and were inhibited similarly by CaCl2, organic solvents, Triton X-100, Tween 20, and dithiothreitol. Ethylene glycol bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid stimulated both activities similarly. With 1,2-dioleoyl-sn-glycerol, the cholinephosphotransferase activity had an apparent Km for CDP-choline of 23.9 micronM and a V max of 8.54 nmol/min/mg. CDP-ethanolamine and CDP were competitive inhibitors of the cholinephosphotransferase activity (apparent Kl values of 227 micronM and 360 micronM, respectively). With 1,2-dioleoyl-sn-glycerol, the ethanolaminephosphotransferase activity had an apparent Km of 18.3 micronM for CDP-ethanolamine and a V max of 1.14 nmol/min/mg. CDP-choline appeared to be a noncompetitive inhibitor of the ethanolaminephosphotransferase activity (apparent Kl of 1620 micronM). Inhibition of the ethanolaminephosphotransferase activity by CDP appeared to be of a mixed type. The dependences on diacylglycerols containing fatty acids 6 to 18 carbons in length were investigated...     

The supply of both CDP-choline and diacylglycerol can regulate the rate of phosphatidylcholine synthesis in HeLa cells

The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was measured in HeLa cells permeabilized with 0.125 mg digitonin/mL. The rate of phosphatidylcholine formation was influenced by the concentration of CDP-choline in the medium. The CDP-choline:1,2-diacylglycerol cholinephosphotransferase in permeabilized cells showed a Km of 88 microM for CDP-choline. A similar Km value of 104 microM was found for cholinephosphotransferase in microsomes isolated from HeLa cells when assayed in the presence of 2.4 mM dioleoylglycerol. In the absence of added diacylglycerol, the Km for CDP-choline for the microsomal cholinephosphotransferase was only 38 microM. The incorporation of [methyl-14C]CDP-choline into phosphatidylcholine was stimulated by the supply of diacylglycerol in both HeLa cells and isolated microsomes. A 2.4 mM dioleoylglycerol suspension increased cholinephosphotransferase activity fourfold in microsomes. The digitonin-treated cells were impermeable to the dioleoylglycerol suspension. Incubation of permeabilized cells with 150 microM acyl-CoA and 0.8 mM glycero-3-phosphate tripled cellular diacylglycerol levels, causing a doubling in the rate of phosphatidylcholine synthesis. A similar incubation of microsomes with acyl-CoA stimulated phosphatidylcholine synthesis twofold. Furthermore, incubation of microsomes with [3H]diacylglycerol and [14C]CDP-choline showed that both of the substrates were incorporated into phosphatidylcholine at the same rate. This result suggests that the stimulatory effects on cholinephosphotransferase arise from increases in the availability of substrates rather than activation of the enzyme. These results suggest that both in the permeabilized cells and in isolated membranes, the biosynthesis of phosphatidylcholine can be limited by both CDP-choline and diacylglycerol.     

Cholinephosphotransferase in rat lung. In vitro formation of dipalmitoylphosphatidylcholine and general lack of selectivity using endogenously generated diacylglycerol

Diacylglycerol was generated in vitro in rat lung microsomes by forming phosphatidic acid via sn-glycerol-3-phosphate acyltransferase followed by the hydrolysis of the phosphatidic acid by phosphatidate phosphohydrolase. Diacylglycerol concentrations of 35 to 50 nmol/mg of microsomal protein were obtained. Cholinephosphotransferase activity was determined in microsomes by measuring the conversion of endogenously generated [14C]diacylglycerol to phosphatidylcholine. Reaction rates of 14 to 16 nmol/min/mg of protein were obtained with a 30-s reaction. Diacylglycerol which was primarily dipalmitoylglycerol was produced when palmitic acid was used in the sn-glycerol-3-phosphate acyltransferase reactions. Dipalmitoylphosphatidylcholine was formed via cholinephosphotransferase from the dipalmitoylglycerol with an apparent maximal velocity of 20 nmol/min/mg of protein. When oleic acid was used instead of palmitic acid, the apparent maximal velocity for cholinephosphotransferase was 26 nmol/min/mg of protein. The apparent Km values for the two different diacylglycerol substrates were the same (28.5 nmol/mg of protein). Diacylglycerols, with different molecular species composition, were generated using a variety of fatty acids and fatty acid mixtures. The phosphatidylcholine formed from these diacylglycerols had the same molecular species profiles as the diacylglycerol used as the substrate. The relative reaction rates with the different diacylglycerols were essentially the same except when 20:4 and 22:6 fatty acids were used individually, in which case the rates were lower. We conclude that cholinephosphotransferase readily forms dipalmitoylphosphatidylcholine from endogenously generated dipalmitoylglycerol and that the cholinephosphotransferase reaction is generally nonselective for the diacylglycerol substrate.     

Activity of cholinephosphotransferase, lysolecithin: lysolecithin acyltransferase and lysolecithin acyltransferase in the developing mouse lung.

1. The present study presents the activity profiles of cholinephosphotransferase, lysolecithin:lysolecithin acyltransferase and lysolecithin acyltransferase at different stages of development of the mouse lung. 2. The specific activity of cholinephosphotransferase, a key enzyme in the de novo synthesis of phosphatidylcholine, increases during the later stages of fetal development until it reaches a maximal value at a gestational age of 17 days, i.e. 2 days before term. Thereafter, the activity of the enzyme declines again until around term. 2. The specific activity of lysolecithin:lysolecithin acyltransferase which catalyzes the transesterification between two molecules of 1-acyl-sn-glycero-3-phosphocholine, appears to be much lower than that of cholinephosphotransferase at gestational ages below 18 days. However, around day 18, the specific activity of lysolecithin:lysolecithin acyltransferase increases dramatically until it almost equals the maximal activity of cholinephosphotransferase measured on day 17. 4. The specific activity of lysolecithin acyltransferase, which catalyzes the direct acylation of 1-acyl-sn-glycero-3-phosphocholine, does not change significantly during the prenatal development and is lower than that of either lysolecithin:lysolecithin acyltransferase or cholinephosphotransferase at all stages of development. 5. These results are discussed in view of the possible role of these enzymes in the biosynthesis of pulmonary 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.