Douglas A. Feldman

EVIDENCE FOR A REGULATORY ROLE OF CTP : CHOLINE PHOSPHATE CYTIDYLYLTRANSFERASE IN THE SYNTHESIS OF PHOSPHATIDYLCHOLINE IN FETAL LUNG FOLLOWING PREMATURE BIRTH

The sequence of reactions which function to incorporate choline into phosphatidylcholine was investigated in lung from fetuses following premature delivery. The rate of [methyl-14C]choline incorporation by rat lung slices into phosphatidylcholine increases following premature delivery at both 20 and 21 days gestation. The increase in choline incorporation is primarily due to an increased specific activity of phosphorylcholine resulting from a decreased pool size of phosphorylcholine. The decrease in the concentration of phosphorylcholine following premature delivery is apparently caused by an increased activity of cytidylyltransferase which leads to an increase in the conversion of phosphorylcholine to phosphatidylcholine. The total activity of choline kinase, cytidylyltransferase, cholinephosphotransferase and phosphatidate phosphohydrolase did not change significantly. However, the cytidylyltransferase activity in the microsome fraction increased following premature delivery at 20 and 21 days gestation. The amount of cytidylyltransferase in the H form in the cytosol fraction increased following premature delivery at 21 days gestation but not at 20 days gestation. The results are interpreted to indicate that the active form of cytidylyltransferase in lung cells is the membrane-bound enzyme and this form increases following birth resulting in an increased synthesis of phosphatidylcholine.

Characterization of cytosolic forms of CTP: choline-phosphate cytidylyltransferase in lung, isolated alveolar type II cells, A549 cell and Hep G2 cells

The subcellular forms of cytidylyltransferase (EC 2.7.7.15) in rat lung, rat liver, Hep G2 cells, A549 cells and alveolar Type II cells from adult rats were separated by glycerol density centrifugation. Cytosol prepared from lung, Hep G2 cells, A549 cells and alveolar Type II cells contained two forms of the enzyme. These species were identical to the L-Form and H-Form isolated previously from lung cytosol by gel filtration. Liver cytosol contained only the L-Form. Rapid treatment of Hep G2 cells with digitonin released all of the cytoplasmic cytidylyltransferase activity. The released activity was present in both H-Form and L-Form. The molecular weight of L-Form was determined from sedimentation coefficients and Stokes radius values to be 97,690 +/- 10,175. Thus, the L-Form appears to be a dimer of the Mr 45,000 catalytic subunit. The f/f degrees value of 1.5 indicated that the protein molecule has an axial ratio of 10, assuming a prolate ellipsoid shape. The estimated molecular weight of the H-Form was 284,000 +/- 25,000. The H-Form was dissociated into L-Form by incubation of cytosol at 37 degrees C. Triton X-100 (0.1%) and chlorpromazine (1.0 mM) also dissociated the H-Form into L-Form. Western blot analysis indicated that both forms contained the catalytic subunit. An increase in Mr 45,000 subunit coincided with the increase in cytidylyltransferase activity in L-Form, which resulted from the dissociated of H-Form. The L-Form was dependent on phospholipid for activity. The H-Form was active without lipid. Phosphatidylinositol was present in the H-Form isolated from Hep G2 cells. The phosphatidylinositol dispersed when the H-Form was dissociated into L-Form. Phosphatidylinositol and phosphatidylglycerol cause L-Form to aggregate into a form similar to H-Form. Phosphatidylcholine/oleic acid (1:1 molar ratio) and oleic acid also aggregated the L-Form. Phosphatidylcholine did not produce aggregation. We conclude that the H-Form is the active form of cytidylyltransferase in cytoplasm. The H-Form appears to be a lipoprotein consisting of an apoprotein (L-Form dimer of the Mr 45,000 subunit) complexed with lipids. A change in the relative distribution of H-Form and L-Form in cytosol would alter the cellular activity and thus may be important in the regulation of phosphatidylcholine synthesis.

Activation of CTP : Phosphocholine cytidylyltransferase in rat lung by fatty acids

CTP : phosphocholine cytidylyltransferase activity exists in both the microsome and cytosol fractions of adult lung, 36 and 59%, respectively. Although these enzyme activities are stimulated in vitro by added lipid activators (i.e. phosphatidylglycerol), there are significant levels of activity in the absence of added lipid. We have removed endogenous lipid material from microsome and cytosol preparations of rat lung by rapid extraction with isopropyl ether. The extraction procedure did not cause any loss of cytidylyltransferase activity in the cytosol. After the extraction the enzyme was almost completely dependent upon added lipid activator. Isopropyl ether extraction of microsome preparations produced a loss of 40% of the cytidylyltransferase activity, when measured in the presence of added phosphatidylglycerol. Lipid material extracted into isopropyl ether restored the cytidylyltransferase activity in cytosol. The predominant species of enzyme activator in the isopropyl ether extracts was fatty acid. A variety of naturally occurring unsaturated fatty acids stimulated the cytidylyltransferase to the same extent as phosphatidylglycerol. Saturated fatty acids were inactive.

COMPARISON OF THE PHOSPHOLIPID REQUIREMENTS AND MOLECULAR FORM OF CTP : PHOSPHOCHOLINE CYTIDYLYLTRANSFERASE FROM RAT LUNG, KIDNEY, BRAIN AND LIVER

The cytidylyltransferase activity in fresh cytosol from different tissues of the rat was measured in the absence and presence of phosphatidylglycerol. In all cases addition of this lipid produced large increases in enzyme activity. Agarose gel (A-5.0) filtration profiles of the enzyme activities indicated that the L-form of the enzyme (190 000 molecular weight) predominated in liver, brain, kidney, and fetal lung. However, adult lung cytosol contained 70--80% of the activity in the H-form (molecular weight greater than or equal to 5 x 10(6)). Removal of phospholipid material from the alveolar spaces by lavage produced a significant reduction of the H-form of the enzyme in the cytosol fraction. The L-form of the cytidylyltransferases from fetal lung and adult liver, kidney, and brain all possess the same specificities for activation by phospholipids in vitro. In all cases, phosphatidylglycerol was the most potent activator at 0.2 mM. Lysophosphatidylethanolamine stimulated enzyme activity, whereas lysophosphatidylglycerol was a potent inhibitor. These studies implicate the role of acidic phospholipids in the regulation of cytidylyltransferase activity in vivo and the existence of a common L-form of the enzyme in serveral tissues of the rat.

[29] Choline-phosphate cytidylyltransferase

Publisher Summary Choline-phosphate cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase) catalyzes a major rate-determining step in the biosynthesis of phosphatidylcholine in mammalian cells. Both cytosolic and membrane fractions contain choline-phosphate cytidylyltransferase activity. Enzyme activity is determined by measuring the formation of radioactive CDPcholine from phospho[methyl- 14 C]choline. Two methods have been used to separate CDPcholine from phosphocholine: adsorption of CDPcholine by charcoal or separation of CDPcholine from phosphocholine by thin-layer chromatography. The charcoal adsorption method is rapid, sensitive, and reproducible. The ability to perform many assays (50–100) per day is an additional advantage. A variety of lipids have been used by investigators for the assay of cytidylyltransferase. It has been a common practice to include lipid in assays involving soluble forms of cytidylyltransferase. The molecular and kinetic properties of cytidylyltransferase are discussed this chapter.

Dexamethasone increases the activity but not the amount of choline-phosphate cytidylyltransferase in fetal rat lung.

The activity of choline-phosphate cytidylyltransferase is increased by glucocorticoids in late gestation fetal lung in association with increased phosphatidylcholine biosynthesis. Previous indirect data had suggested that the stimulatory effect of the hormone was due to activation of existing enzyme rather than synthesis of new cytidylyltransferase protein. Using a rabbit antibody raised against purified rat liver choline-phosphate cytidylyltransferase, we have now quantitated the amount of the enzyme in fetal rat lung explants cultured with and without dexamethasone. Our results show that the hormone increased the activity of the enzyme but not the amount of cytidylyltransferase protein. Thus the stimulatory effect of dexamethasone on cytidylyltransferase is due to activation of existing enzyme rather than induction of enzyme synthesis.

Regulation of CTP : phosphocholine cytidylyltransferase in HepG2 cells: effect of choline depletion on phosphorylation, translocation and phosphatidylcholine levels

We studied the effect of choline depletion on the biosynthesis of phosphatidylcholine (PC) and the distribution and phosphorylation of cytidylyltransferase (CT) in HepG2 cells. Phosphocholine concentrations decreased within 24 h of choline depletion to values less than 2% of controls. The incorporation of [3H]glycerol into PC was reduced in choline-depleted (CD) cells. The apparent turnover of PC was similar in CD and choline-supplemented (CS) cells (T1/2 = 20 h). The methylation pathway for PC synthesis increased nearly 10-fold in CD cells. Cell growth was similar in CD and CS cells. Over 95% of CT activity in CS cells was in the soluble pool. Choline depletion resulted in a progressive decrease in CT activity and immunodetected enzyme in the soluble pool and a corresponding increase in membrane CT over a 48-h period. Choline supplementation of CD cells caused a rapid release of membrane CT (complete release by 3 h). Two phosphorylated forms of CT were identified. One form contained a higher level of phosphorylation (HPCT) than the other form (LPCT). HPCT migrated slightly slower than LPCT on SDS gels. CD cells contained only LPCT in both soluble and membrane pools. CS cells contained only HPCT. During choline depletion PC content decreased nearly 20% but CT binding did not occur until LPCT was generated in cytosol. Conversely, choline supplementation released LPCT into cytosol and HPCT was formed only after the release. We conclude that both the induction of binding sites, perhaps by depletion of PC and dephosphorylation of HPCT to LPCT, are required for CT translocation to membranes. The release of CT from membranes is initiated by changes in membrane binding sites followed by trapping of the CT in the soluble pool by phosphorylation of LPCT to HPCT.

Calcium binding properties of rat heart plasma membrane and inhibition by structural analogues of dl-propranolol.

The isolation and characterization of a plasma membrane preparation from rat heart is described. Enzymatic, chemical, and electron microscopic analysis revealed a relative lack of contami- nation with nuclear, mitochondrial, ribosomal, and sarcoplasmic reticulum membrane. One calcium binding site (I& = 265 pM, B,,, = 65 nmoIes/mg protein) was detected by equilibrium diaiysis. Mono- valent metal ions exhibited ItSlOO-fold less inhibition potency than divalent metal ions when analyzed by competitive inhibition of calcium binding. The range of K, values found for divalent metal ions was similar to the K, value for calcium. La+3 produced a potent non-competitive inhibition, A large variety of structural analogues of d,l-propranolol, many of which have been shown to lack B-adrenergic blocking activity, were competitive inhibitors of the calcium binding activity, with Ki values ranging from 40-W pM. Electrophilic, hydrophobic, and diamino substituents greatly increased the inhibitory activity. There was no significant difference between related tertiary and quaternary amines. The experi- mental antiarrhythmic agent UM 272 had the least ability to inhibit calcium binding to the cardiac plasma membrane preparation (Ki = 795 PM). However, UM 424, another experimental antiarrhythmic agent, had an inhibitory activity similar to df-propranolol (Ki = 115pM and 108pM, respectively),

Microsomal CTP: choline phosphate cytidylyltransferase: kinetic mechanism of fatty acid stimulation

Fatty acids are known to cause an increase in the incorporation of radioactive choline into phosphatidylcholine. A coincident increase in membrane cytidylyltransferase activity is well documented. The purpose of the present studies was to determine the direct effects of oleic acid on the kinetic properties of membrane cytidylyltransferase. An examination of the reaction characteristics of membrane cytidylyltransferase revealed that membranes from adult rat lung contained high CTPase activity. This activity prevented the determination of reaction velocities at low CTP concentrations. The CTPase activity was blocked by the addition of ADP or ATP to the reaction. The addition of 6.0 mM ADP to the assay mixture enabled us to determine the effect of oleate on the CTP Km. Oleate (122 microM) caused a significant decrease in CTP Km for microsomal cytidylyltransferase (0.99 mM to 0.33 mM) and H-Form cytidylyltransferase (1.04 mM to 0.27 mM). Oleate did not decrease the CTP Km for L-Form cytidylyltransferase. Oleate had no effect on the choline phosphate Km in microsomal, H-Form or L-Form cytidylyltransferase. Oleate also increased the Vmax for cytidylyltransferase. The increase was dependent upon the concentration of oleate with a maximal increase of 50-60% at 100-130 microM oleate. We conclude that oleate has a direct stimulatory effect on cytidylyltransferase when it is in the active form (membrane bound or H-Form lipoprotein complex). We suggest that the kinetic effects operate synergistically with other regulatory mechanisms such as translocation or conversion of inactive to active species. The direct effect of oleate on the cytidylyltransferase may be an important regulatory mechanism when CTP concentrations are limiting.

Cytidylyltransferase-binding Protein Is Identical to Transcytosis-associated Protein (TAP/p115) and Enhances the Lipid Activation of Cytidylyltransferase

We previously identified a protein from rat liver that binds CTP:phosphocholine cytidylyltransferase (CT). We have now purified this protein (cytidylyltransferase-binding protein (CTBP)) from rat liver. The purification involved precipitation at pH 5 and extraction of the precipitate with buffer, followed by sequential chromatography on DEAE-Sepharose and butyl-agarose. Final purification was accomplished by either preparative electrophoresis or hydroxylapatite chromatography. Amino acid sequences from six peptides derived from pure CTBP matched sequences in transcytosis-associated protein (TAP) with 98% identity. Thus, CTBP was positively identified to be TAP. Purified CTBP increased the activity of purified CT measured with phosphatidylcholine (PC)/oleic acid. In the absence of PC/oleic acid, CTBP did not stimulate CT activity. Dilution of CT to reduce the Triton X-100 concentration produced a loss of CT activity. The lost activity was recovered by the addition of CTBP plus PC/oleic acid to the assay, but not by the addition of either PC/oleic acid or CTBP alone. Removal of CTBP from purified preparations by immunoprecipitation with CTBP antibodies eliminated the activation of CT. Both CT and CTBP were shown to bind to PC/oleic acid liposomes. The formation of complexes between CT and CTBP in the absence of PC/oleic acid liposomes could not be demonstrated. These results suggest that CTBP functions to modify the interaction of CT with PC/oleic acid liposomes, resulting in an increase in the catalytic activity perhaps by the formation of a ternary complex between CT, CTBP, and lipid. Overall, these results suggest that CTBP (TAP) may function to coordinate the biosynthesis of phosphatidylcholine with vesicle transport.