Inhibition of phosphatidylcholine synthesis by vasopressin and angiotensin in rat hepatocytes.

The addition of 1 microM-vasopressin or -angiotensin to isolated rat hepatocytes induced a fast transient inhibition of the rate of incorporation of [Me-3H]choline into phosphatidylcholine. The cationophore A23187 induced a similar inhibition of phosphatidylcholine synthesis. The addition of micromolar Ca2+ to rat liver microsomes inhibited the activity of CDP-choline: 1,2-diacylglycerol cholinephosphotransferase. This inhibition is due a decrease in the Vmax. of the enzyme without affecting the Km for CDP-choline. It is concluded that Ca2+ regulates phosphatidylcholine synthesis in rat liver.     

Isolation and characterization of a yeast mutant defective in cholinephosphotransferase

A yeast mutant defective in cholinephosphotransferase (cpt) was isolated as a revertant from a choline-sensitive mutant, which exhibited lowered phosphatidylinositol synthesis. A block at the cholinephosphotransferase step in the mutant was indicated by the enzyme defect and the accumulation of CDP-choline in the cells with a decrease in phosphatidylcholine synthesis. The defect was due to a single recessive mutation in a nuclear gene. The residual activity in the mutant showed an increased apparent Km for CDP-choline and an altered sensitivity to Tween 20. Thus the structural gene may be affected in the mutant. The occurrence of an intact ethanolaminephosphotransferase in the mutant indicates the distinctness of the genes encoding cholinephosphotransferase and ethanolaminephosphotransferase in yeast. The present selection method was also effective for isolating mutants defective in the other steps of the CDP-choline pathway and choline transport.     

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.     

Maternal administration of dexamethasone stimulates choline-phosphate cytidylyltransferase in fetal type II cells.

Administration of dexamethasone to pregnant rats at 19 days gestation increased phosphatidylcholine synthesis (45%) from radioactive choline in type II cells. This enhanced synthesis of phosphatidylcholine was accompanied by an increased conversion of choline phosphate into CDP-choline. Similar results were obtained by incubating organotypic cultures of 19-day-fetal rat lung with cortisol. The increased conversion of choline phosphate into CDP-choline correlated with an enhanced choline-phosphate cytidylyltransferase activity (31% after dexamethasone treatment; 47% after cortisol exposure) in the cell homogenates. A similar increase (26% after dexamethasone treatment; 39% after cortisol exposure) was found in the microsomal-associated enzyme. No differences in cytosolic enzyme activity were observed. The specific activity of the microsomal enzyme was 3-4 times that of the cytosolic enzyme. Most of the enzyme activity was located in the microsomal fraction (58-65%). The treatments had no effect on the total amount of enzyme recovered from the cell homogenates. These results, taken collectively, are interpreted to indicate that the active form of cytidylyltransferase in type II cells is the membrane-bound enzyme and that cytidylyltransferase activation in type II cells from fetal rat lung after maternal glucocorticoid administration occurs by binding of inactive cytosolic enzyme to endoplasmic reticulum.     

Hepatic mitochondrial inner membrane properties and carnitine palmitoyltransferase A and B. Effect of diabetes and starvation.

Intact mitochondria and inverted submitochondrial vesicles were prepared from the liver of fed, starved (48 h) and streptozotocin-diabetic rats in order to characterize carnitine palmitoyltransferase kinetics and malonyl-CoA sensitivity in situ. In intact mitochondria, both starved and diabetic rats exhibited increased Vmax., increased Km for palmitoyl-CoA, and decreased sensitivity to malonyl-CoA inhibition. Inverted submitochondrial vesicles also showed increased Vmax. with starvation and diabetes, with no change in Km for either palmitoyl-CoA or carnitine. Inverted vesicles were uniformly less sensitive to malonyl-CoA regardless of treatment, and diabetes resulted in a further decrease in sensitivity. In part, differences in the response of carnitine palmitoyltransferase to starvation and diabetes may reside in differences in the membrane environment, as observed with Arrhenius plots, and the relation of enzyme activity and membrane fluidity. In all cases, whether rats were fed, starved or diabetic, and whether intact or inverted vesicles were examined, increasing membrane fluidity was associated with increasing activity. Malonyl-CoA was found to produce a decrease in intact mitochondrial membrane fluidity in the fed state, particularly at pH 7.0 or less. No effect was observed in intact mitochondria from starved or diabetic rats, or in inverted vesicles from any of the treatment groups. Through its effect on membrane fluidity, malonyl-CoA could regulate carnitine palmitoyltransferase activity on both surfaces of the inner membrane through an interaction with only the outer surface.     

Effect of Glucose Starvation on Glucose Transport in Neuronal Cells in Primary Culture from Rat Brain

The regulation of glucose transport into cultured brain cells during glucose starvation was studied. On glucose deprivation for 40 h, 2-deoxy-D-glucose (2-DG) uptake was stimulated twofold in neuronal cells but was not changed significantly in astrocytes. On refeeding, the increased activity of neuronal cells rapidly returned to the basal level, an observation indicating that the effect of glucose starvation was reversible. The increase was due solely to change in the Vmax, a finding suggesting that the number of glucose transporters on the plasma membrane is increased in starved cells. Cycloheximide inhibited this increase. In the presence of cycloheximide, the activity of 2-DG uptake of starved cells remained constant for 12 h and then slowly decreased, whereas that of fed cells decreased rapidly. These findings suggest that glucose starvation regulates glucose transport by changing the rate of net synthesis of the transporter in neuronal cells in culture.     

Regulation of CTP: phosphocholine cytidylyltransferase activity in type II pneumonocytes.

Phosphatidylcholine synthesis by rat type II pneumonocytes was altered either by depleting the cells of choline or by exposing the cells to extracellular lung surfactant. Effects of these experimental treatments on the activity of a regulatory enzyme, CTP:phosphocholine cytidylyltransferase, were investigated. Although choline depletion of type II pneumonocytes resulted in inhibition of phosphatidylcholine synthesis, cytidylyltransferase activity (measured in cell homogenates in either the absence or presence of added lipids) was greatly increased. Activation of cytidylyltransferase in choline-depleted cells was rapid and specific, and was quickly and completely reversed when choline-depleted cells were exposed to choline (but not ethanolamine). Choline-dependent changes in enzymic activity were apparently not a result of direct actions of choline on cytidylyltransferase and they were largely unaffected by cyclic AMP analogues, oleic acid, linoleic acid or cycloheximide. The Km value of cytidylyltransferase for CTP (but not phosphocholine) was lower in choline-depleted cells than in choline-repleted cells. Subcellular redistribution of cytidylyltransferase also was associated with activation of the enzyme in choline-depleted cells. When measured in the presence of added lipids, 66.5 +/- 5.0% of recovered cytidylyltransferase activity was particulate in choline-depleted cells but only 34.1 +/- 4.5% was particulate in choline-repleted cells. An increase in particulate cytidylyltransferase also occurred in type II pneumonocytes that were exposed to extracellular surfactant. This latter subcellular redistribution, however, was not accompanied by a change in cytidylyltransferase activity even though incorporation of [3H]choline into phosphatidylcholine was inhibited by approx. 50%. Subcellular redistribution of cytidylyltransferase, therefore, is associated with changes in enzymic activity under some conditions, but can also occur without a resultant alteration in enzymic activity.     

Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis. Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum

After a 3-h incubation of Krebs II ascitic cells in the presence of phospholipase C from Clostridium welchii under nonlytic conditions, the incorporation of [3H] choline into phosphatidylcholine was increased 1.7-fold as compared to untreated cells. The total amounts of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were unchanged up to 3 h of incubation. The limiting step in phosphatidylcholine biosynthesis was the formation of CDP-choline catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) as monitored by the decrease in phosphocholine labeling following phospholipase C treatment of cells prelabeled with [3H]choline. The specific activity of homogenate cytidylyltransferase was increased about 1.6-fold in phospholipase C-treated cells. Specific activity of the membrane fraction was increased 2-fold, whereas cytosolic specific activity decreased in phospholipase C-treated cells. The activation of cytidylyltransferase was concomitant with translocation of the enzyme from the cytosol to the membrane fraction. The latter was further fractionated using a Percoll gradient that allowed an efficient separation between endoplasmic reticulum and other subcellular membranes. In control cells, particulate cytidylyltransferase activity co-migrated with the endoplasmic reticulum and ribosome markers and not with the plasma membrane. Also, in treated cells, the stimulation of cytidylyltransferase activity occurred at the endoplasmic reticulum level and did not involve either the external cell membrane or other cellular organelles including the Golgi apparatus, lysosomes, or mitochondria. Thus, our results demonstrate that a stimulus acting on the plasma membrane promotes the translocation of the soluble form of cytidylyltransferase specifically to the endoplasmic reticulum.     

Altered phosphatidylcholine metabolism in C3H10T1/2 cells transfected with the Harvey-ras oncogene

The effect of expression of the Harvey-ras oncogene on phosphatidylcholine metabolism in C3H10T1/2 mouse fibroblast cells was examined. There were multiple changes in the CDP-choline pathway for phosphatidylcholine biosynthesis in the ras-expressing cells. The activity of the first enzyme in the pathway, choline kinase, was stimulated 1.9-fold, while the activity of the second enzyme, CTP:phosphocholine cytidylyltransferase, was decreased by one-half. High levels of intracellular phosphocholine measured in the ras cells were consistent with the altered activities of choline kinase and cytidylyltransferase. The overall rate of phosphatidylcholine synthesis appeared to be increased because the turnover rate of phosphocholine from the intracellular pool was higher in the ras-transfected cells. There also appeared to be an increased rate of phosphatidylcholine degradation in ras-expressing C3H10T1/2 cells. Very high levels of glycerophosphocholine (6-fold increased over control cells) suggested that phospholipase A was activated in these cells. These results indicate that the ras oncogene product directly or indirectly causes an increased turnover of phosphatidylcholine in C3H10T1/2 cells.     

Properties of particulate and solubilized phosphatidylserine synthase activity from Saccharomyces cerevisiae. Inhibitory effect of choline in the growth medium

When radiolabeled serine is incubated with a particulate fraction from Saccharomyces cerevisiae, radioactivity is incorporated initially into phosphatidylserine and gradually appears in phosphatidylethanolamine. Because decarboxylation of phosphatidylserine is blocked by hydroxylamine, phosphatidylserine synthase can be assayed separately. The yeast phosphatidylserine synthase activity 1) exhibits a divalent cation requirement; 2) is stimulated by exogenous CDP-diolein (apparent Km = 0.17 mM); 3) has an apparent Km = 4 mM for L-serine; 4) has a neutral pH optimum; 5) is inhibited by p-hydroxymercuribenzoate; and 6) is reversible in the presence of 5'-CMP, but not 2'-CMP, 3'-CMP, or 5'-AMP. The phospholipid-synthesizing activity is solubilized with Triton X-100 and the enzymatic parameters have been compared with the particulate form of the enzyme. Detergent extracts catalyze the conversion of exogenous purified [31P]CDP-diglyceride to [32P]phosphatidylserine in the presence of Mn2+ and L-serine. Enzyme preparations from cells grown in the presence of choline, that have reduced phospholipid methylation activity (Waechter, C. J., Steiner, M. R., and Lester, R. L. (1969) J. Biol. Chem. 244, 3419-3422), also have substantially less phosphatidylserine synthase activity compared to identical preparations grown in the absence of choline. When choline, phosphocholine, CDP-choline, and phosphatidylcholine are present in vitro, there is no direct inhibitory effect on phosphatidylserine synthase activity. While the inclusion of choline in the growth medium caused a significant reduction in phosphatidylserine synthase activity, it did not appreciably effect the apparent Km values for L-serine and CDP-diglyceride. These results are consistent with choline-grown cells containing less phosphatidylserine synthase activity because of lower amounts of enzyme present or perhaps less active enzyme due to covalent modification.     

Avian fatty acid synthetase: Evidence for short-term regulation of activity

Liver biopsies were performed on starved chicks at 0 and 4 h after refeeding a fat-free diet. Fatty acid synthetase activity increased after refeeding, and administration of cycloheximide did not prevent the rise of enzyme activity. Incorporation of [carboxyl-¹⁴C]leucine into fatty acid synthetase was measured in enzyme purified from the livers of starved chicks, starved-refed (4 h) chicks, and starved-refed chicks injected with cycloheximide. The data suggest that the synthesis of enzyme protein was inhibited in starved and cycloheximide-treated refed chicks in comparison with refed chicks. Liver cytosol from fed or starved chicks was filtered through centrifuge ultrafiltration membranes and the residues were suspended in the same or opposite filtrates. Fatty acid synthetase activity in residues from starved chicks was stimulated when suspended in filtrates from fed chicks. The evidence is consistent with the hypothesis that a portion of the fatty acid synthetase in the liver of starved chicks is present as an inactive form which can be activated upon refeeding.     

Uptake and utilization of CDP-choline in primary brain cell cultures from fetal brain

The utilization of double-labeled CDP-choline by cultured brain cells has been studied. CDP-choline is demonstrated to be rapidly hydrolysed into CMP and choline phosphate. The fragments, or their hydrolysis products, penetrate into the cells and are utilized for lipid synthesis. At short times after the isotope administration a rapid labeling of phosphatidylcholine was detected, when cells were incubated with CDP-choline. The same was not seen when cells were incubated with labeled choline. From these observations it can be inferred that either CDP-choline can penetrate the cell membrane or that some mechanism involving CDP-choline and leading to phospholipid synthesis can work at the external surface of the plasma membranes.     

A phospholipid serine base exchange enzyme

A membrane bound L-serine exchange enzyme which catalyzes the exchange reaction between L-serine and phospholipid-base was solubilized and separated from the ethanolamine-exchange enzyme by Sepharose 4B and DEAE-cellulose column chromatography. The separated fraction was purified approximately 37-fold with a yield of 2--5%. This fraction did not possess ethanolamine or choline exchange activity. The optimal pH was approx. 8.0, the incorporation rate of L-serine into phospholipid was linear up to 20 min incubation time and the activity was maximum at 10 mM CaCl2. The calculated Km value for L-serine was 0.4 mM. Ethanolamine phospholipid was the most effective acceptor for L-serine incorporation, particularly ethanolamine plasmalogen. The Km values obtained were: 0.25 mM for ethanolamine plasmalogen, 0.25mM for pig liver phosphatidylethanolamine and 0.66 mM for egg yolk phosphatidylethanolamine. These observations suggest that the hydrophobic moiety in ethanolamine phospholipid, as well as the base moiety, is important for the affinity of the L-serine exchange enzyme. Neither ethanolamine nor choline inhibited the L-serine exchange activity. There was no detectable conversion of phosphatidylcholine or phosphatidylethanolamine to phosphatidic acid by the partially purified enzyme.     

Membrane lipid biosynthesis in Chlamydomonas reinhardtii: ethanolaminephosphotransferase is capable of synthesizing both phosphatidylcholine and phosphatidylethanolamine

Phosphatidylethanolamine, but not phosphatidylcholine, is found in Chlamydomonas reinhardtii. A cDNA coding for diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase (EPT) was cloned from C. reinhardtii. The C. reinhardtii EPT appears phylogenetically more similar to mammalian aminoalcoholphosphotransferases than to those of yeast and the least close to those of plants. Similar membrane topography was found between the C. reinhardtii EPT and the aminoalcoholphosphotransferases from mammals, yeast, and plants. A yeast mutant deficient in both cholinephosphotransferase and ethanolaminephosphotransferase was complemented by the C. reinhardtii EPT gene. Enzymatic assays of C. reinhardtii EPT from the complemented yeast microsomes demonstrated that the C. reinhardtii EPT synthesized both PC and PE in the transformed yeast. The addition of either unlabeled CDP-ethanolamine or CDP-choline to reactions reduced incorporation of radiolabeled CDP-choline and radiolabeled CDP-ethanolamine into phosphatidylcholine and phosphatidylethanolamine. EPT activity from the transformed yeast or C. reinhardtii cells was inhibited nearly identically by unlabeled CDP-choline, CDP-ethanolamine, and CMP when [14C]CDP-choline was used as the primary substrate, but differentially by unlabeled CDP-choline and CDP-ethanolamine when [14C]CDP-ethanolamine was the primary substrate. The Km value of the enzyme for CDP-choline was smaller than that for CDP-ethanolamine. This provides evidence that C. reinhardtii EPT, similar to plant aminoalcoholphosphotransferase, is capable of catalyzing the final step of phosphatidylcholine biosynthesis, as well as that of phosphatidylethanolamine in the Kennedy pathway.     

An increase of CDP-choline: diglyceride phosphocholine transferase reaction in microsome fraction and its decrease in nuclear membranes of the rat liver after treatment with hydrocortisone

The availability of CDP-choline: diaglyceride phosphocholine transpherase activity in the rat liver nuclear membrane fraction is shown. It is established that the enzyme activity in nuclear membrane fraction makes up less than 1/3 of the analogous activity in microsomes. The hydrocortisone treatment of animals leads to an increase of the enzyme activity in microsomes and to a decrease in the nuclear membranes. The obtained data indicate the existence of the own enzyme of phosphatidyl choline synthesis in the nuclear membranes. This enzyme is, probably, responsible for the nuclear membrane structure when the functional status of cell changes.     

Size and transforming activity of deoxyribonucleic acid in Diplococcus pneumoniae during thymidine starvation.

The transforming activity and the molecular structure of DNA from cells of Diplococcus pneumoniae during thymidine starvation have been analyzed and the effects of thymidine starvation have been compared with the effects of single-strand breaks produced by deoxyribonucleases in DNA of unstarved cells. The decrease in transforming activity of lysates from starved cells as a function of the size of DNA particles, measured by centrifugation in neutral and alkaline sucrose gradients, does not follow the kinetics observed after enzymatic degradation of DNA of unstarved cells. Moreover, a strain lacking exo- and endonuclease activities is not protected from thymineless death. These results suggest that the basic lethal mechanism of thymidine starvation might have an origin other than the activation of nucleases.     

Stimulation of lactase synthesis induced by starvation in the jejunum of adult rat

The effects of actinomycin D and of cycloheximide administration have been investigated on the enzyme activities of the jejunal brush border membrane in adult rats after a 48-hour period of starvation. The modifications in the protein and enzyme patterns of the brush border membrane and the incorporation of radiolabelled amino acid in the protein band corresponding to lactase have been studied in the nourished and in the starved animal. The results show that actinomycin D administration did not modify the stimulation of lactase activity caused by starvation whereas cycloheximide completely inhibited this process. The stimulation of lactase activity, in the starved animal, is related to a quantitative increase of the corresponding protein band and with enhanced incorporation of L-[3H]valine in this protein band after separation of brush border proteins by gel electrophoresis. It is concluded that the stimulation of lactase activity observed during starvation is the consequence of de novo synthesis of lactase molecules and that this process is regulated at a translational level. A general hypothesis is proposed in order to clear up partly the mechanism involved in the stimulation of lactase activity by food deprivation in the adult rat.     

Reduction of phosphatidylcholine turnover in a Nb 2 lymphoma cell line after prolactin treatment. A novel mechanism for control of phosphatidylcholine levels in cells

Phosphatidylcholine metabolism was investigated in Nb 2 rat node lymphoma cells, a cell line which is dependent on prolactin for growth in culture. Treatment of stationary cultures with prolactin stimulated the incorporation of [methyl-3H]choline into phosphatidylcholine (1.7-fold after 4 h) and its aqueous precursors, mainly phosphocholine (1.9-fold after 4 h and 2.7-fold after 10 h). These effects were blocked by cycloheximide. Pulse-chase studies demonstrated that the reaction catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) was rate-limiting for phosphatidylcholine synthesis in Nb 2 cells and that the rate of this reaction was not altered by prolactin treatment. The cell-free activity of choline kinase (EC 2.7.1.32) was found to increase in correspondence with the increase in choline incorporation. This induction of choline kinase was also blocked by cycloheximide. The activities of the other enzymes of phosphatidylcholine synthesis were unchanged. These results suggest that phosphatidylcholine biosynthesis was not altered in Nb 2 cells after prolactin treatment. However, phosphatidylcholine levels increased in prolactin-treated cells (1.4-fold after 16 h). Turnover of labeled phosphatidylcholine was markedly reduced in prolactin-treated cells. Calculated turnover rates for phosphatidylcholine averaged 4.2-fold lower in prolactin-treated cells, whereas the synthetic rates were similar in prolactin-treated and stationary cells. Thus, Nb 2 cells utilize a novel mechanism, reduction of turnover, to regulate the cellular levels of phosphatidylcholine during growth.     

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.