Phosphorylcholine Cytidyltransferase Research Articles

Value of 11C-choline PET/CT for lung cancer diagnosis and the relation between choline metabolism and proliferation of cancer cells

The aim of this study was to investigate the efficacy of 11C-choline PET/CT imaging for lung cancer and the correlation between choline uptake of lung cancer tissue and the expression of choline kinase (ChoK), phosphorylcholine-cytidyl transferase and Ki-67 index. Between March 2008 and June 2010, 53 patients diagnosed or suspected of having lung cancer underwent integrated 11C-choline PET/CT and contrast-enhanced CT scans before surgery. After surgery, specimens from 42 patients diagnosed with lung cancer were used to detect the expression of ChoK, phosphorylcholine-cytidyl transferase and the Ki-67 index. The PET/CT results were analyzed using visual methods and the standardized uptake value (SUV) of lesions was measured using semi-quantitative methods. Finally, the analyzed results were compared to the histopathological results. The accuracy of the 11C-choline PET/CT for diagnosing lung cancer was 81.13% (43/53), compared with 71.70% (38/53) for CT scanning. The difference was not statistically significant (P=0.61). The accuracy of 11C-choline PET/CT for diagnosing lymph nodes was 83.76% (227/271), compared with 66.79% (181/271) for CT scanning. This difference was statistically significant (P=0.04); the SUVmean value of lesions correlated positively with the Ki-67 index (r=0.51, p=0.002). Of the 35 patients with positive 11C-choline PET results, 29 (82.86%) overexpressed ChoK, 26 (74.29%) overexpressed phosphorylcholine-cytidyl transferase. The seven patients with negative 11C-choline PET results did not exhibit overexpression of ChoK or phosphorylcholine-cytidyl transferase; the SUVmean value correlated positively with the expression of both ChoK and phosphorylcholine-cytidyl transferase (r=0.52, p=0.001; r=0.37, p=0.029). In conclusion, compared with contrast-enhanced CT, 11C-choline PET offers nodal staging with higher accuracy. The SUV value of PET is correlated with the proliferation of tumor cells and the mechanism of PET imaging is associated with the overexpression of ChoK and phosphorylcholine-cytidyl transferase.

Sublethal hydrogen peroxide inhibits alveolar type II cell surfactant phospholipid biosynthetic enzymes

Alveolar type II cell injury by phagocytic cell-derived reactive oxygen metabolites represents a potential mechanism for the altered surfactant metabolism found in patients with the adult respiratory distress syndrome (ARDS). Previous studies demonstrated altered surfactant phospholipid metabolism after sublethal oxidant exposure. In this study, we measured intracellular ATP levels and the activities of several enzymes involved in surfactant phospholipid biosynthesis after sublethal H2O2 exposure of cultured rat alveolar type II cells. Intracellular ATP levels were reduced by 46.6% after exposure to 75 microM H2O2. The activity of CTP:phosphorylcholine cytidyltransferase was unchanged after H2O2 exposure when measured in whole cell homogenates. However, when measured in the microsomal fraction, cytidyltransferase activity significantly fell after exposure of type II cells to 75 microM H2O2. Activity in the cytosolic fractions remained unchanged. Similarly, microsomal cholinephosphotransferase was reduced after H2O2 exposure. We conclude that H2O2 decreases surfactant phosphatidylcholine biosynthesis independently of its ability to deplete intracellular ATP content. These deleterious effects may partially explain the diminished alveolar surfactant observed in patients with ARDS.

Localization of phosphatidylcholine synthesizing enzymes in etiolated bean seedlings ( Phaseolus vulgaris L.)

The enzymes of the CDP-choline pathway of lecithin biosynthesis are present in etiolated hypocotyl hooks of bean seedlings ( Phaseolus vulgaris L.). The subcellular distribution of the enzymes was determined by sucrose density gradient centrifugation. The enzyme choline kinase (EC 2.7.1.32) proved to be soluble protein while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine-diglyceride transferase (EC 2.7.8.2) were associated with particulate fractions. Phosphorylcholine-cytidyl transferase activity was found in microsomal fractional (density 1 · 10 g cm −3) whereas phosphorylcholine-diglyceride transferase activity was exclusively localized in the microsomal fraction.

A novel method for determining equilibrium constants. CTP:phosphorylcholine cytidyltransferase

A novel method for the determination of equilibrium constants for reversible reactions is described. The method is based on the measurement of initial velocities of isotope transfer for a given substrate-product pair of both the forward and reverse reactions as a function of the mass action of reactants. The reciprocal values of these initial velocities are plotted against the mass action ratios of reactants. The observed K eq is the abscissa of the intersection point of these reciprocal plots, i.e. the mass action ratio at which the initial velocities of isotope transfer for both the forward and reverse reaction are identical, that is, when isotope exchange is occurring. In this manner, an observed K eq of 0.2 was obtained from CTP:phosphorylcholine cytidyltransferase (CTP:cholinephosphate cytidyltransferase, EC 2.7.7.15) at 37°C and pH 7.5 under physiological conditions 1.0 mM free Mg 2+ and 0.15 M salt concentration. A comparison of this value with the in vivo mass action of reactants calculated from published data indicates that this reaction is rate-limiting in the rat liver (Infante, J.P. (1977) Biochem. J. 167, 847–849).

Development and intracellular localization of lipase activity in rapessed (Brassica napus L.) cotyledons

In homogenates of resting rapeseeds no lipase activity (glycerolester hydrolase, EC 3.1.1.3) could be detected using a titrimetric assay procedure. Following a 30-h lag-phase after imbibition, lipase activity increased sharply, reaching its maximum at day 4 after sowing. Simultaneously triglyceride content of the cotyledons decreased sharply. At any time during the 11-day period of seedling growth examined, only an alkaline lipase activity with a pH optimum around 9 was present. White light had essentially no effect on the development of lipase activity. However, the disappearance of lipase activity from the cotyledons after fat utilization was found to depend on nitrogen nutrition of the seedlings. The activities of the glyoxysomal enzymes catalase and malate synthetase showed the usual rise and fall patterns with peak activities at day 4 after sowing, independently of the mineral nutrition of the seedlings.About 90% of the lipase activity was associated with a microsomal membrane fraction. Resolution of this fraction by sucrose density gradient centrifugation (62,000 g for 14 h) yielded three distinct membrane fractions. Maximum activities of membrane marker enzymes were recovered from the gradients at following densities: The major portion of microsomal protein and lipase activity at 1.085 kg/l; microsomal malate synthetase and phosphorylcholineglyceride transferase at 1.116 kg/l; NADH-cytochrome c reductase and phosphorylcholinecytidyl transferase at 1.133 kg/l. Evidently in rapeseed cotyledons lipase activity is associated only with a discrete microsomal membrane fraction which sediments differently from membrane fractions of the endoplasmic reticulum.

Phosphatidylcholine- Lysophosphatidylcholine Cycle Pathway Enzymes in Rabbit Lung. II. Marked Differences in the Effect of Gestational Age on Activity Compared to the CDP-Choline Pathway

Lysophosphatidylcholine (lysoPC), a breakdown product of phosphatidylcholine (PC), might be important in pulmonary PC synthesis through fatty acid exchange reactions. This study defines the levels of three of the enzymes of the PC-lysoPC cycle pathway (lysophospholipase (LPL) (EC. 3.1.1.5), lysophosphatidylcholine-lysophosphatidylcholine acyltransferase (LAT), and acyl-CoA lysophosphatidylcholine acyltransferase (acryl-CoA LAT) (EC. 2.3.1.23)) in developing fetal rabbit lung and compared them with the enzymes of the CPD-choline synthetic pathway (choline kinase (CK) (EC. 2.7.1.32), phosphorylcholine cytidyl transferase (CyT) (EC. 2.7.7.15), and phosphorylcholine glyceride transferase (PCGT) (EC. 2.7.8.2). Lung homogenates of fetal rabbits of known gestation, newborn, and adult rabbits were used for the enzyme, protein, and phospholipid analyses. Total lung phospholipid, PC, and protein increased with gestational age. Thirty days' gestation, newborn, and maternal lung activities of CK, CyT, and PCGT had decreased to only 50% of their activities at 22-26 days' gestation. In contrast, LPL and LAT activities increased 4-5-fold from 22-26 days to 30 days' gestation, and increased further in the newborn lung, finally to a level matching maternal lung (about 8-10-fold higher than the 22-26 days' gestation activities). The microsomal acyl-CoA LAT also showed a similar increasing activity with gestational age. In fetal lung, enzymic activities for the apparent major PC synthetic pathway decreased. In contrast, the marked increases in LPL, LAT, and acyl-CoA LAT activities with increasing gestational age and at birth suggests importance of the PC lysoPC cycle pathway in regulating synthesis and turnover with maturation.

Dependence of Wound-induced Respiration in Potato Slices on the Time-restricted Actinomycin-sensitive Biosynthesis of Phospholipid

Actinomycin D prevents the full development in a 24-hour period of both wound respiration and cyanide resistance only when given in the first 10 to 12 hours following the cutting of potato tuber (Solanum tuberosum var. Russet) slices. The capacity for choline incorporation into phosphatidylcholine increases with slice aging and is inhibited by actinomycin D in the same time-restricted way. The time-restricted effectiveness of actinomycin D applies to the cutting-elicited enhanced synthesis of three critical enzymes of phosphatidylcholine synthesis, namely phosphorylcholine-glyceride transferase, phosphorylcholine-cytidyl transferase, and phosphatidylphosphatase. By contrast, actinomycim D given at any time is without effect on the measurable levels after 24 hours of a selection of glycolytic and mitochondrial respiratory enzymes. Neither succinic dehydrogenase nor cytochrome oxidase activity increases with time in aging potato slices in the presence or absence of chloramphenicol. The foregoing observations emphasize the central role of phospholipid, and ultimately membrane biosynthesis, in the development of wound-induced respiration.

Stimulation of CDP-choline biosynthesis by enantiomeric lysophosphatidylcholines in rat intestinal mucosa

The phosphorylcholine cytidyltransferase activity of the cytosol fractions of rat intestinal crypt and villus cells was observed to be stimulated by addition of 3- sn-lysophosphatidylcholine. A 15- to 20-fold increase in activity was seen following a prior removal of phospholipids with acetone and butanol. 3- sn-Lysophosphatidylcholines of C 16 and C 18 carbon fatty acids were three to four times more effective than those with fatty chains of shorter length. The 3- sn-phosphatidyl and 3- sn-lysophosphatidyl ethanolamine, inositol, and serine were ineffective. 3- sn-Lysophosphatidic acid and 3- sn-glycerophosphorylcholine did not stimulate the transferase activity indicating that both fatty acyl and phosphorylcholine moieties of the 3- sn-lysophosphatidylcholine molecule were required for the observed effect. Addition of the enantiomeric 1- sn-lysophosphatidylcholine had an effect comparable to that of 3- sn-lysophosphatidylcholine on the phosphorylcholine cytidyltransferase activity when assayed in rat intestinal mucosa and in pigeon liver. It is suggested that the stimulatory effect of lysophosphatidylcholine may be related to a specific detergent property dependent upon the peculiar balance of hydrophilic and hydrophobic components in the molecule.

Phospholipid-synthesizing Enzymes Associated with Golgi Dictyosomes from Pea Tissue

Golgi dictyosomal membranes isolated from pea (Pisum sativum) stem tissue, using a combination of rate zonal and isopycnic sucrose density centrifugation, were shown to bear cytidine diphosphate-choline:diglyceride phosphorylcholinetransferase, CDP-ethanolamine:diglyceride phosphorylethanolaminetransferase, and CTP:phosphorylcholine cytidyltransferase activities. Although the majority of the activity of the phospholipid-synthesizing enzymes was associated with the endoplasmic reticulum, the activity found in the Golgi system was about 25% of the total activity. These results suggest that Golgi dictyosomes probably synthesize at least part of the membrane phospholipids that they may need for their secretory function and for dictyosomal proliferation during cell growth, rather than importing this material entirely from the endoplasmic reticulum.

The Enzymes of Lecithin Biosynthesis in Human Neonatal Lungs. IV. Phosphorylcholine Cytidyltransferase

Phosphorylcholine cytidyltransferase, the enzyme which catalyzes the transfer of phosphorylcholine to cytidine 5'-triphosphate to form CDP-choline, was studied for the first time in human neonatal lung. The assay of product synthesis was linear for 10-20 min and up to 12 mg protein. The pH optimum was 6-6.5. The Km of CTP was 2.0 times 10- minus 3 M, and the Km of phosphorylcholine was 0.25 times 10- minus 3 M. The true Vmax was 10 nmol CDP-choline/mg protein/10 min. The enzyme was stable under frozen conditions. Oxygen had no apparent affect on enzyme activity.

Choline phosphokinase, phosphorylcholine cytidyltransferase and CDP-choline: 1,2-diglyceride cholinephosphotransferase activity in developing rat lung.

CHIDA, N., HIRONO, H., NISHIMURA, Y. and ARAKAWA, Ts., Choline Phosphokinase, Phosphorylcholine Cytidyltransferase and CDP-choline-1, 2-diglyceride Cholinephosphotransferase Activity in Developing Rat Lung. Tohoku J. exp. Med., 1973, 110 (3), 273-282-Incorporation of choline-methyl-14C into phosphorylcholine, CDP-choline, and phosphatidylcholine by lung tissue of developing rats was studied. Choline phosphokinase, phosphorylcholine cytidyltransferase, and CDP-choline: 1, 2-diglyceride choline phosphotransferase were determined in the lung tissue of developing rats. Results indicated that phosphorylcholine cytidyltransferase was the rate-limiting enzyme in the reactions-choline→phosphoryl-choline→CDP-choline→phosphatidylcholine

Intracellular distribution of enzymes of the cytidine diphosphate choline pathway in castor bean endosperm.

The occurrence and subcellular distribution of enzymes of the cytidine diphosphate choline pathway of lecithin synthesis have been examined. Choline kinase (EC 2.7.1.32) was completely soluble, while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine-glyceride transferase (EC 2.7.8.2) were associated with particulate fractions. Although components sedimenting at 10,000 to 100,000 x g contained both enzymes, phosphorylcholine-cytidyl transferase and particularly phosphorylcholine-glyceride transferase were present in the 10,000 x g pellet, which contained the major organelles, mitochondria, and glyoxysomes. When the crude homogenate was centrifuged on a sucrose density gradient, four major bands of particulate protein were recovered. A band at density 1.24 g/cm(3) contained the glyoxysomes and was devoid of phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase activity. Enzyme activity was barely detectable in the mitochondria, at density 1.18 g/cm(2). Phosphorylcholine-glyceride transferase was found almost exclusively in a sharp band at density 1.12 g/cm(3), and phosphorylcholinecytidyl transferase was found in the uppermost band at density 1.08 g/cm(3). Thus, for the synthesis of lecithin in their membranes, the glyoxysomes and mitochondria depend on enzymes elsewhere in the cell; the final two steps in lecithin formation occur, apparently exclusively, in separate particulate cell components.

Lecithin Biosynthetic Enzymes of Onion Stem and the Distribution of Phosphorylcholine-Cytidyl Transferase among Cell Fractions

Enzymatic activities of the cytidine 5'-diphosphate choline pathway for lecithin biosynthesis were demonstrated in homogenates of onion stem (Allium cepa). Choline kinase activity was present in the postmicrosomal supernatant, with less than 3% sedimenting with the particulate fractions. Phosphorylcholine-cytidyl transferase was distributed among all fractions, and phosphorylcholine-glyceride transferase was predominantly found in the particulate fraction.The phosphorylcholine-cytidyl transferase activity of onion stem required a divalent ion (Mg(2+) or Mn(2+)) for activity, was inhibited by Ca(2+), and was specific for cytidine triphosphate, with optimal activity in the range pH 6 to 7. To evaluate the distribution among cell fractions, conditions of pH, cofactors, substrate, and assay were optimized for each fraction. One-third of the transferase activity sedimented with the mitochondria-proplastids fraction, and one-third was in the microsomal supernatant. The dictyosome fraction contained about 10% of the total activity but showed a greater specific activity than the other fractions. Similar results were obtained with homogenates from rat liver, in that purified Golgi apparatus fractions contained the highest phosphorylcholine-cytidyl transferase activity on a protein basis when compared with other cell fractions at pH 7.2.

Biosynthesis of phosphatidylcholine in the fat body of Phormia regina larvae

The biosynthesis of phosphatidylcholine from choline by the intact fat body of larval Phormia regina as well as by cell-free preparations from the fat body has been demonstrated. ATP and CTP stimulate this synthesis. O-phosphorylcholine and cytidine diphosphate choline are intermediates and time course studies indicate that the pathway proceeds in the following sequence: choline→O-phosphorylcholine→cytidine diphosphate choline→phosphatidycholine, as has been demonstrated in other animals. Studies with subcellular fractions indicate that choline kinase (ATP: choline phosphotransferase, E.C. 2.7.1.32) and phosphorylcholine cytidyltransferase (CTP: choline phosphate cytidyltransferase, E.C. 2.7.7.14) are located in the 100,000 g supernatant whereas phosphorylcholine glyceride transferase (cytidine diphosphate choline: 1,2: diglyceride cholinephosphotransferase, E.C. 2.7.8.2) appears in the particulate fraction.

The Role of Phospholipids in Stimulating Phosphorylcholine Cytidyltransferase Activity

Abstract The phosphorylcholine cytidyltransferase activity of fresh rat liver supernatant fractions was observed to be stimulated by boiled, previously incubated particulate fractions of rat liver. This stimulation was shown to be mediated by the phospholipids present in the latter. Treatment of rat liver supernatant fractions with acetone and butanol to remove phospholipids resulted hi very low transferase activity which could be increased up to 25-fold by the addition of suitable phospholipid preparations. Lipid extracts of fresh rat liver homogenates did not stimulate transferase activity unless they were exposed to a continuous flow of air for 15 hours leading to lipid oxidation. Fractionation of the phospholipids of fresh and incubated rat liver homogenates showed that lysophosphatides were present in higher concentrations in the latter and were the most active phospholipids in stimulating transferase activity. Similarly, highly purified egg lecithin showed little ability to stimulate transferase activity whereas egg lysolecithin was highly active. None of the individual isolated phospholipids stimulated to the same degree as unfractionated rat liver lipid preparations. The stimulation of transferase activity by degraded phospholipids is suggested to represent a positive feedback mechanism for the control of phospholipid synthesis.

Metabolism of phospholipids VIII. Biosynthesis of phosphatidylcholine in the intestinal mucosa

1. 1. The occurrence of the enzyme cholinephosphotransferase (CDPcholine: 1,2-diglyceride cholinephosphotransferase, EC 2.7.8.2) has been demonstrated in the intestinal mucosa of the cat, guinea pig and rabbit. 2. 2. The characteristics of the enzyme with respect to substrate concentrations, pH optimum, Mg 2+ requirement, inhibition by Tween 20 and stability to freezing and thawing were studied, using a mixed particulate subcellular fraction of cat intestinal mucosa as source of the enzyme and 1,2-diglyceride and [ 32P]CMPcholine as substrates. 3. 3. A fractionation of homogenates of the small intestinal mucosa of the cat and guinea pig showed that the enzyme is largely concentrated in the microsomal fraction, though comparison using enzyme markers indicated that it is also a true mitochondrial constituent. 4. 4. Further subfractionation of the microsomal fraction from cat small intestinal mucosa into two smooth- vesicle and one rough-vesicle fractions revealed that the cholinephosphotransferase was ahnost exactly divided between the rough-vesicle fraction and one of the smooth-vesicle fractions, whereas the second smooth-vesicle fraction contained negligible amounts of this enzyme. 5. 5. The enzyme cholinephosphate cytidyltransferase (CTP: phosphorylcholine cytidyltransferase, EC 2.7.7.15) also occurs in the intestinal mucosa of the cat indicating that the CDPcholine-dependent biosynthetic pathway for phosphatidylcholine is operating in the intestinal mucosa. 6. 6. The possibility of alternative pathways for the biosynthesis of phosphatidylcholine in the small intestinal mucosa were examined in studies in vivo using cats injected with inorganic 32P. From activity-time curves of the individual phospholipids it was concluded that neither the methylation of phosphatidylethanolamine nor the Ca 2+-activated exchange reaction of free choline with preformed phospholipid could be a major pathway for the formation of phosphatidylcholine. 7. 7. The results are discussed in relation to fat absorption and formation of chylomicrons.