Ethanolamine Kinase Research Articles

Complete co-purification of choline kinase and ethanolamine kinase from rat kidney and immunological evidence for both kinase activities residing on the same enzyme protein(s) in rat tissues

Choline kinase and ethanolamine kinase were completely co-purified from rat kidney cytosol through acid treatment, ammonium sulfate fractionation, DEAE-cellulose column chromatography, Sephadex G-150 gel filtration followed by choline-Sepharose affinity chromatography. The final preparation appeared to be highly homogeneous with respect to both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ishidate K., Nakagomi K. and Nakazawa Y. (1984) J. Biol. Chem. 259, 14706–14710). Throughout the purification steps, the ratio of ethanolamine kinase activity to choline kinase activity was almost constant in a range of 0.3–0.4, which strongly indicated that, in rat kidney, both activities could reside on a single enzyme protein. The rabbit polyclonal antibody raised against highly purified rat kidney choline (ethanolamine) kinase protein inhibited both choline and ethanolamine kinase activities in a parallel manner in crude enzyme preparations not only from rat kidney, but also from rat liver, lung and intestinal cytosols. The results, together with our previous findings, suggested strongly that, in rat tissues, at least large portions of both kinase activities are present on the same enzyme protein(s). The kinetic properties of both kinase reactions with the highly purified kidney enzyme were compared in some detail and the overall result suggested that choline kinase and ethanolamine kinase activities may not have a common active site on a single enzyme protein.

Evidence for the existence of multiple forms of choline (ethanolamine) kinase in rat tissues

In order to characterize the form of choline kinase in rat tissues, both electrophoretic and gel chromatographic patterns of choline kinase activity were compared in the liver, kidney, lung, whole intestine and carbon tetrachloride-induced liver cytosols. Kinetic parameters of the reaction were also compared for the main forms of choline kinase protein from these tissues. The overall results suggested strongly that choline kinase does not exist in one particular active form but exists in multiple forms in rat tissues. In the study present here, the electrophoretic patterns of both choline kinase and ethanolamine kinase activities were compared in rat liver, kidney, lung and intestinal cytosols. The results strongly supported the view that both kinase activities are represented on the same enzyme protein(s) in each of the rat tissues examined.

Ethanolamine accumulation by photoreceptor cells of the rabbit retina.

The rabbit retina accumulates ethanolamine by an overall process that has a high affinity for ethanolamine. This process is different from the choline uptake, since ethanolamine accumulation was unaffected by high choline concentrations. Autoradiography identified the major site of high-affinity uptake as the perinuclear region of the photoreceptor cells. Ethanolamine accumulated by the high-affinity uptake was not used for neurotransmission by photoreceptor cells but was used to synthesize phosphatidylethanolamine. However, only a small percentage of the accumulated ethanolamine was converted into phospholipid. The rate of phosphorylation may contribute to control of phospholipid synthesis, since choline kinase activity is much greater than ethanolamine kinase activity in the rabbit retina.

Biosynthesis of major phospholipids of Microsporum gypseum

The biosynthesis of major phospholipids of Microsporum gypseum was examined by identification of different enzymes and incorporation of specific radioactively labelled lipid substrates. The presence of choline kinase and phosphatidylserine synthetase was demonstrated in this fungus, whereas ethanolamine kinase was absent. Phosphatidylcholine is largely synthesised through the cytidine pathway, whereas phosphatidylethanolamine appears to be synthesised by either phosphatidylserine decarboxylase or ethanolamine-exchange reactions.

Choline regulates phosphatidylethanolamine biosynthesis in isolated hamster heart.

The effect of choline on phosphatidylethanolamine biosynthesis in the isolated hamster heart was investigated. Hamster hearts were perfused with [2-14C]ethanolamine in the presence of 0-0.5 mM choline. Incorporation of label into phosphatidylethanolamine was decreased 28% at 0.2-0.5 mM choline. Analysis of the ethanolamine-containing metabolites of the CDP-ethanolamine pathway revealed that choline (0.2-0.5 mM) in the perfusate had no effect on the labeling of ethanolamine. However, a 28-30% reduction in the labeling of phosphoethanolamine and CDP-ethanolamine was observed. Concurrently, there was a 2-fold increase in the intracellular choline level. Although the formation of CDP-ethanolamine is regarded as the rate-limiting step for phosphatidylethanolamine biosynthesis, choline was found to inhibit ethanolamine kinase, but did not have any effect on phosphoethanolamine cytidylyltransferase. Hence, choline may provide an additional mechanism for the regulation of phosphatidylethanolamine biosynthesis.

Lung Development in the Chick Embryo II. Choline and Ethanolamine Kinase Activity in the Developing Lungs of Normal and Hypophysectomized Chick Embryos

Lung Development in the Chick Embryo II. Choline and Ethanolamine Kinase Activity in the Developing Lungs of Normal and Hypophysectomized Chick Embryos

Choline and ethanolamine phosphorylation in rodents as a function of age

The activities of choline kinase and ethanolamine kinase in rat and mouse liver and mouse kidney were evaluated at different stages of postnatal development. The activities were calculated as units per g tissue, units per mg protein and as units in the organ mass corresponding to 100 g body weight. Developmental variations were observed in the enzyme activities and in the ratio of ethanolamine kinase to choline kinase activity with age of animal. The weaning of animals was not accompanied by any marked and consistent changes in the kinase activities by all parameters in the liver of rat and mouse or kidney of mouse. The results obtained also support the possible distinction between the proteins catalyzing the phosphorylation of choline and ethanolamine.

Ethanolamine kinase from Culex pipiens fatigans

Ethanolamine kinase was partially purified from the larvae of Culex pipiens fatigans and its properties were studied. The enzyme was separated from choline kinase by acetic acid precipitation at pH 5.0 of a 13,000 g supernatant of the larval homogenate. Alkaline phosphatase activity was removed from the enzyme preparation by the acid treatment followed by ammonium sulfate fractionation. The enzyme was localized in the cytosolic fraction and had a requirement for Mg 2+ as a cofactor. The K m values for ethanolamine and ATP were 4 × 10 −4 and 1.54 × 10 −4 m, respectively. The affinity of the enzyme for nucleotide triphosphates was in the order, ATP > ITP > GTP while UTP and CTP were poorly utilized. p-Chloromercuribenzoate and N-ethylmaleimide inhibited the enzyme activity and reduced glutathione protected the enzyme from their inhibition. Choline and serine had no effect on the enzyme activity. The enzyme had a molecular weight of 44, 000 daltons as determined by gel filtration chromatography. Eggs contained the highest specific activity of the enzyme while adult insects had the highest total enzyme activity.

Alteration in enzyme activities of de novo phosphatidylcholine biosynthesis in rat liver by treatment with typical inducers of microsomal drug-metabolizing system

1. 1.The activities of choline kinase, ethanolamine kinase, cholinephosphate cytidylyltransferase(s) and cholinephosphotransferase were compared in the liver subcellular fractions after the treatment of rats for two successive days with phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls. 2. 2. The administration of phenobarbital resulted in a significant decrease in choline kinase activity while not affecting ethanolamine kinase activity. Both 3-methylcholanthrene and poly chlorinated biphenyls caused considerable enhancement of choline kinase activity concomitantly with ethanolamine kinase activity. 3. 3. The activity of cytosolic cytidylyltransferase was not affected by any of the inducers while the microsomal activity was significantly depressed by the administration of either phenobarbital or poly chlorinated biphenyls. 4. 4. The activity of microsomal cholinephosphotransferase decreased significantly after the treatment with both 3-methylcholanthrene and polychlorinated biphenyls and increased slightly after phenobarbital administration. 5. 5. The observed opposite effects of phenobarbital and 3-methylcholanthrene on the enzymes in de novo phosphatidylcholine synthesis indicate that there exist a possible relation between induction of microsomal drug-metabolizing system and modulation of phosphatidylcholine biosynthesis in animal liver.

Ethanolamine kinase activity and compositions of diacylglycerols, phosphatidylcholines and phosphatidylethanolamines in livers of choline‐deficient rats

These experiments were performed to find the reasons for the increased concentrations of docosahexaenoyl phosphatidylethanolamines (PE) in livers of choline-deficient rats. We measured the activity of ethanolamine kinase, which catalyzes the first step in PE formation. We also measured the compositions of PE and phosphatidylcholines (PC) and concentrations and fatty acid compositions of diacylglycerols (DG), which are precursors of PE. Young male rats were fed for one week a low-methionine, choline-deficient diet, or the same diet supplemented with choline. Ethanolamine kinase activity was measured in liver cytosol (100,000 g supernatant). Fatty acids were measured in total liver diacylglycerols and in microsomal PE and PC. Ethanolamine kinase activities were equal in choline-deficient and choline-supplemented rats. Concentrations of DG were elevated 6-fold by choline deficiency. The percentage of docosahexaenoic acid (22:6n-3) in microsomal PE was nearly doubled by choline deficiency. Although the increased concentrations of PE in choline-deficient livers cannot be attributed to increased activity of ethanolamine kinase, the rate of PE formation probably was increased by increases in concentrations of its precursors, including DG. The disproportionate increase in 22:6n-3 PE probably was caused by a selective formation of PE from DG that contain 22:6n-3.

Lipid metabolism in germinating seeds: Purification of ethanolamine kinase from soya bean

Ethanolamine kinase has been purified to homogeneity from germinating soya bean ( Glycine max L.) seeds. The purified enzyme had a molecular weight of 17–19 000 as estimated by gel filtration and sodium dodecyl sulphatepolyacrylamide gel electrophoresis. It would not phosphorylate choline, had a K m for ethanolamine of 8 μM and utilised Mg-ATP. The kinase could be purified in a 37 000 molecular weight form (dimer) which would easily dissociate on storage. In contrast to ethanolamine kinase whose activity was unaffected by the presence of choline in the assay system, soya bean choline kinase, although not phosphorylating ethanolamine, was competitively inhibited by the latter. The purification of specific choline and ethanolamine kinases from germinating soya bean confirmed in vivo observations which had indicated separate enzymes.

Influence of choline and ethanolamine administration on choline and ethanolamine phosphorylating activities of mouse liver and kidney

The administration of ethanolamine to adult male mice resulted in a significant increase in ethanolamine kinase activity in liver and kidney. Similarly, choline administration resulted in a significant increase in choline kinase activity in liver and kidney. The administration of ethanolamine resulted in enhancement of choline kinase activity concomitantly with ethanolamine kinase activity in liver and kidney. The administration of choline, however, did not result in any significant increase in ethanolamine kinase activity in liver or kidney. Cycloheximide administration along with choline-ethanolamine prevented the increase in kinase activity in liver and kidney. The results obtained have been discussed in relation to the regulatory role of choline kinase and ethanolamine kinase by de novo synthesis in response to enhanced substrate concentration, the secondary nature of choline kinase induction on ethanolamine administration, and possible distinction between choline kinase and ethanolamine kinase.

Co-ordinate regulation of ethanolamine kinase and phosphoethanolamine cytidylyltransferase in the biosynthesis of phosphatidylethanolamine in rat liver.

Essential-fatty acid deficiency produces a 52% increase in the rate of phosphatidyl-ethanolamine synthesis in rat liver as calculated from results obtained in vivo [Trewhella & Collins (1973) Biochem. Biophys. Acta 296, 34--50]. This flux change was used to test the possible regulatory roles of ethanolamine kinase and of phosphoethanolamine cytidylyltransferase, which are rate-limiting enzymes of the cytidine pathway for the synthesis of phosphatidylethanolamine [Infante (1977) Biochem. J. 167, 847--849]. The results show that essential-fatty acid deficiency produces 50% and 53% increases respectively in the specific activity of these enzymes, accounting for the increased rate of phosphatidylethanolamine synthesis produced by this dietary insufficiency. This evidence leads to the conclusion that ethanolamine kinase and phosphoethanolamine cytidylyl-transferase have co-ordinated regulatory roles in the flux control of the cytidine pathway, and its sphinganine 1-phosphate lyase branch reaction, for the synthesis of phosphatidylethanolamine.

Choline kinase and ethanolamine kinase activity in the cytosol of nerve endings from rat forebrain.

Both choline kinase and ethanolamine kinase are present in the cytosol of nerve endings prepared from rat brain are the products of their action, phosphocholine (84 nmol/g fresh wt. of brain) and phosphoethanolamine (190 nmol/g fresh wt. of brain). In contrast with the enzymes from the cytosol of whole brain, both are as equally active at pH 7.5 as 9.0. Determination of kinase activity in membrane-containing tissue samples at pH9 gives low values because of the activity of alkaline phosphatase. Choline kinase, but not ethanolamine kinase, requires Mg2+ in excess of that required for the formation of the MgATP complex and is inhibited by an excess of free ATP. The Km for choline is 2.6mM and for ethanolamine is 2.2mM. The differing requirements for ATP and Mg2+ and the inhibition of choline kinase, but not ethanolamine kinase, by hemicholinium-3 suggest either the presence of two separate enzymes or two different active sites on the same enzyme.

Choline kinase of Culex pipiens fatigans

Choline kinase has been identified in homogenates of whole mosquito, Culex pipiens fatigans and some of the properties of the enzymes were studied. The enzyme activity when expressed relative to protein content was present in highest amounts in eggs followed by third instar larvae. In larvae, it was localized largely in the cytosolic and nuclear fractions. The enzyme from the cytosolic fraction showed two pH optima, at 8.0 and 9.0. Mg 2+ and ATP were essential for activity. K m values for choline and ATP were 5.6 × 10 −6 M and 9.5 × 10 −4 M respectively. ADP and p- chloromercuribenzoate inhibited enzyme activity. The insect preparations also possessed ethanolamine kinase activity. Added ethanolamine and serine had no significant inhibitory effect on the choline kinase activity suggesting that choline kinase and ethanolamine kinase were distinct enzymes in the insect.

Evidence for the existence of a single enzyme catalyzing the phosphorylation of choline and ethanolamine in primate lung

Choline kinase (ATP: choline phosphotransferase, EC 2.7.1.32) has been isolated and purified 1000-fold from adult African Green monkey lung with a yield of 10%. The purified enzyme also phosphorylated ethanolamine (ratio of ethanolamine kinase to choline kinase = 0.30). This ratio remained constant throughout the purification procedure. The K m for choline (3.0 · 10 −5 M) was lower than that of ethanolamine (1.2 · 10 −3 M). Choline was also found to inhibit ethanolamine kinase activity by 50% at a concentration of 0.005 mM, while ethanolamine inhibited choline only at very high concentrations (100–150 mM). When the enzyme was subjected to inactivation by heat, hemicholinium-3, trypsin digestion, and p-hydroxymercuribenzoate, both ethanolamine kinase and choline kinase activities were destroyed at the same rate. Freezing and thawing in the absence of glycerol also destroyed both activities at the same rate. Based on these findings, we conclude that in adult African Green monkey lung tissue, there is only one enzyme for the phosphorylation of ethanolamine and choline, and that choline phosphorylation predominates.

Effect of choline deficiency on the enzymes that synthesize phosphatidylcholine and phosphatidylethanolamine in rat liver.

Activities have been determined in subcellular fractions of livers from choline-deficient and normals rats for the enzymes that convert choline and ethanolamine to phosphatidylcholine and phosphatidylethanolamine respectively, that methylate phosphatidylethanolamine to yield phosphatidylcholine, and that oxidize choline to betaine. The activities of ethanolamine kinase, phosphoethanolamine cytidylyltransferase, and CDP-ethanolamine: 1,2-diacylglycerol phosphoethanolaminetransferase are not changed in the livers from choline-deficient rats for at least 18 days. Similarly, the activities of choline kinase and CDP-choline: 1,2-diacylglycerol phosphocholine transferase were unaffected by choline depletion. A decrease of 30-41% was observed, however, in the mitochondrial oxidation of choline to betaine. Also, the activity of the phosphocholine cytidylyltransferase was reduced in the choline-deficient livers to 60% olf the control values. The only observed increase in enzyme activity was a 62% elevation of the phosphatidylethanolamine-S-adenosylmethionine methyltransferase activity after 2 days of choline deficiency. This increased activity was maintained for at least 18 days of choline deprivation. The results suggest a lack of adaptive change in the levels of these phospholipid biosynthetic enzymes as a result of choline deficiency.

Search for sex-dependent and gestation-induced changes in choline and ethanolamine phosphorylating activities

Choline kinase and ethanolamine kinase of liver, brain and kidney had nearly the same activity in 4-month-old male and virgin female mice. Ethanolamine kinase activity was almost doubled in the liver and brain of mice in advanced pregnancy compared with the virgin, while choline kinase activity was unaltered.

Effect of oral ethanol administration on choline and ethanolamine phosphorylating activities in liver and brain of mice.

The effect of intake of ethanol on choline- and ethanolamine kinase activities in liver and brain tissues of mice was studied over a period of 31 days. With a single dose of ethanol there was an initial slight increase in hepatic kinase activity, while brain kinase activity decreased. The activities returned to the normal level after 21 h. Intragastric intubation of 1 ml of 30% aqueous ethanol daily for 1 month to mice led to a decrease in activity of both enzymes in both tissues.

Rate-limiting steps in the cytidine pathway for the synthesis of phosphatidylcholine and phosphatidylethanolamine.

An analysis of the available data on the cytidine pathway for the synthesis of phosphatidylcholine and phosphatidylethanolamine, by the logic derived from the theoretical principles of metabolic regulation, shows that the first two reactions catalysed by choline (ethanolamine) kinase and phosphocholine (phosphoethanolamine) cytidylyltransferase are rate-limiting, whereas the phosphocholine (phosphoethanolamine) transferase step is near equilibrium in rat liver.