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Increased phosphoethanolamine (PE) has been observed in tumors almost as consistently as increased phosphocholine (PC), but the role of PE in cancer is relatively unexplored. Our ongoing studies have shown that choline kinase (ChK)-α, the enzyme that forms PC from choline, has a dual choline/ethanolamine kinase activity but ChK-β has no significant role in maintaining PE levels in vivo. We observed that ethanolamine kinase (EtnK)-1siRNA significantly reduces PE levels in triple negative MDA-MB-231 human breast cancer cells. We further investigated the role of ethanolamine kinase-2 (EtnK-2) in contributing to the increased PE observed in cancers, and the effect of various small interfering RNA (siRNA) combinations downregulating ChK and EtnK on cell viability, as potential therapeutic strategies in these triple negative breast cancer cells. Cells were cultured in RPMI-1640 medium supplemented with 20μM choline and 50μM ethanolamine. While 50nM siRNA was used in all individual siRNA treatment, for combination siRNA treatment 50nM of each specific siRNA was used. Silencing of 80-90% message was confirmed using qRT-PCR for the siRNAs used. Approximately 40 million cells were harvested 48h post transfection and cell extracts were prepared using a dual-phase extraction method based on methanol/chloroform/water (1/1/1; v/v/v). 31P MR spectra were acquired on water soluble fraction and metabolites were quantitated. For assaying cell viability, 4000 cells per well were plated in a 96 well plate and transfected with 50nM siRNA and viability assessed 96h post transfection. PC decreased consistently when cells were treated with Chk-α siRNA or in the presence of high levels of ethanolamine in the medium showing dual choline/ethanolamine kinase activity of ChK-α. In cells treated with EtnK-1 siRNA, a significant reduction in PE levels was observed. ChK-β or EtnK-2 siRNA treatment did not affect PC or PE levels. These results indicate that ChK-β and EtnK-2 do not have significant choline or ethanolamine kinase activity in these breast cancer cells. Etnk-1 is the major contributor to PE levels in these breast cancer cells with some contribution from Chk-α. ChK-β siRNA did not have a significant effect on cell viability whereas ChK-α and EtnK1 siRNA resulted in ∼ 60% and 50% reduction of cell viability respectively. Combined treatment with ChK-α+ EtnK-1 siRNA was not more effective than treatment with the individual siRNA alone, suggesting that both siRNA act through the same path in decreasing cell viability. The reduction of cell viability caused by treatment with EtnK-1 siRNA and its major contribution to elevated PE levels in tumors indicates its therapeutic potential that warrants further investigation. Additionally, like radiolabeled choline, radiolabeled ethanolamine may be useful in detecting breast and other cancers with PET imaging. Acknowledgements: Supported by P50 CA103175. Citation Format: Tariq Shah, Balaji Krishnamachary, Flonne Wildes, Jannie Wijnen, Kristine Glunde, Zaver M. Bhujwalla. Ethanolamine kinase-1 and phosphoethanolamine are potential diagnostic markers and therapeutic targets in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1195. doi:10.1158/1538-7445.AM2015-1195

Fatty acids are precursors of potent lipid signaling molecules. They are stored in membrane phospholipids and released by phospholipase A2 (PLA2). Lysophospholipid acyltransferases (ATs) oppose PLA2 by re-esterifying fatty acids into phospholipids, in a biochemical pathway known as the Lands Cycle. Drosophila Lands Cycle ATs oys and nes, as well as 7 predicted PLA2 genes, are expressed in the male reproductive tract. Oys and Nes are required for spermatid individualization. Individualization, which occurs after terminal differentiation, invests each spermatid in its own plasma membrane and removes the bulk of the cytoplasmic contents. We developed a quantitative assay to measure individualization defects. We demonstrate that individualization is sensitive to temperature and age but not to diet. Mutation of the cyclooxygenase Pxt, which metabolizes fatty acids to prostaglandins, also leads to individualization defects. In contrast, modulating phospholipid levels by mutation of the phosphatidylcholine lipase Swiss cheese (Sws) or the ethanolamine kinase Easily shocked (Eas) does not perturb individualization, nor does Sws overexpression. Our results suggest that fatty acid derived signals such as prostaglandins, whose abundance is regulated by the Lands Cycle, are important regulators of spermatogenesis.

Ethanolamine kinase catalyzes the first step in the CDP-ethanolamine pathway for the formation of the major membrane phospholipid phosphatidylethanolamine (PtdEtn). In this work, the predicted Etnk2 cDNA was established as a soluble protein with ethanolamine-specific kinase activity that was most highly expressed in liver. Mice with an inactivated Etnk2 gene were derived, and its absence reduced the rate of PtdEtn synthesis from exogenous ethanolamine in hepatocytes. PtdEtn is a major precursor to phosphatidylcholine in liver; however, Etnk2(-/-) mice did not have reduced amounts of either PtdEtn or phosphatidylcholine or an altered phospholipid molecular species distribution. The knock-out animals were able to adapt to a choline-deficient diet. The Etnk2(-/-) mice exhibited a maternal-specific intrauterine growth retardation phenotype that resulted in a 33% reduction in litter size and frequent perinatal death. Histological analysis of pregnant Etnk2(-/-) females showed that fetal development failed at the late stage of pregnancy in a significant percentage of embryos because of the appearance of extensive placental thrombosis. These results illustrate a non-redundant role for EtnK2 expression in regulating placental hemostasis.

Ethanolamine kinase (EKI) is the first committed step in phosphatidylethanolamine (PtdEtn) biosynthesis via the CDP-ethanolamine pathway. We identify a human cDNA encoding an ethanolamine-specific kinase EKI1 and the structure of the EKI1 gene located on chromosome 12. EKI1 overexpression in COS-7 cells results in a 170-fold increase in ethanolamine kinase-specific activity and accelerates the rate of [3H]ethanolamine incorporation into PtdEtn as a function of the ethanolamine concentration in the culture medium. Acceleration of the CDP-ethanolamine pathway does not result in elevated cellular PtdEtn levels, but rather the excess PtdEtn is degraded to glycerophosphoethanolamine. EKI1 has negligible choline kinase activity in vitro and does not influence phosphatidylcholine biosynthesis. Acceleration of the CDP-ethanolamine pathway also does not change the rate of PtdEtn formation via the decarboxylation of phosphatidylserine. The data demonstrate the existence of separate ethanolamine and choline kinases in mammals and show that ethanolamine kinase can be a rate-controlling step in PtdEtn biosynthesis.

For characterization of the detailed gene structure of human muscle type carnitine palmitoyltransferase I (M-CPTI), we analyzed the 5'-upstream region of the M-CPTI transcripts. As a result, we found a cDNA clone containing a nucleotide sequence unexpected from the reported M-CPTI gene structure in the upstream region of its 5' end. Comparison of this nucleotide sequence with that of genomic DNA showed that this sequence was derived from the 3'-untranslated region of the gene encoding choline/ethanolamine kinase-beta (CK/EK-beta) located upstream of the M-CPTI gene. Southern blot analysis showed that there was no other region homologous to the CK/EK-beta gene in the whole human genome. Thus, the overlapping transcript was concluded to be produced from the functional genes of CK/EK-beta and M-CPTI. Furthermore, cDNAs containing both exons of these genes were detected by the polymerase chain reaction using the cDNA of human heart M-CPTI obtained by specific reverse transcription from its 3'-untranslated region as a template. From these results, the production and organization of these overlapping transcripts are discussed.

Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1. 82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The gene encoding ethanolamine kinase (EKI1) was identified from the Saccharomyces Genome Data Base (locus YDR147W) based on its homology to the Saccharomyces cerevisiae CKI1-encoded choline kinase, which also exhibits ethanolamine kinase activity. The EKI1 gene was isolated and used to construct eki1Delta and eki1Delta cki1Delta mutants. A multicopy plasmid containing the EKI1 gene directed the overexpression of ethanolamine kinase activity in wild-type, eki1Delta mutant, cki1Delta mutant, and eki1Delta cki1Delta double mutant cells. The heterologous expression of the S. cerevisiae EKI1 gene in Sf-9 insect cells resulted in a 165,500-fold overexpression of ethanolamine kinase activity relative to control insect cells. The EKI1 gene product also exhibited choline kinase activity. Biochemical analyses of the enzyme expressed in insect cells, in eki1Delta mutants, and in cki1Delta mutants indicated that ethanolamine was the preferred substrate. The eki1Delta mutant did not exhibit a growth phenotype. Biochemical analyses of eki1Delta, cki1Delta, and eki1Delta cki1Delta mutants showed that the EKI1 and CKI1 gene products encoded all of the ethanolamine kinase and choline kinase activities in S. cerevisiae. In vivo labeling experiments showed that the EKI1 and CKI1 gene products had overlapping functions with respect to phospholipid synthesis. Whereas the EKI1 gene product was primarily responsible for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, the CKI1 gene product was primarily responsible for phosphatidylcholine synthesis via the CDP-choline pathway. Unlike cki1Delta mutants, eki1Delta mutants did not suppress the essential function of Sec14p.

The functional characteristics of enzymes depends upon their environment, and within physiologically intact cells, many metabolic pathways are thought to involve multienzyme complexes and other enzyme–enzyme interactions that increase efficiency and specificity by mechanisms such as channeling of intermediates. A disease such as cataract may change the intracellular environment, but the effects of these changes on enzyme–enzyme interactions can be observed only in relatively intact cells, and in enzymes that have unambiguously different properties in different environments. In intact rat lenses, choline and ethanolamine are phosphorylated independently, with no competition between the two compounds, as the first step of phospholipid biosynthesis. However, disruption of lens structure and intracellular interactions by homogenization leads to a paradoxical change in enzymic properties, causing choline and ethanolamine to become competing alternative substrates of a single enzyme that resembles the purified choline/ethanolamine kinase from liver and other tissues. The properties of ethanolamine kinase in intact cataractous lenses from rats fed a 50% galactose diet for 7–14 days were intermediate between those of intact control lenses and those in lens homogenates. In monolayers of human and dog lens epithelial cells and human retinal pigment epithelial cells ethanolamine kinase was similar to that in intact tissue, showing that the kinetic differences between intact lenses and homogenates result from the intracellular environment, not from artifacts of diffusion, and that they are not exclusive to rats or to lens cells. Results with intact lenses from monkeys, rabbits, pigs and dogs showed some differences between species, but in every case, choline had little or no effect on the phosphorylation of radiolabeled ethanolamine. Further studies will be necessary to determine how the changes in intracellular environment during cataractogenesis affect other enzymes and whether other model systems for cataractogenesis cause similar changes.

Ethanolamine (Etn), as well as its N-methyl (MeEtn) and N,N-dimethyl (Me2Etn) analogues, were recently shown to potentiate the stimulatory effect of insulin on DNA synthesis in serum-starved NIH 3T3 fibroblasts. In the present work we assessed the impact of the co-mitogenic effects of Etn and its methyl analogues on cell proliferation and cell survival, and examined whether the cell growth regulatory effects of these ethanolamines involve an Etn-kinase-mediated phosphorylation step. For this purpose, NIH 3T3 sublines highly overexpressing Drosophila Etn kinase and an appropriate vector control line were utilized and the effects of Etn, MeEtn, Me2Etn, methylamine (MeNH2), and dimethylamine (Me2NH) were studied. 31P-NMR analysis of the water-soluble cell metabolites revealed that both MeEtn and Me2Etn, but not choline, are excellent substrates for the expressed Etn kinase. The methylated ethanolamines (MeEtn and Me2Etn) and methylamines (MeNH2, Me2NH) were used as Etn models that can or cannot be phosphorylated, respectively. In serum-starved vector control cells, both MeNH2 (1 mM) and Me2NH (1 mM) were more effective than Etn in enhancing insulin-induced DNA synthesis, and both were almost as effective as MeEtn and Me2Etn. However, in the Etn kinase overexpressor cells the potentiating effects of Etn, MeEtn and Me2Etn, but not those of MeNH2 and Me2NH, were significantly reduced. Moreover, in the overexpressor cells, lower concentrations of Etn (50-200 microM) inhibited the combined mitogenic effects of Me2NH (1 mM) and insulin. These data are consistent with a mechanism in which the phosphorylated and non-phosphorylated ethanolamines are negative and positive regulators of insulin-induced mitogenesis, respectively. After incubating the cells for 13 days in serum-free medium in 96-well microplates, there was a steady decrease in cell numbers in both cell lines. However, between 6-13 days, 0.1-1 mM MeEtn and, particularly, Me2Etn provided significant protection against cell death in the Etn kinase overexpressor cells. In vector control cells, only Me2Etn in combination with insulin had similar effects on cell survival. The data suggest that phosphorylated ethanolamines may function as promoters of cell survival.

The present report describes, for the first time, a mammalian kinase highly specific for ethanolamine (Km = 41 microM). Ethanolamine kinase catalyzes the initial step in the CDP-ethanolamine pathway for phosphatidylethanolamine synthesis. Although plant and protozoan kinases are known to exhibit remarkable specificity for ethanolamine, no equivalent mammalian kinase has been characterized previously. The easily shocked (eas) mutant of Drosophila has been characterized and found to lack ethanolamine kinase activity while still possessing normal choline kinase activity (Pavlidis, P., Ramaswami, M., and Tanouye, M.A. (1994) Cell 79, 23-33). The gene compensating this mutation encodes a sequence resembling that of choline kinase. In the present report, the eas gene product, expressed in Escherichia coli as fusion protein, is found to have highly specific ethanolamine kinase activity. Anti-eas antibody revealed a protein with a molecular mass of approximately 86 kDa in rat tissues and human HeLa cells by Western blotting, but did not bind rat choline kinase isozymes. Rat liver kinase activity specific for ethanolamine was separated chromatographically and by an immunological procedure using anti-choline kinase antibody, associated with the 86 kDa protein, and immunoprecipitated by anti-eas antibody. This 86 kDa protein is characterized as ethanolamine kinase. Relations with previously reported kinases are discussed.

Growing evidence suggests an involvement of brain membrane phospholipid metabolism in a variety of neurodegenerative and psychiatric conditions. This has prompted the use of drugs (e.g., CDPcholine) aimed at elevating the rate of neural membrane synthesis. However, no information is available regarding the human brain enzymes of phospholipid synthesis which these drugs affect. Thus, the objective of our study was to characterize the enzymes involved, in particular, whether differences existed in the relative affinity of substrates for the enzymes of phosphatidylethanolamine (PE) compared to those of phosphatidylcholine (PC) synthesis. The concentration of choline in rapidly frozen human brain biopsies ranged from 32–186 nmol/g tissue, a concentration similar to that determined previously for ethanolamine. Since human brain ethanolamine kinase possessed a much lower affinity for ethanolamine (K m=460 μM) than choline kinase did for choline (K m=17 μM), the activity of ethanolamine kinase in vivo may be more dependent on substrate availability than that of choline kinase. In addition, whereas ethanolamine kinase was inhibited by choline, and to a lesser extent by phosphocholine, choline kinase activity was unaffected by the presence of ethanolamine, or phosphoethanolamine, and only weakly inhibited by phosphocholine. Phosphoethanolamine cytidylyl-transferase (PECT) and phosphocholine cytidylyltransferase (PCCT) also displayed dissimilar characteristics, with PECT and PCCT being located predominantly in the cytosolic and particulate fractions, respectively. Both PECT and PCCT exhibited a low affinity for CTP (Km approximately 1.2 mM), suggesting that the activities of these enzymes, and by implication, the rate of phospholipid synthesis, are highly dependent upon the cellular concentration of CTP. In conclusion, our data indicate different regulatory properties of PE and PC synthesis in human brain, and suggest that the rate of PE synthesis may be more dependent upon substrate (ethanolamine) availability than that of PC synthesis.

The nucleotide pathway is the major pathway of phosphatidylethanolamine (PE) headgroup biosynthesis by plants (Sparace, et al., 1981). It is catalyzed in sequence by ethanolamine kinase (EK), ethanolaminephosphate cytidylyltransferase (ECT) and ethanolamine phosphotransferase (EPT). EK has been characterized and purified from spinach leaves (Macher and Mudd, 1976) and soybean seeds (Williams and Harwood, 1994), EPT has been characterized in several plants (10), but little information is available about ECT. The only plant tissue in which ECT has been characterized is castor bean endosperm, where approximately 80% of the activity is in the outer mitochondria) membrane and the remainder in the endoplasmic reticulum (ER; Wang and Moore, 1991). ECT is soluble in mammals and yeast (Sundler, 1975).

We have characterized easily shocked (eas), a Drosophila "band-sensitive" paralytic mutant. Electrophysiological recordings from flight muscles in the giant fiber pathway of adult eas flies reveal that induction of paralysis with electrical stimulation results in a brief seizure, followed by a failure of the muscles to respond to giant fiber stimulation. Molecular cloning, germline transformation, and biochemical experiments show that eas mutants are defective in the gene for ethanolamine kinase, which is required for a pathway of phosphatidylethanolamine synthesis. Assays of phospholipid composition reveal that total phosphatidylethanolamine is decreased in eas mutants. The data suggest that eas bang sensitivity is due to an excitability defect caused by altered membrane phospholipid composition.

Ethanolamine kinase (EC 2.7.1.82) catalyses the reaction of ethanolamine and Mg^2+-ATP to produce phosphoethanolamine and Mg^2+ -ADP. For spinach (Spinacia oleracea) the activity of ethanolamine kinase is increased in leaf extracts of salinized plants. A comparison of ethanolamine kinase activity between extracts from control and salinized plants after native polyacrylamide gel electrophoresis shows that ethanolamine kinase activity migrates to the same position on a gel. This observation suggests that salinization does not induce the activity of a novel ethanolamine kinase isozyme. Ethanolamine kinase has been purified 6,537 fold to apparent homogeneity from spinach leaves by ammonium sulphate fractionation and sequential fractionation by both open-bed and HPLC chromatography, using ion-exchange and hydrophobic interaction matrices. The enzyme has an estimated molecular weight of 80,000 D by gel filtration chromatography and a subunit size of 38,000 D by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Ethanolamine kinase has a broad pH optimum between pH 7 and 9 and the optimal ratio of Mg^2+:ATP for the reaction is 1: 1 at 5 mM. The apparent K^m value for the substrate ethanolamine is 16 μM and the V^max is 438 nmol • min^-1 •mg^-l protein. Monomethylethanolamine and dimethylethanolamine serve as substrates for. ethanolamine kinase but not trimethylethanolamine (ie choline). Enzyme activity is slightly stimulated by NaCl and KCl and inhibited to varying degrees by phosphate, ammonium, phosphoethanolamine and phosphodimethylethanolamine. Not surprisingly enzyme activity is also inhibited by ADP and to varying degrees by the divalent cations Mn^2+, Ca^2+, Co^2+, Ba^2+ and Ni^2+. This work is the first purification and biochemical characterization of ethanolamine kinase in spinach and is the first step towards understanding the contribution ethanolamine kinase makes towards the synthesis of choline. Thesis Master of Science (MSc)

Choline metabolism was examined in spinach (Spinacia oleracea L.) plants growing under nonsaline and saline conditions. In spinach, choline is required for phosphatidylcholine synthesis and as a precursor for the compatible osmolyte glycine betaine (betaine). When control (nonsalinized) leaf discs were incubated for up to 2 h with [1,2–14C]ethanolamine, label appeared in the N-methylated derivatives of phosphoethanolamine including phosphomono-, phosphodi-, and phosphotri- (i.e. phosphocholine) methyl-ethanolamine, as well as in choline and betaine, whereas no radioactivity could be detected in the mono- and dimethylated derivatives of the free base ethanolamine. Leaf discs from salinized plants showed the same pattern of labeling, although the proportion of label that accumulated in betaine was almost 3-fold higher in the salinized leaf discs. Enzymes involved in choline metabolism were assayed in crude leaf extracts of plants. The activites of ethanolamine kinase and of the three S-adenosylmethionine:phospho-base N-methyltransferase enzymes responsible for N-methylating phosphoethanolamine to phosphocholine were all higher in extracts of plants salinized step-wise to 100, 200, or 300 mM NaCI compared with controls. In contrast, choline kinase, phosphocholine phosphatase, and cytidine 5[prime]-triphosphate: phosphocholine cytidylyltransferase activities showed little variation with salt stress. Thus, the increased diversion of choline to betaine in salt-stressed spinach appears to be mediated by the increased activity of several key enzymes involved in choline biosynthesis.

In the hamster heart, exogenous ethanolamine is taken up by the heart and utilized for the biosynthesis of phosphatidylethanolamine. The role of the exogenous supply of ethanolamine on phosphatidylethanolamine biosynthesis was examined by perfusing hamster heart with various concentrations of labelled ethanolamine. Analysis of the radioactivity distributed in the ethanolamine-containing metabolites indicated that at low exogenous ethanolamine concentrations (≤ 0.1 μM), the conversion of phosphoethanolamine to CDP-ethanolamine was rate-limiting for phosphatidylethanolamine biosynthesis. However, perfusion with higher concentrations of ethanolamine (≥ 0.4 μM) resulted in the phosphorylation of ethanolamine becoming rate-limiting. Since the intracellular ethanolamine levels remained unchanged, the alterations in radioactivity distribution suggested that the newly imported ethanolamine was preferentially utilized for phosphatidylethanolamine biosynthesis. The effects of ethanolamine analogues on ethanolamine uptake and subsequent conversion to phosphatidylethanolamine at physiological concentrations of exogenous ethanolamine were examined. Monomethylethanolamine was found to inhibit ethanolamine uptake, the conversion of ethanolamine to phosphoethanolamine and incorporation of radioactivity into phosphatidylethanolamine. The accumulation of radioactivity in the ethanolamine fraction by monomethylethanolamine, despite of the inhibition of ethanolamine uptake, further confirms the rate-limiting role of ethanolamine kinase in the biosynthesis of phosphatidylethanolamine. (Mol Cell Biochem 116: 69–73, 1992)

Publisher Summary Choline kinase is the initial enzyme in the CDPcholine pathway for phosphatidylcholine biosynthesis in mammalian cells. It catalyzes the Mg 2+ -ATP-dependent phosphorylation of choline. Choline kinase is rate limiting for phosphatidylcholine formation in several cells and tissues. The enzyme exists as at least two isoelectric forms in all tissues examined; a third form is inducible with polycyclic aromatic hydrocarbons and hepatoxic agents in liver. Both constitutive isoforms in liver have ethanolamine kinase activity, and kinetic and immunological data show that both kinase activities are catalyzed by the same enzymes. These results on the regulation and enzymatic activities of choline kinase indicate that the enzyme plays an important role in the long-term regulation of the CDPcholine pathway and may be involved in the coordinate regulation of phosphatidylethanolamine biosynthesis. To study choline and ethanolamine phosphorylation with a purified kinase preparation and to investigate the nature of the isoforms, a purification protocol for rat liver choline-ethanolamine kinase is developed. With the use of size-exclusion chromatography as the final step, a preparation of very high specific activity can be obtained. To obtain homogeneous, though less active, enzyme, chromatofocusing is used as the final step.

Publisher Summary Choline kinase (ATP:choline phosphotransferase) and ethanolamine kinase (ATP:ethanolamine O-phosphotransferase) catalyze the phosphorylation of choline/ethanolamine by ATP in the presence of Mg 2+ , yielding phosphocholine/phosphoethanolamine and ADP. This enzyme step commits choline (ethanolamine) to the CDPcholine (CDPethanolamine) pathway for the biosynthesis of phosphatidylcholine (phosphatidylethanolamine) in all animal cells. The chapter discusses different methods to assay choline kinase activity. The purification of choline/ethanolamine kinase from rat kidney is also discussed. Purification is started with the high-speed supernatant fraction from fresh kidneys (∼200 g wet weight). All procedures are carried out below 5 ° C. One of the most important features of choline/ethanolamine kinase is its inducibility in various experimental systems. Although the physiological significance of the inducibility has not yet been fully understood, the induction of choline/ethanolamine kinase by several means suggests that more than one mechanism could be involved in this process. Distinct mechanisms of choline kinase induction have been demonstrated between rooster liver by estrogens and rat liver by certain hepatotoxins. A new isozyme of choline/ethanolamine kinase appears to be preferentially induced in the latter case.

Publisher Summary Choline kinase or ATP:choline phosphotransferase catalyzes the phosphorylation of choline with ATP yielding phosphorylcholine and ADP. This is the initial enzyme of the CDPcholine pathway, which serves as the principal pathway of phosphatidylcholine synthesis in animal tissues. Choline kinase has been highly purified from lung, kidney, liver and brain. The brain is one of the richest sources of choline kinase. The enzyme of the soluble fraction from brain has been purified to homogeneity and shown to mediate the phosphorylation of choline, N,N -dimethylethanolamine, N -monomethylethanolamine, and ethanolamine. Choline kinase activity is assayed by determining the rate of formation of either phosphorylcholine isotopically or ADP spectrophotometrically. In the first method, [ 14 C]choline is used as substrate and the phosphorylcholine formed is separated from unreacted choline by solvent extraction and then counted. In the second method, choline kinase is coupled with the pyruvate kinase–lactate dehydrogenase system, so that the ADP formed can be determined spectrophotometrically by measuring the decrease in NADH.

Ethanolamine kinase was purified from human liver to apparent homogeneity and its properties were studied. The minimum molecular weight of the purified enzyme was estimated by SDS-PAGE to 42,000 +/- 2000 Da. The molecular weight of the native enzyme determined by Superose 12 (FPLC) gel filtration was 87,000 +/- 4000 Da, suggesting that ethanolamine kinase in human liver consists of two chains of the same or almost the same molecular weight. The titration of the active site normality showed 1.03 +/- 0.05 active sites/chain. The Km value for ethanolamine was 0.25 +/- 0.01 mM.

A blue-dye column separated rat brain choline kinase (EC 2.7.1.32) into two peaks, very likely corresponding to distinct isozymes. The major-peak enzyme was purified 15000-fold to homogeneity. The final specific activity was approx. 40 μmol • min−1 • mg−1. This is 10-times higher than that reported for the enzymes from lung and kidney. The purified enzyme gave a single 44 kDa protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Analytical gel-filtration showed that the native enzyme had a molecular weight of 90000 and a Stokes radius of 4.2 nm. The sedimentation coefficient was deduced to be 4.8 S and the molecular weight 87600 by sucrose-density-gradient centrifugation. Hence, the native enzyme appears to be a dimer. The apparent Km values for ATP and choline were 1.0 mM and 14 μM, respectively. At high choline concentrations, the enzyme showed deviation from Michaelis-Menten kinetics. The enzyme was active in a high pH range and utilized a variety of amino alcohols structurally related to choline, including ethanolamine, N-methylethanolamine and N, N-dimethylethanolamine as substrates. Spermine and spermidine stimulated the enzyme by decreasing the apparent Km for ATP and increasing Vmax. Although less efficiently, monovalent cations such as NH+4, K+, Li+ and Na+ and quaternary amines such as carpronium, chlorocholine and acetylcholine were also stimulatory.

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.

On subcellular fractionation of isoosmotic homogenates in 0.25 m sucrose, the entire choline kinase activity of liver and brain of rat and mouse was recovered in the microsomal fraction, whereas the entire ethanolamine kinase activity was associated with the cytosol fraction. In goat liver and brain, both the activities were present in the cytosol fraction. Storage of isoosmotic homogenates of rat and mouse liver and of brain for 6 h under ice-cold conditions resulted in a significant increase in assayable choline kinase activity but a decrease in ethanolamine kinase activity. The two kinases present in the postmitochondrial supernatants of rat and mouse liver could be separated by ammonium sulfate fractionation. A phylogenetic study of the kinase activities in liver and brain of a number of vertebrates revealed choline kinase activity in all. Ethanolamine was not phosphorylated by toad brain under the experimental conditions employed, but all other tissues possessed ethanolamine kinase activity. Among the various tissues of rat tested, brain and intestinal mucosa appeared to be the richest sources for kinase activities. When pregnancy ensued in the mouse, ethanolamine kinase activity was significantly increased in liver, but choline kinase activity remained unaltered. The results have been interpreted as pointing to distinct enzymes catalyzing the phosphorylation of choline and ethanolamine.

Choline kinase was purified from rat kidney to apparent homogeneity with respect to both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme showed a minimum molecular weight of 42,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the other hand, the molecular size of 75,000-80,000 was estimated through Sephadex G-150 gel filtration, indicating that the enzyme in rat kidney exists most likely in a dimeric form. Specific antibody was raised in rabbit against the highly purified rat kidney choline kinase protein, then immunochemical cross-reactivity was investigated between rabbit antiserum and choline kinase preparations from various rat tissues. The antiserum inhibited choline kinase activity almost completely in the crude preparation not only from kidney but also from lung, intestine, and normal untreated liver cytosol, but it could inhibit only partially the activity from either 3-methylcholanthrene- or carbon tetrachloride-induced rat liver cytosol. The overall results demonstrated that, although choline kinase protein appears to exist in multiple forms in rat tissues, most of them are immunochemically identical, and that either 3-methylcholanthrene- or carbon tetrachloride-inducible form(s) of choline kinase in rat liver could be quite different from a form or forms existing in normal untreated rat liver cytosol.

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 13,000g 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 Mg2+ as a cofactor. The Km 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.

Highly purified rat brain myelin showed a significant level of ethanolamine kinase, amounting to 17% of the specific activity of whole brain homogenate. This kinase level in myelin was an order of magnitude higher than that of lactate dehydrogenase, a marker for cytosol. Subcellular distribution studies revealed that in addition to myelin, this kinase was present in the P1, P2, P3, and cytosolic fractions with highest relative specific activity in the latter. The possibility that myelin activity resulted from adsorption of the soluble enzyme was unlikely since activity was retained in myelin that had been washed with buffered sodium chloride or taurocholate. Mixing experiments and repeated purification further indicated that the enzyme is intrinsic to myelin. Kinetic studies indicated similar Km values for ethanolamine in the microsomal, cytosolic, and myelin fractions but a significantly lower apparent Km for ATP in myelin. This and other differences suggested the possible existence of isozymes. Establishment of the presence of this kinase completes the list of phospholipid synthesizing enzymes needed to synthesize phosphatidylethanolamine from diacylglycerol within the myelin membrane.

The effects of choline, ethanolamine and its N-methyl analogs, different fatty acids, and L-methionine on phospholipid biosynthesis via the CDP-ester pathways and the methylation pathway were studied in rat hepatocytes. Phosphatidylethanolamine synthesis was stimulated severalfold by 0.02 to 0.1 mM ethanolamine, especially in the presence of long chain unsaturated fatty acids. At higher concentrations of ethanolamine, phosphorylethanolamine accumulated but the level of CDP-ethanolamine and the rate of phosphatidylethanolamine synthesis did not increase further. The rate of phosphatidylcholine synthesis via the CDP-ester pathway responded in a way analogous to that of phosphatidylethanolamine synthesis upon the addition of choline and fatty acid, except that a 10- to 20-fold higher concentration of choline was required for maximal stimulation, probably due to the rapid oxidation of choline to betaine. Phospholipids containing N-monomethyl- or N,N-dimethylethanolamine were efficiently formed from the corresponding free bases in the absence of ethanolamine and choline. Ethanolamine, but not other bases, inhibited completely phospholipid formation from N-monomethylethanolamine, probably as a result of competition at the level of CDP-ester formation. The data indicate that the cytidylytransferase reactions are rate-limiting steps in the synthesis of phosphatidylethanolamine and probably also phosphatidylcholine. In addition, the availability of diacylglycerol and its fatty acid composition may significantly affect the rate of phospholipid synthesis. The rate of phosphatidylcholine formation via phospholipid N-methylation approximately doubled when L-methionine was added at concentrations similar to that in rat plasma. Under these conditions the rate of phosphatidylcholine synthesis via this pathway was 20 to 40 percent of that via diacylglycerols and CDP-choline. The methylation of phosphatidylethanolamine to phosphatidylcholine remained essentially constant when the rate of phosphatidylethanolamine synthesis was varied 8-fold, but was significantly reduced when the formation of N-monomethyl- or N,N-dimethylphospholipid was stimulated by addition of the corresponding base. These phospholipids not only replaced phosphatidylethanolamine as the substrate for methylation but also increased the rate of phosphatidylcholine formation via this pathway. A method for the determination of nanomole amounts of different ethanolamine compounds is described.

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.

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.

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. Mg2+ and ATP were essential for activity. Km 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.

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.

Ethanolamine kinase was purified 60-fold by fractionation with ammonium sulfate, freeze-thawing, and gel filtration from a 100,000g supernatant from spinach leaf. The 100,00g supernatant preparation was stable for weeks, but the partially purified preparation lost half of the ethanolamine kinase activity in 10–14 days at 0–4 °C or −20 °C. A molecular weight of 110,000 was estimated by gel filtration on Sephadex G-200. The reaction required ethanolamine (Km, 42 μ m ), MgATP (Km, 63 μ m ), and free magnesium ions. The enzyme was inhibited by MgATP, with an apparent Ki of 6.7 m m . Ethanolamine kinase was inhibited by calcium (in the presence of magnesium) and o-phenanthroline. EDTA above 0.9 m m inhibited the formation of phosphorylethanolamine and EGTA stimulated at low concentrations (0.4-0.9 m m ) and inhibited at 1.8 m m . Ethanolamine kinase was inhibited by monomethylethanolamine and dimethylethanolamine, but not by choline (5 m m ). The ethanolamine kinase and choline kinase activities of the 100,000g supernatant preparation could be separated by gel electrophoresis

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 suphate-polyacrylamide gel electrophoresis. It would not phosphorylate choline, had a Km for ethanolamine of 8 microM 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.

Choline and ethanolamine kinases are located in the high speed supernatant of lactating bovine mammary gland. Maximum activities of choline and ethanolamine kinases were observed at pH 9.2 and 8.0, respectively, with the rate of ethanolamine phosphorylation being 1/15 that of choline phosphorylation. Activation energies of 29 joules (Q10--1.5) and 31 joules (Q10 = 1.5) were calculated between 3.4 and 31.3 C for choline kinase and ethanolamine kinase, respectively. Above 31.3 C, the Arrhenius plot deviated from linearity for both enzymes, suggesting that denaturation was occurring. An apparent Km of 0.25 mM for choline was obtained for choline kinase activity. The apparent Km of ethanolamine kinase for ethanolamine was unusually high (17 mM), the activity was not linear with increasing protein concentration. Activity was tripled and the Km decreased to 2.5 mM when the enzyme preparation was washed with butanol: benzene mixture, suggesting the presence of an endogenous competitive inhibitor(s), with respect to ethanolamine. Choline kinase was not affected by the solvent wash. Substrate competition studies revealed that choline kinase was slightly inhibited competitively by ethanolamine (apparent Ki = 19-21 mM), whereas choline was a potent competitive inhibitor of ethanolamine kinase (apparent Ki = 0.33-0.50 mM). The results indicated that these two kinase activities were mediated by two distinct active sites, possibly on a single protein. The significance of choline in the regulation of phosphatidylethanolamine synthesis is discussed.

Using a mutant defective in choline kinase (Hosaka, K., and Yamashita, S. (1980) J. Bacteriol. 143, 176-181; Hosaka, K., and Yamashita, S. (1987) Eur. J. Biochem. 162, 7-13), the structural gene (CKI) for choline kinase of the yeast, Saccharomyces cerevisiae, was isolated by means of genetic complementation. Within its sequence there was an open reading frame capable of encoding 582 amino acids with a calculated molecular weight of 66,316. The primary translation product contained a segment closely related to the phosphotransferase consensus sequence (Brenner, S. (1987) Nature 329, 21). A yeast transformant carrying CKI in multiple copies exhibited very high choline kinase activity as well as ethanolamine kinase activity. In-frame insertion of the CKI coding frame into lacZ' on the pUC19 vector led to efficient expression of choline kinase in Escherichia coli cells in the presence of a lac inducer, isopropylthiogalactoside, proving that CKI is the structural gene for choline kinase. Concomitantly, ethanolamine kinase activity was also expressed. When the CKI locus in the wild-type yeast genome was inactivated by its replacement with the in vitro disrupted cki gene, the yeast cells lost virtually all of the choline kinase activity and most of the ethanolamine kinase activity. Thus, it is concluded that choline kinase is mono-cistronic and that the ethanolamine kinase activity is a second activity of choline kinase in the yeast.

Summary 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 24 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.

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.

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.

Dimethylethylcholine, dimethyl-n-propylcholine, dimethyl-i-propyl choline, and 2-dimethylaminoethanol are substrates for choline kinase, partially purified from the fat-body of Phormia regina larvae by ammonium sulphate fractionation. Choline analogues and related compounds, which have been previously reported to be effective substitutes for dietary choline, are effective competitive inhibitors of choline phosphorylation. Some additional compounds, triethylcholine, diethylcholine, and 3-trimethylamino-1-propanol, are effective inhibitors of choline phosphorylation yet inadequate as substitutes for dietary choline. Further evidence for separate choline and ethanolamine kinases is provided by studies of both competitive and non-competitive inhibitors. The criteria for an effective inhibitor of ethanolamine phosphorylation are the presence of only one N-alkyl group (except dimethylaminoethanol) and a 2-carbon distance between the nitrogen atom and the hydroxyl group. The I50 values for the inhibition of choline and ethanolamine kinases by Cu2+ are 2.2 and 8.0 × 10−3 M respectively.

The distribution of phospholipids in the mollusc Helix lactea was investigated. The results showed that ethanolamine and choline phosphoglycerides are the major phospholipids in this species, amounting to 49% and 26% of the total lipid phosphorus. The amount of ceramide 2-aminoethylphosphonate was 9% of the total phospholipids and this compound was the only phosphonolipid detected in this species. Ethanolamine and choline kinase activities were shown to be present in the high-speed supernatant fraction of snail digestive gland. The maximum activity of choline kinase was found to be higher than that of ethanolamine kinase in the same enzyme preparation. The enzyme CTP:phosphorylethanolamine cytidyltransferase was also demonstrated in the high-speed supernatant fraction of snail digestive gland. The enzymic reaction product was identified as CDP-ethanolamine by paper chromatography and radioautography. The enzymes phosphorylcholine cytidyltransferase and aminoethylphosphonate cytidyltransferase were not detected under the conditions employed in the phosphorylethanolamine cytidyltransferase assays.

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.

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.

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.

The administration to rats of polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, 3,4-benzo(a) pyrene and β-naphthoflavone caused a significant elevation of hepatic choline kinase activity. On the other hand, phenobarbital-type inducers (phenobarbital, 1,1,1-trichloro 2,2-bis (ρ-chlorophenyl) ethane (DDT) and hexachlorobenzene) did not stimulate the activity at all. The administration of either cycloheximide or actinomycin D completely depressed the elevation of choline kinase activity induced by polycyclic aromatic hydrocarbons, indicating that the elevated activity by these chemicals could be due to the change in the enzyme level. These results strongly suggest that induction of choline kinase are involved in the sequence of events leading to the induction of hepatic drug metabolism by polycyclic aromatic hydrocarbons.

Choline accumulation was studied in rat lenses incubated in TC-199 medium containing radiolabeled choline. Choline entered the lens and was rapidly phosphorylated. Phosphorylcholine did not readily escape the lens and continued to accumulate throughout 24 hr of incubation. Accumulation of choline displayed saturation kinetics and this saturability appeared to be a property of transport rather than a reflection of the properties of choline kinase. Countertransport of labeled choline from lenses preloaded with radiolabeled choline indicates that choline transport in rat lens is carrier mediated. The existence of a choline carrier would also be consistent with the kinetic data. Ethanolamine competed for the choline carrier, however a component of ethanolamine uptake was non-saturable at concentrations of ethanolamine or choline up to 5 m m . Choline and ethanolamine appeared to be phosphorylated by separate kinases in lens.

Mature mammalian erythrocytes lack the enzymatic machinery for synthesizing lipids and there is no net synthesis of major phospholipids (PL). The turnover can be attributed to two main processes: the exchange of PL between plasma lipoproteins and membrane, or the acylation of lyso-phosphatidylcholine, originating from the surrounding plasma and possibly from membrane (1). The synthesis of phosphatidylcholine (PC) by N-methylation of phosphatidylethanolamine (PE) is of minor importance in supplying the cellular PC requirements, but could play a role in transmitting signals acting via cell surface receptors (2).

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.