A rapid accurate assay for choline kinase
Abstract
A new assay system for the enzyme, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32), has been developed. The procedure employed [14C]choline as the substrate. The product, [14C]phosphorylcholine, was separated from the substrate by a simple 30-min paper chromatography step. After development, the area of the chromatogram which contained the phosphorylcholine was cut out, placed directly into a scintillation cocktail, and counted. This assay was tested on a preparation of partially purified yeast choline kinase. The optimum concentrations of ATP and Mg2+ and the Km for choline were determined and found to agree with published values obtained by other assay methods. The new assay was also shown to be useful in measuring choline kinase activity in primate lung tissue homogenates.
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Cited by (20)
Multiple isoforms of choline kinase from Caenorhabditis elegans: Cloning, expression, purification, and characterization
2003, Biochimica et Biophysica Acta - Proteins and ProteomicsCholine kinase is the first enzymatic step in the CDP–choline pathway for phosphatidylcholine biosynthesis. The genome of the nematode, Caenorhabditis elegans, contains seven genes that appear likely to encode choline and/or ethanolamine kinases. We cloned five and expressed four of these genes, and purified or partially purified three of the encoded enzymes. All expressed proteins had choline kinase activity; those that most closely resemble the mammalian choline kinases were the most active. CKA-2, a very active form, was purified to near homogeneity. The Km values for CKA-2 were 1.6 and 2.4 mM for choline and ATP, respectively, and kcat was 74 s−1. CKA-2 was predominantly a homodimer as assessed by glycerol gradient sedimentation and dynamic light scattering. CKB-2, which was less similar to mammalian choline kinases, had Km values for choline and ATP of 13 and 0.7 mM, and kcat was 3.8 s−1. Both of these highly purified enzymes required magnesium, had very alkaline pH optima, and were much more active with choline as substrate than with ethanolamine. These results provide a foundation for future studies on the structure and function of choline kinases, as well as studies on the genetic analysis of the function of the multiple isoforms in this organism.
Early increase in choline kinase activity upon induction of the H-ras oncogene in mouse fibroblast cell lines
1995, Archives of Biochemistry and BiophysicsThe effects of expression of the H-rasoncogene on phosphatidylcholine metabolism were examined in C3H10T1/2 and NIH3T3 cells expressing ras constitutively or under the control of inducible promoters. Cell lines expressing ras under the control of the mouse metallothionein promoter and the Escherichia coli lac operator/repressor system and an NIH3T3 cell line stably transfected with the ras oncogene were studied. Phosphocholine levels were elevated in the cells constitutively expressing ras and were increased 4–6 h upon induction in the inducible cell lines. Glycerophosphocholine, which is elevated five- to sixfold in constitutively transfected ras cells, did not increase at early times of induction, suggesting the absence of increased phosphatidylcholine degradation via a phospholipase A. Choline kinase activity increased within 4–6 h upon induction and correlated well with the increase in phosphocholine levels. This increase in phosphocholine levels chould be prevented by the addition of hemicholinium-3, a competitive inhibitor of choline kinase. Expression of activated c-raf or v-raf also increased choline kinase activity, suggesting that the induction of choline kinase by ras is downstream of the ras/raf interaction. Long-term and short-term labeling experiments failed to detect evidence for increased phospholipase C activity. These results suggest that the increase in choline kinase activity observed in cells expressing ras is an early, integral part of ras transformation and is the main contributor to increased phosphocholine levels accompanying morphological changes.
Rat lens choline and ethanolamine kinases: Independent kinetics in intact tissue-competition in homogenates
1994, Biochimica et Biophysica Acta (BBA)/Lipids and Lipid MetabolismIn the committed step of phosphatidylcholine and phosphatidylethanolamine synthesis, choline and ethanolamine are phosphorylated to form phosphocholine and phosphoethanolamine. Studies of rat tissues in several laboratories attribute these two kinase activities to a single enzyme for which choline and ethanolamine are competing alternative substrates. However, in this study of intact cultured lenses, choline and ethanolamine were phosphorylated independently, and neither compound inhibited phosphorylation of the other, even at high lenticular concentrations. In contrast, choline kinase in lens homogenates was competitively inhibited by ethanolamine (Ki = 2.3 mM), and choline strongly inhibited ethanolamine kinase activity. The results suggest a fragile metabolic compartmentation or organization of kinase enzyme(s) or substrates within the intact, physiologically integrated lens which results in striking changes in kinetic characteristics when the biological organization of the lens is disrupted.
Effect of nitrogen dioxide inhalation on surfactant phosphatidylcholine synthesis in rat alveolar type II cells
1993, Biochimica et Biophysica Acta (BBA)/Lipids and Lipid MetabolismAfter exposure of rats to NO2 (10 ppm, 72 h) type II pneumocytes were isolated and compared to cells from control animals in order to determine whether nitrogen dioxide inhalation affects surfactant phospholipid synthesis. (1) Exposed cells contained more DNA, protein and phospholipid than type II cells from controls. (2) Choline kinase, CTP : cholinephosphate cytidylyltransferase, and cholinephosphotransferase showed higher specific activities in the exposed cells. (3) In correspondence with this finding, the incorporation rates of choline into intermediate metabolic products were also higher in the NO2-exposed cells. (4) The pool sizes of the intermediate metabolic products of the CDPcholine-pathway for the synthesis of phosphatidylcholine were also higher in the cells isolated from exposed animals. This suggests that acute nitrogen dioxide exposure leads to an enhanced phospholipid synthesis that may be responsible for the higher amount of phospholipid detectable in lung lavage.
Effect of xylose on the synthesis of phosphorylcholine and phosphorylethanolamine in rat lenses
1993, Experimental Eye ResearchCholine, an essential phospholipid precursor, enters the lens by a facilitated transport system and is phosphorylated to form phosphorylcholine (P-choline). Intact lenses incubated with [3H]choline accumulate both [3H]choline and P-[3H]choline. The rate and extent of this accumulation have been used to study the effects of osmotic or oxidative cataractogenic stress, and also to test the ability of compounds to protect lenses from stress-related damage. The initial effect of oxidative stress on choline metabolism is decreased choline transport, but the mechanism by which osmotic stress affects the accumulation of [3H]choline is not understood.
The effects of osmotic and oxidative stress on choline influx and metabolism were compared in rat lenses incubated in TC-199 medium. After osmotic stress by incubation with 30 mM xylose for up to 24 hr, lenses accumulated the same amount of radiolabel as controls during a 30 min pulse with [3H]choline. However, if the lenses were exposed to [3H]choline for 6 hr so that accumulation of radiolabel in the lenses was limited by the rate of P-choline synthesis. xylose treated lenses accumulated less choline than controls. Separation of the lenticular radiolabel into [3H]choline and P-[3H]choline confirmed that xylose decreased synthesis of P-choline, although adequate unphosphorylated [3H]choline was available in the lenses. A decrease in P-[3H]ethanolamine synthesis was also seen in xylose-treated lenses incubated with [3H]ethanolamine. Ethanolamine can enter lenses by a non-saturable process which is not dependent upon a transporter. Although xylose decreased P-choline synthesis in intact lenses, neither xylose nor xylitol inhibited choline kinase in lens homogenates, Xylose also decreased transport of αaminoisobutyric acid, which enters lenses by a different transporter than choline. In contrast to osmotic stress from xylose, oxidative stress from singlet oxygen decreased choline influx. Thus, the superficially similar effects of osmotic and oxidative stresses on accumulation of radiolabeled choline by cultured rat lenses actually occur by different mechanisms and reflect different types of damage to the lens.
Choline/ethanolamine kinase from rat kidney
1992, Methods in EnzymologyCholine kinase (ATP:choline phosphotransferase) and ethanolamine kinase (ATP:ethanolamine O-phosphotransferase) catalyze the phosphorylation of choline/ethanolamine by ATP in the presence of Mg2+, 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.