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PubMedDate not supplied

[Rapid method for the determination of choline kinase activity. Choline kinase in marine invertebrates].

A quick sensitive test is suggested to determine choline kinase (EC 2.7.1.32). The method includes incubation of a substrate with enzyme in microvolume and separation of the formed phosphorylcholine from the initial choline by high-performance thin-layer silica gel chromatography. The procedure of determination is simple, reproducible and takes no more than 30 min. The method reveals high activity of choline kinase in some marine invertebrates. Certain features of the enzyme from the intestine of strongylocentrotus intermedius are described.

Animals↗
PubMed1966

Effect of Cycocel derivatives and gibberellin on choline kinase and choline metabolism.

Cycocel stimulated the activity of partial purified choline kinase from spinach or squash leaves, but it inhibited the activity of yeast choline kinase. The activity of different Cycocel analogs on plant growth corresponded to their stimulatory effect on the isolated choline kinase. Cycocel had no effect upon the activity of a plant phosphatase which hydrolyzed phosphorylcholine nor upon adenosine triphosphatase from wheat roots or leaves. Gibberellin A(3) inhibited choline kinase activity and reversed the stimulatory effect of Cycocel on the kinase. Total choline kinase activity per squash plant was not greatly increased by Cycocel treatment. However, on the basis of fresh weight, total kinase activity was increased by Cycocel treatment. Gibberellin A(3) partially reversed these increases. Treatment with Cycocel plus indoleacetic acid resulted in a large increase in choline kinase activity. The same distribution of tracer among phosphorylcholine, choline and betaine was observed when either phosphorylcholine-C(14) or choline-C(14) was fed to barley or wheat roots. Cycocel stimulated the incorporation of choline-C(14) into the insoluble fraction and into lipids. Cycocel inhibited phosphorylcholine uptake by roots. Thus Cycocel stimulated choline kinase activity and the utilization of choline-C(14). The effect of Cycocel upon kinase activity in vivo and in vitro was reversed by gibberellin A(3).

Choline↗
PubMed1987

Effects of extracellular choline concentration and K+ depolarization on choline kinase and choline acetyltransferase activities in superior cervical sympathetic ganglia excised from rats.

The activities of choline kinase (CK) and choline acetyltransferase (ChAT) were examined in vitro in superior cervical sympathetic ganglia (SCG) excised from rats following aerobic incubation for 1 h in a medium containing various choline concentrations, with and without application of a high KCl level (70 mM). Ganglionic CK activity was strongly inhibited (by approximately 75%) at low extracellular choline concentrations (1-5 microM) but rose as the choline concentration was raised to 10-50 microM in the incubation medium, then fell and rose again with further increases in choline concentration. A similar but moderate accelerative effect on ganglionic CK activity was also observed after addition of acetylcholine (ACh; 1 mM) without eserine. Whereas specific CK activity did not change significantly in axotomized SCG, in which the ratio of glial cells to neurons is greatly increased for a week after the operation., it was remarkably increased after denervation, in which the preganglionic cholinergic nerve terminals had degenerated. When either a high KCl level or hemicholinium-3 (HC-3; 50 microM) was added to the medium in the presence or absence of choline, ganglionic CK activity was markedly inhibited. On the other hand, ChAT activity in the SCG remained at a significantly high level during incubation with low choline concentrations (1-10 microM), but the enhanced enzyme activity became inhibited as the extracellular choline concentration was raised to 50-100 microM in the medium. Addition of HC-3 to the medium did not alter ganglionic ChAT activity at low choline concentrations. However, application of quinacrine (10 microM) considerably reduced ganglionic CK activity and also suppressed ChAT activity induced by high KCl levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Partial purification of two forms of choline kinase and separation of choline kinase from sphingosine kinase of rat brain.

Choline kinase of rat brain was purified approximately 200,000 fold using acid precipitation, ammonium sulphate fractionation, Q-Sepharose, Octyl-Sepharose and AH-Sepharose chromatography. The ability of this enzyme to catalyze the phosphorylation of choline, ethanolamine (Etn), monomethylethanolamine (MeEtn), dimethylethanolamine (Me2Etn) and sphingosine was investigated. Choline kinase was separated from sphingosine kinase. The fraction with highly purified choline kinase had four major polypeptides with different molecular masses and possessed activities towards choline, Etn, MeEtn and Me2Etn. Two forms of choline kinase were obtained when the enzymatically active fractions eluted from the Q-Sepharose column were subjected to a horizontal isoelectrofocusing electrophoresis. One form focused around pH 4.7 and is able to phosphorylate choline, Etn, MeEtn and Me2Etn. The other form focused around pH 10 and possessed only choline kinase activity. The latter form of choline kinase did not display classical Michaelis-Menten's mechanism but revealed a positive co-operative pattern for two choline binding sites. This form was purified to apparent homogeneity with a approximate molecular mass of 14.4 kDa.

Animals↗
PubMed1984

Complete purification of choline kinase from rat kidney and preparation of rabbit antibody against rat kidney choline kinase.

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.

Animals↗
PubMed1994

Regulation of choline kinase R: analyses of alternatively spliced choline kinases and the promoter region.

The previous report described the cloning kinase R1 cDNA [Uchida, T. and Yamashita, S. (1992) J. Biol. Chem. 267, 10156-10162]. A new cDNA, for choline kinase R2, was isolated from rat liver cDNA libraries. This transcript was thought to be generated by alternative splicing. Ribonuclease protection analysis revealed the presence of a third choline kinase. Three transcripts were detected in all of the examined rat tissues at different levels. Southern blot analysis demonstrated a single copy of choline kinase R gene. The genomic DNA containing the first exon and its flanking regions of choline kinase R gene was isolated and characterized. A number of transcription start sites, determined by ribonuclease protection and primer extension analyses, were found. The most 3' site, 193 base pairs upstream of the initiation codon and common in liver and testis, was the main site in liver. Some transcription start sites were detected only in testis. Choline kinase R gene showed features not only of a typical housekeeping gene but also of a gene regulated through a variety of putative cis-acting motifs. 3-Methylcholanthrene and carbon tetrachloride increased all of three transcripts to various levels, and enhanced transcription from the same start site, which scarcely gave a detectable product in normal liver.

Alternative Splicing↗
PubMed1984

Regulation of phosphatidylcholine metabolism in mammalian cells. Isolation and characterization of a Chinese hamster ovary cell pleiotropic mutant defective in both choline kinase and choline-exchange reaction activities.

By means of an in situ autoradiographic assay for the base-exchange reaction of phospholipids with L-serine in Chinese hamster ovary cell colonies immobilized on filter paper ( Esko , J.D. and Raetz , C.R.H. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1190-1193), a mutant (designated 89.1) was isolated in which the specific activity of the serine-exchange enzyme was about 2-fold less than in the parent. Unexpectedly, it was demonstrated that in extracts of the mutant the specific activities of both ATP:choline phosphotransferase (choline kinase) (EC 2.7.1.32) and the enzyme that catalyzes the base-exchange of phospholipids with choline (choline-exchange enzyme) were strikingly reduced (3- to 4-fold and 10- to 15-fold, respectively), while the specific activities of other enzymes of phosphatidylcholine synthesis were normal. Several lines of evidence presented here suggested that the partial defect of serine-exchange activity in this mutant was due to a decrease of acceptor phospholipid(s) for the reaction. The growth rates and phospholipid compositions of the mutant and parent were quite similar. However, mutant 89.1 exhibited a significant defect in its ability in vivo to synthesize phosphatidylcholine. The fact that the mutant was also defective in phosphorylcholine biosynthesis in vivo, together with the finding of an enzymatic lesion of the mutant in choline kinse in vitro as described above, clearly demonstrated that with respect to the reduced phosphatidylcholine biosynthesis the primary defect was at the level of choline kinase. In addition to the decreased synthetic rate of phosphatidylcholine, the turnover rate of phosphatidylcholine was also reduced approximately 2-fold in this mutant. These decreased rates of both synthesis and degradation of phosphatidylcholine probably account for the identical phosphatidylcholine contents between the mutant and parent. As a conclusion, it may be given that strain 89.1 is a pleiotropic mutant which possesses several alterations in phosphatidylcholine metabolism, and such mammalian mutants have not been isolated previously.

Animals↗
PubMed1990

Repression of choline kinase by inositol and choline in Saccharomyces cerevisiae.

The regulation of choline kinase (EC 2.7.1.32), the initial enzyme in the CDP-choline pathway, was examined in Saccharomyces cerevisiae. The addition of myo-inositol to a culture of wild-type cells resulted in a significant decrease in choline kinase activity. Additional supplementation of choline caused a further reduction in the activity. The coding frame of the choline kinase gene, CK1, was joined to the carboxyl terminus of lacZ and expressed in Escherichia coli as a fusion protein, which was then used to prepare an anti-choline kinase antibody. Upon Western (immuno-) and Northern (RNA) blot analyses using the antibody and a CK1 probe, respectively, the decrease in the enzyme activity was found to be correlated with decreases in the enzyme amount and mRNA abundance. The molecular mass of the enzyme was estimated to be 66 kilodaltons, in agreement with the value predicted previously from the nucleotide sequence of the gene. The coding region of CK1 was replaced with that of lacZ, and CK1 expression was measured by assaying beta-galactosidase. The expression of beta-galactosidase from this fusion was repressed by myo-inositol and choline and derepressed in a time-dependent manner upon their removal. The present findings indicate that yeast choline kinase is regulated by myo-inositol and choline at the level of mRNA abundance.

Antibodies↗
PubMed1982

Induction of choline kinase by polycyclic aromatic hydrocarbons in rat liver. I. A comparison of choline kinases from normal and 3-methylcholanthrene-induced rat liver cytosol.

Choline kinase in rat liver has been shown to be induced up to 2-fold by the administration of polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene and 3,4-benzo[a]pyrene (Ishidate, K., Tsuruoka, M. and Nakazawa, Y., (1980) Biochem. Biophys. Res. Commun. 96, 946-952). In order to characterize the nature of choline kinase induction by these carcinogens, the 3-methylcholanthrene-induced form as well as the normal form of choline kinase were partially purified from rat liver cytosol through acid treatment, (NH4)2SO4 precipitation and DEAE-cellulose column chromatography with linear KCl-gradient elution, and the catalytic properties were compared between the two preparations. Both enzyme activities were purified about 17-fold with a yield of 50% through the purification steps and there appeared no detectable difference in the elution pattern from either DEAE-cellulose column or Sephadex G-200 gel filtration. On the other hand, some differences were observed in catalytic properties between the two enzyme preparations; (1) the induced form showed a higher apparent Km value for choline (0.19 mM) when compared to the normal form (0.11 mM) and (2) the addition of polyamines caused a considerable increase in the maximum reaction velocity for the normal form whereas no remarkable change for the induced form, when the activities were plotted as a function of choline concentration. The overall results suggest that the 3-methylcholanthrene-induced form of choline kinase in rat liver could be different from the normal form, or that there exist several isoenzymes of choline kinase in rat liver, and one or some of them are inducible by the administration of polycyclic aromatic hydrocarbons.

Adenosine Triphosphate↗
PubMed2003

The crystal structure of choline kinase reveals a eukaryotic protein kinase fold.

Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 A crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline binding site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.

Adenosine Triphosphate↗
PubMed2025

Phenomics-Based Discovery of Novel Orthosteric Choline Kinase Inhibitors.

Choline kinase alpha (CHKA) is a central mediator of cell metabolism linked to cancer and immune regulation. Cellular and clinical evaluation of CHKA has been hampered by challenges in the development of drug-like choline kinase inhibitors. Here, we identify CHKA as an unexpected off-target of histone methyltransferase inhibitors using an integrated phenomic approach. We confirm CHKA as a direct protein target of the aminoquinazolines UNC0638 and UNC0737 using a combination of chemoproteomic, biochemical, cellular, and metabolic profiling assays, possibly explaining the previously reported discrepancies observed for different G9a/GLP inhibitor scaffolds in cellular assays. Using primary human cell model systems, we discover that CHKA modulation impairs IgG secretion and B-cell maturation consistent with the notion that choline metabolism plays an important role in immune signalling. Co-crystal structures of UNC0638 and UNC0737 with CHKA unravel an unexpected binding mode and suggest the inhibitors as attractive starting points for the development of selective chemical tools to further explore the biological role of CHKA in cancer and immune metabolism.

Humans↗
PubMed1988

Phosphatidylcholine synthesis in castor bean endosperm: characteristics and reversibility of the choline kinase reaction.

Choline kinase (EC 2.7.1.32) was measured in concentrated 100,000gav supernatants from castor bean endosperm (Ricinus communis L. var. Hale). Initial velocity analysis, along with competitive inhibitor (hemicholinium-3) and product inhibition (ADPMg2+) studies suggested that the forward reaction followed a sequentially ordered mechanism with ATPMg2+ binding to the enzyme first, followed by choline and then activation of the ternary complex by free Mg2+. The kinetic constants of the forward reaction are reported. A reverse reaction was measured which had a pH optimum of 6.5 and produced 1 mol of ATP for every mole of choline phosphate. The estimated maximum possible Keq at 7.25 was 5 X 10(-3) which suggested that this reaction is highly reversible in this tissue. The possible physiological significance of this is discussed.

Ricinus communis↗
PubMed1997

Choline kinase from yeast.

Choline kinase, the initial enzyme of the CDP-choline pathway, mediates the conversion of choline to phosphorylcholine and is localized in the supernatant fraction of cells. The enzyme also catalyzes the phosphorylation of ethanolamine, functioning as the initial enzyme of the CDP-ethanolamine pathway as well. Yeast choline kinase is encoded by a single structural gene, CKI, which was cloned by the genetic complementation of the choline kinase mutation cki. The deduced sequence comprises 582 amino acid residues with a molecular mass of 66316 Da and bears local sequence similarity to various protein kinases and bacterial antibiotic phosphotransferases. The expression of yeast choline kinase is transcriptionally repressed by myo-inositol and choline in a coordinate manner with other phospholipid-synthesizing enzymes in yeast.

Amino Acid Sequence↗
PubMed1999

Phosphorylation and regulation of choline kinase from Saccharomyces cerevisiae by protein kinase A.

The CKI1-encoded choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) from Saccharomyces cerevisiae was phosphorylated in vivo on multiple serine residues. Activation of protein kinase A activity in vivo resulted in a transient increase in the phosphorylation of choline kinase. This phosphorylation was accompanied by a stimulation in choline kinase activity. In vitro, protein kinase A phosphorylated choline kinase on a serine residue with a stoichiometry (0.44 mol of phosphate/mol of choline kinase) consistent with one phosphorylation site/choline kinase subunit. The major phosphopeptide derived from the enzyme phosphorylated in vitro by protein kinase A was common to one of the major phosphopeptides derived from the enzyme phosphorylated in vivo. Protein kinase A activity was dose- and time-dependent and dependent on the concentrations of ATP (Km 2.1 microM) and choline kinase (Km 0.12 microM). Phosphorylation of choline kinase with protein kinase A resulted in a stimulation (1.9-fold) in choline kinase activity whereas alkaline phosphatase treatment of choline kinase resulted in a 60% decrease in choline kinase activity. The mechanism of the protein kinase A-mediated stimulation in choline kinase activity involved an increase in the apparent Vmax values with respect to ATP (2.6-fold) and choline (2.7-fold). Overall, the results reported here were consistent with the conclusion that choline kinase was regulated by protein kinase A phosphorylation.

Alkaline Phosphatase↗
PubMed2002

Phosphorylation of Saccharomyces cerevisiae choline kinase on Ser30 and Ser85 by protein kinase A regulates phosphatidylcholine synthesis by the CDP-choline pathway.

The Saccharomyces cerevisiae CKI-encoded choline kinase is phosphorylated on a serine residue and stimulated by protein kinase A. We examined the hypothesis that amino acids Ser(30) and Ser(85) contained in a protein kinase A sequence motif in choline kinase are target sites for protein kinase A. The synthetic peptides SQRRHSLTRQ (V(max)/K(m) = 10.8 microm(-1) nmol min(-1) mg(-1)) and GPRRASATDV (V(max)/K(m) = 0.15 microm(-1) nmol min(-1) mg(-1)) containing the protein kinase A motif for Ser(30) and Ser(85), respectively, within the choline kinase protein were substrates for protein kinase A. Choline kinase with Ser(30) to Ala (S30A) and Ser(85) to Ala (S85A) mutations were constructed alone and in combination by site-directed mutagenesis and expressed in a cki1Delta eki1Delta double mutant that lacks choline kinase activity. The mutant enzymes were expressed normally, but the specific activity of choline kinase in cells expressing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 44, 8, and 60%, respectively, when compared with the control. In vivo labeling experiments showed that the extent of phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 70, 17, and 83%, respectively. Phosphorylation of the S30A, S85A, and S30A,S85A mutant enzymes by protein kinase A in vitro was reduced by 60, 7, and 96%, respectively, and peptide mapping analysis of the mutant enzymes confirmed the phosphorylation sites in the enzyme. The incorporation of (3)H-labeled choline into phosphocholine and phosphatidylcholine in cells bearing the S30A, S85A, and S30A,S85A mutant enzymes was reduced by 56, 27, and 81%, respectively, and by 58, 33, and 84%, respectively, when compared with control cells. These data supported the conclusion that phosphorylation of choline kinase on Ser(30) and Ser(85) by protein kinase A regulates PC synthesis by the CDP-choline pathway.

Amino Acid Motifs↗