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PubMed1994

Immunologically and enzymatically distinct rat choline kinase isozymes.

The purification and characterization of choline kinase R1 expressed in Escherichia coli, using expression plasmid pKCK(H) constructed with rat liver choline kinase cDNA [Uchida, T. and Yamashita, S. (1992) J. Biol. Chem. 267, 10156-10162], were carried out. Choline kinase R1 was chromatographically, enzymatically, and immunologically distinct from rat brain choline kinase [Uchida, T. and Yamashita, S. (1990) Biochim. Biophys. Acta 1043, 281-288]. This report, for the first time, definitively demonstrates the presence of two types of choline kinase isozymes. Rat choline kinase was immunologically classified into choline kinases P and R. Choline kinase P was purified from rat brain as described previously. Choline kinase R was designated as the choline kinase generated from the choline kinase R gene for choline kinase R1. Choline kinases from cytosol were chromatographically separated into two fractions, that is, one passed through a Blue-Toyopearl column and the other was retained on it. The choline kinase retained on the Blue-Toyopearl column was highly purified from rat liver cytosol and characterized in this study, being found to comprise isoforms of choline kinase R, and a choline kinase which was immunologically similar to choline kinase P. Choline kinases P and R are co-distributed in rat tissues. The carcinogen, 3-methylcholanthrene, and hepatotoxic carbon tetrachloride increased the enzyme activity, and the transcript and protein levels of choline kinase R in rat liver. The relation of choline kinase isozymes to previously purified enzymes was discussed.

Animals↗
PubMed2004

Identification of critical residues of choline kinase A2 from Caenorhabditis elegans.

Choline kinase catalyzes the phosphorylation of choline by ATP, the first committed step in the CDP-choline pathway for phosphatidylcholine biosynthesis. To begin to elucidate the mechanism of catalysis by this enzyme, choline kinase A-2 from Caenorhabditis elegans was analyzed by systematic mutagenesis of highly conserved residues followed by analysis of kinetic and structural parameters. Specifically, mutants were analyzed with respect to K(m) and k(cat) values for each substrate and Mg(2+), inhibitory constants for Mg(2+) and Ca(2+), secondary structure as monitored by circular dichroism, and sensitivity to unfolding in guanidinium hydrochloride. The most severe impairment of catalysis occurred with the modification of Asp-255 and Asn-260, which are located in the conserved Brenner's phosphotransferase motif, and Asp-301 and Glu-303, in the signature choline kinase motif. For example, mutation of Asp-255 or Asp-301 to Ala eliminated detectable catalytic activity, and mutation of Asn-260 and Glu-303 to Ala decreased k(cat) by 300- and 10-fold, respectively. Additionally, the K(m) for Mg(2+) for mutants N260A and E303A was approximately 30-fold higher than that of wild type. Several other residues (Ser-86, Arg-111, Glu-125, and Trp-387) were identified as being important: Catalytic efficiencies (k(cat)/K(m)) for the enzymes in which these residues were mutated to Ala were reduced to 2-25% of wild type. The high degree of structural similarity among choline kinase A-2, aminoglycoside phosphotransferases, and protein kinases, together with the results from this mutational analysis, indicates it is likely that these conserved residues are located at the catalytic core of choline kinase.

Adenosine Triphosphate↗
PubMed1998

Expression, purification, and characterization of choline kinase, product of the CKI gene from Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) is the product of the CKI gene. Choline kinase catalyzes the committed step in the synthesis of phosphatidylcholine by the CDP-choline pathway. The yeast enzyme was overexpressed 106-fold in Sf-9 insect cells and purified 71.2-fold to homogeneity from the cytosolic fraction by chromatography with concanavalin A, Affi-Gel Blue, and Mono Q. The N-terminal amino acid sequence of purified choline kinase matched perfectly with the deduced sequence of the CKI gene. The minimum subunit molecular mass (73 kDa) of purified choline kinase was in good agreement with the predicted size (66.3 kDa) of the CKI gene product. Native choline kinase existed in oligomeric structures of dimers, tetramers, and octomers. The amounts of the tetrameric and octomeric forms increased in the presence of the substrate ATP. Antibodies were raised against the purified enzyme and were used to identify choline kinase in insect cells and in S. cerevisiae. Maximum choline kinase activity was dependent on Mg2+ ions (10 mM) at pH 9.5 and at 30 degrees C. The equilibrium constant (0.2) for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 6.26 kcal/mol, and the enzyme was labile above 30 degrees C. Choline kinase exhibited saturation kinetics with respect to choline and positive cooperative kinetics with respect to ATP (n = 1.4-2.3). Results of the kinetic experiments indicated that the enzyme catalyzes a sequential Bi Bi reaction. The Vmax for the reaction was 138.7 micromol/min/mg, and the Km values for choline and ATP were 0.27 mM and 90 microM, respectively. The turnover number per choline kinase subunit was 153 s-1. Ethanolamine was a poor substrate for the purified choline kinase, and it was also poor inhibitor of choline kinase activity. ADP inhibited choline kinase activity (IC50 = 0.32 mM) in a positive cooperative manner (n = 1.5), and the mechanism of inhibition with respect to ATP and choline was complex. The regulation of choline kinase activity by ATP and ADP may be physiologically relevant.

Adenosine Diphosphate↗
PubMed2006

Molecular characterization and localization of Plasmodium falciparum choline kinase.

Generation of phosphocholine by choline kinase is important for phosphatidylcholine biosynthesis via Kennedy pathway and phosphatidylcholine biosynthesis is essential for intraerythrocytic growth of malaria parasite. A putative gene (Gene ID PF14_0020) in chromosome 14, having highest sequence homology with choline kinase, has been identified by BLAST searches from P. falciparum genome sequence database. This gene has been PCR amplified, cloned, over-expressed and characterized. Choline kinase activity of the recombinant protein (PfCK) was validated as it catalyzed the formation of phosphocholine from choline in presence of ATP. The K(m) values for choline and ATP are found to be 145+/-20 microM and 2.5+/-0.3 mM, respectively. PfCK can phosphorylate choline efficiently but not ethanolamine. Southern blotting indicates that PfCK is a single copy gene and it is a cytosolic protein as evidenced by Western immunoblotting and confocal microscopy. A model structure of PfCK was constructed based on the crystal structure of choline kinase of C. elegans to search the structural homology. Consistent with the homology modeling predictions, CD analysis indicates that the alpha and beta content of PfCK are 33% and 14%, respectively. Since choline kinase plays a vital role for growth and multiplication of P. falciparum during intraerythrocytic stages, we can suggest that this well characterized PfCK may be exploited in the screening of new choline kinase inhibitors to evaluate their antimalarial activity.

Adenosine Triphosphate↗
PubMed1976

Equilibrium constants under physiological conditions for the reactions of choline kinase and the hydrolysis of phosphorylcholine to choline and inorganic phosphate.

The observed equilibrium constants (Kobs) of the P-choline hydrolysis reaction have been determined under physiological conditions of temperature (38 degrees) and ionic strength (0.25 M) and physiological ranges of pH and free [Mg2+]. Using sigma and square brackets to indicate total concentrations: (see article.) The value of Kobs has been found to be relatively insensitive to variations in pH and free [Mg2+]. At pH 7.0 and taking the standard state of liquid water to have unit activity ([H2O] = 1), Kobs = 26.6 M at free [Mg2+] = 0 [epsilon G0obs = -2.03 kcal/mol(-8.48 kJ/mol)], 26.8 M at free [Mg2+] = 10(-3) M, and 28.4 M at free [Mg2+] = 10(-2) M. At pH 8.0, Kobs = 18.8 M at free [Mg2+] = 0, 19.2 M at free [Mg2+] = 10(-3), and 22.2 M at free [Mg2+] = 10(-2) M. These values apply only to situations where choline and Pi concentrations are both relatively low (such as the conditions found in most tissues). At higher concentrations of phosphate and choline, the value of Kobs becomes significantly increased since HPO42- complexes choline weakly (association constant = 3.3 M-1). The value of K at 38 degrees and I = 0.25 M is calculated to be 16.4 +/- 0.3 M [epsilonG0 = 1.73 kcal/mol (-7.23 kJ/mol)]. The K for the P-choline hydrolysis reaction has been combined with the K for the ATP hydrolysis reaction determined previously under physiological conditions to calculate a value of 4.95 X 10(-3 M [deltaG0 j.28 kcal/mol (13.7 kJ/mol] for the K of the choline kinase reaction (EC 2.7.1.32), an important step in phospholipid metabolism: (see article.) Likewise, values for Kobs for the choline kinase reaction at 38 degrees, pH 7.0, and I = 0.25 M have been calculated to be 5.76 X 10(4) [deltaG0OBS = -6.77 KCAL/MOL (-28.3 KJ/mol)] at [Mg2+] = 0; 1.24 X 10(4) [deltaG0obs = -5.82 kcal/mol (-24.4 kJ/mol)] at [Mg2+] = 10(-3) M and 8.05 X 10(3) [delta G0obs = -5.56 kcal/mol (-23.3 kJ/mol)] at [Mg2+ = 10(-2) M. Attempts to determine the Kobs of the choline kinase reaction directly were unsuccessful because of the high value of the constant. The results indicate that in contrast to the high deltaG0obs for the hydrolysis of the ester bond of acetylcholine, the deltaG0obs for the hydrolysis of the ester bond of P-choline is quite low, among the lowest known for phosphate ester bonds of biological interest.

Binding Sites↗
PubMed1987

Substrate specificity of choline kinase.

The substrate specificity of choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) from brewer's yeast has been examined using multiple analogs of choline, most of which have been reported to be a substrate of one or another choline-using system from other sources. In contrast to many such systems, choline kinase from brewer's yeast has been found to have relatively stringent and straight-forward structural requirements for its substrates. It is hypothesized that there are at least four points of interaction of the substrate with the enzyme--one for the hydroxyalkyl side chain and one for each of the three substituents on the quaternary nitrogen. Of the latter, one site seems relatively more sterically hindered than the other two. Short, single or double alkyl substitutions on the quaternary nitrogen are possible without a large loss of substrate capacity of the analog. Thus N,N-dimethyl-N-propylethanolamine had a relative Vmax of 116% and a relative Vmax 96% that of choline and a Km of 68 +/- 15 microM [nearly four times that of choline itself (18 microM)]. However, N-butyl-N,N-dimethylethanolamine and N,N,N-triethylethanolamine were very poor substrates. Analogs with substituents on the quaternary nitrogen of longer chain length were without activity as were aromatic derivatives. None of the bisquaternary compounds of the general structure HOCH2CH2N+(CH3)2-(CH2)n-N+(CH3)2CH2CH2OH (n = 2-10) showed any substrate capacity, as well. Restrictions on the hydroxyethyl side chain were also severe. One additional methylene group in this chain greatly reduced substrate capacity of the analog and two additional ones eliminated it entirely, as did almost any substituent on the beta carbon. A single (but not a double) substituent on the alpha carbon was moderately tolerated, however. Thus alpha-methylcholine and N-methyl-2-hydroxymethylpiperidine were substrates (although the latter one was a poor one) but beta-methylcholine and N-methyl-3-hydroxypiperidine were not. Such information may be of use toward designing cholinergic probes targeting specific enzyme or metabolic functions.

Allosteric Site↗
PubMed1972

Choline kinase in Cuscuta reflexa.

1. Choline kinase is a mitochondrial enzyme in Cuscuta reflexa. It can be solubilized from the particles by treatment with 350mm-sodium chloride, or by freezing and thawing. 2. Choline kinase of C. reflexa was purified by starting from the crude mitochondrial fraction. A 33-52% recovery of the enzyme, on the basis of the activity in the original homogenate, in 1200-2250-fold enrichment, was effected. 3. The purified preparation of choline kinase had a sigmoid saturation curve with respect to choline, with a Hill number of 2.3, and was inhibited by ADP (competitive in nature and allosteric in binding, with a Hill number of 2.7) and by phosphorylcholine (non-competitive and non-allosteric). The kinetic characteristics of the enzyme were consistent with the K type allosteric model of Monod et al. (1965). 4. The enzyme was desensitized, with respect to choline regulation, by prolonged storage in the cold, was activated significantly on warming before assay and was inactivated by high concentrations of sodium chloride. 5. The significance of allostery in choline kinase in relation to the intracellular regulation of phospholipid synthesis is discussed.

Adenosine Diphosphate↗
PubMed1996

Incorporation of choline into Streptococcus pneumoniae cell wall antigens: evidence for choline kinase activity.

The choline-containing teichoic and lipoteichoic acids play an important part in cell wall metabolism of Streptococcus pneumoniae. We propose that a choline kinase enzyme has a role in the synthesis of these antigens. The presence of this enzyme was demonstrated in cell free extracts of S. pneumoniae by measuring the fall in ATP concentration due to phosphorylation of choline. Genomic DNA of S. pneumoniae hybridised with a probe consisting of an internal fragment of the choline kinase gene of Saccharomyces cerevisiae and one consisting of the choline binding domain of lytA.

Antigens, Bacterial↗
PubMed1989

The effect of polycyclic aromatic hydrocarbons on choline kinase activity in mouse hepatoma cells.

Choline kinase catalyzes the first rate-limiting step in the pathway of biosynthesis of phosphatidylcholine. This enzyme was shown previously to be induced in liver by treatment of rats with polycyclic aromatic hydrocarbons (Ishidate et al. (1980) Biochem. Biophys. Res. Commun. 96, 946-952). The present study was undertaken to determine whether choline kinase in the murine hepatoma cell line, Hepa 1c1c7, is inducible by aromatic hydrocarbons and, if so, whether this induction is mediated by the aromatic hydrocarbon receptor. Treatment of Hepa 1c1c7 cells with 10 microM beta-naphthoflavone resulted in a 1.6-fold increase of choline kinase activity, but no response was seen when the cells were exposed to either 5.0 microM benzo[a]pyrene or 1.0 nM 2.3,7,8-tetrachlorodibenzo-p-doxin, both potent inducers of aryl hydrocarbon hydroxylase. Cell line variants with either deficient or elevated aromatic hydrocarbon receptors showed no increase in choline kinase activity following treatment with any of the polycyclic aromatic hydrocarbons. These results are not consistent with a role for the aromatic hydrocarbon receptor in increased choline kinase activity in Hepa 1c1c7 cells.

Animals↗
PubMed2005

Phosphorylation of the yeast choline kinase by protein kinase C. Identification of Ser25 and Ser30 as major sites of phosphorylation.

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent and dependent on the concentrations of choline kinase (K(m) = 27 microg/ml) and ATP (K(m) = 15 microM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSSQRRHS (V5max/K(m) = 17.5 mm(-1) micromol min(-1) mg(-1)) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway, whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Although the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHSLTRQ) containing Ser30 was a substrate (V(max)/K(m) = 3.0 mm(-1) micromol min(-1) mg(-1)) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C.

Adenosine Triphosphate↗
PubMed1984

Reciprocal regulation of ornithine decarboxylase and choline kinase activities by their respective reaction products in the developing rat cerebellar cortex.

Changes in the activity of choline kinase were measured in the cerebellum during development. Early transient increase was found in the enzyme activity just prior to and during birth. This period of increase did not coincide with the periods of transient elevation in ornithine decarboxylase and choline acetyltransferase previously observed in the developing cerebellum. The effects of the naturally occurring polyamines (putrescine, spermidine, and spermine) on choline kinase and choline acetyltransferase activities, and of phosphorylcholine (the product of the reaction catalyzed by choline kinase) on ornithine decarboxylase and choline acetyltransferase activities, were also examined. Choline acetyltransferase activity was not influenced by either polyamines or phosphorylcholine. However, choline kinase activity from 7-day-old, but not from adult, cerebellum was increased 25% in the presence of 4 mM spermine. In contrast, low spermidine concentrations (less than 2 mM) inhibited choline kinase activity selectively in 7-day-old cerebellum. Ornithine decarboxylase activity from 7-day-old cerebellum was inhibited in a concentration-dependent manner by phosphorylcholine. The present data together with other previous reports suggest that: (a) polyamines may play a role in choline utilization during development via their regulation of choline kinase activity, on the one hand, and of acetylcholinesterase activity on the other; and (b) during development, a reciprocal regulation of choline kinase and ornithine decarboxylase activities by their respective reaction products may exist, whereby choline kinase activity is regulated in a complex manner by polyamines and, in turn, ornithine decarboxylase is inhibited by phosphorylcholine.

Animals↗
PubMed1985

Evidence for the existence of isozymes of choline kinase and their selective induction in 3-methylcholanthrene- or carbon tetrachloride-treated rat liver.

New evidence is provided that rat liver choline kinase exists in several distinct forms (choline kinases I, II and III) which differ in isoelectric point, molecular size and antigenicity against anti-rat kidney choline kinase IgG. Remarkable and selective induction of the choline kinase II and choline kinase III forms of choline kinase was caused similarly by the administration of polycyclic aromatic hydrocarbon carcinogen, 3-methylcholanthrene or hepatotoxic carbon tetrachloride (CCl4). The immunochemical approach further indicated that the elevation in the activity of choline kinase in the 3-methylcholanthrene- or CCl4-treated rat liver was not accompanied by a parallel increase in the amount of choline kinase II enzyme protein, compatible with the induction of either a small amount of new enzyme protein(s) with very high specific activity or another enzyme which might catalyze post-translational modification of choline kinase.

Animals↗
PubMed2005

Choline kinase is a novel oncogene that potentiates RhoA-induced carcinogenesis.

Choline kinase is overexpressed in human breast, lung, colorectal, and prostate tumors, a finding that suggests the involvement of this enzyme in carcinogenesis. Here we show that overexpression of choline kinase induce oncogenic transformation of human embryo kidney fibroblasts and canine epithelial Madin-Darby canine kidney cells. Choline kinase lays downstream of RhoA signaling and is activated through ROCK kinase, one of the best-characterized RhoA effectors. In keeping with this, coexpression of RhoA and choline kinase potentiates both anchorage independent growth and tumorigenesis. Finally, choline kinase-mediated transformation is sensitive to MN58b, a well-characterized specific choline kinase inhibitor. These results provide the definitive evidence that choline kinase has oncogenic properties and that choline kinase inhibition constitutes a novel valid antitumor strategy.

Animals↗
PubMed2006

Inhibition of Plasmodium falciparum choline kinase by hexadecyltrimethylammonium bromide: a possible antimalarial mechanism.

Choline kinase is the first enzyme in the Kennedy pathway (CDP-choline pathway) for the biosynthesis of the most essential phospholipid, phosphatidylcholine, in Plasmodium falciparum. In addition, choline kinase also plays a pivotal role in trapping essential polar head group choline inside the malaria parasite. Recently, Plasmodium falciparum choline kinase (PfCK) has been cloned, overexpressed, and purified. However, the function of this enzyme in parasite growth and survival has not been evaluated owing to the lack of a suitable inhibitor. Purified recombinant PfCK enabled us to identify an inhibitor of PfCK, hexadecyltrimethylammonium bromide (HDTAB), which has a very close structural resemblance to hexadecylphosphocholine (miltefosin), the well-known antiproliferative and antileishmanial drug. HDTAB inhibited PfCK in a dose-dependent manner and offered very potent antimalarial activity in vitro against Plasmodium falciparum. Moreover, HDTAB exhibited profound antimalarial activity in vivo against the rodent malaria parasite Plasmodium yoelii (N-67 strain). Interestingly, parasites at the trophozoite and schizont stages were found to be particularly sensitive to HDTAB. The stage-specific antimalarial effect of HDTAB correlated well with the expression pattern of PfCK in P. falciparum, which was observed by reverse transcription-PCR and immunofluorescence microscopy. Furthermore, the antimalarial activity of HDTAB paralleled the decrease in phosphatidylcholine content, which was found to correlate with the decreased phosphocholine generation. These results suggest that inhibition of choline kinase by HDTAB leads to decreased phosphocholine, which in turn causes a decrease in phosphatidylcholine biosynthesis, resulting in death of the parasite.

Animals↗
PubMed1988

Adenosine inhibits choline kinase activity and decreases the phosphorylation of choline in striatal synaptosomes.

The main objective of these studies was to determine whether adenosine inhibits choline kinase in rat striata, leading to a decreased incorporation of choline into phosphorylcholine, a mechanism that may mediate seizure-induced increases in the levels of free choline in brain. Incubation of particulate and soluble fractions of striatal synaptosomes with adenosine or its metabolically stable analogues significantly inhibited enzyme activity. The inhibition was noncompetitive versus choline and competitive versus MgATP. Inhibitor constants for adenosine, 2-chloroadenosine, and 2',5'-dideoxyadenosine at the MgATP site were 94, 49, and 207 microM, respectively; these values were less than the Michaelis constant for MgATP (340 microM). To determine whether adenosine altered the phosphorylation of choline in an intact preparation, synaptosomes were incubated with [3H]choline in the presence or absence of adenosine or its analogues and the amount of [3H]-phosphorylcholine formed from the [3H]choline taken up was measured. All compounds tested significantly reduced the synthesis of [3H]phosphorylcholine. Results suggest that following seizures or hypoxia, when levels of adenosine increase and the concentration of ATP decreases, inhibition of choline phosphorylation may be manifest, resulting in increased levels of free choline in brain.

2-Chloroadenosine↗
PubMed1989

Cloning and characterization of the yeast CKI gene encoding choline kinase and its expression in Escherichia coli.

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.

Amino Acid Sequence↗
PubMed1982

Choline kinase activity along the rabbit nephron.

Choline kinase catalyzes the phosphorylation of choline to phosphorylcholine which is thus made available for phosphatidylcholine biosynthesis. Choline kinase activity was determined in defined microdissected structures of rabbit nephron with a radiochemical microprocedure. Enzyme activity was present in all segments tested. When referred to tubular length, the highest activities were found in proximal convoluted tubules. Due to the lower protein content of distal structures these segments exhibited higher enzyme activities when referred to micrograms of protein. From this distribution pattern it was concluded that all nephron segments are able to use extracellular choline for phosphatidylcholine biosynthesis.

Animals↗
PubMed1983

Evidence for membrane-associated choline kinase activity in rat striatum.

The distribution of choline kinase (EC 2.7.1.32) activity was investigated in subcellular fractions of rat striatum. Enzyme activity in the crude mitochondrial fraction, determined after dissolution in Triton X-100, was 5.90 mumol/g initial wet weight/h. When a crude mitochondrial preparation was hypoosmotically shocked and fractionated, followed by the addition of Triton X-100, choline kinase activity in the soluble and particulate fractions was 4.58 and 1.40 mumol/g initial wet weight/h, respectively. Enzyme activity in the particulate fraction was not detected in the absence of Triton X-100 or in the presence of NaCl (up to 1.5 M). Subcellular enzyme markers indicated that the membrane-associated activity was not attributable to mitochondrial or microsomal contamination. Kinetic analysis of the activity of soluble and membrane-solubilized choline kinase indicated Km values of 0.74 mM and 0.68 mM, respectively. Results indicate that choline kinase activity may be measured in both the soluble and the particulate fractions of rat striatum, the latter most likely involving enzyme associated with membrane through hydrophobic or covalent interactions. The specific function of the membrane-associated enzyme has not yet been determined.

Adenosine Triphosphate↗