Choline kinase from brewers' yeast. Partial purification, properties, and kinetic mechanism.
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Acetylcholine↗
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The measurement of choline kinase activity in rat brain: the problem of alternate pathways of ATP metabolism.
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Adenosine Diphosphate↗
Partial purification and properties of choline kinase (EC 2. 7. 1. 32) from rabbit brain: measurement of acetylcholine.
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Adenosine Diphosphate↗
Phospholipid-metabolizing enzymes in Alzheimer's disease: increased lysophospholipid acyltransferase activity and decreased phospholipase A2 activity.
Damage to brain membrane phospholipids may play an important role in the pathogenesis of Alzheimer's disease (AD); however, the critical metabolic processes responsible for the generation and repair of membrane phospholipids affected by the disease are unknown. We measured the activity of key phospholipid catabolic and anabolic enzymes in morphologically affected and spared areas of autopsied brain of patients with AD and in matched control subjects. The activity of the major catabolic enzyme phospholipase A2 (PLA2), measured in both the presence and absence of Ca2+, was significantly decreased (-35 to -53%) in parietal and temporal cortices of patients with AD. In contrast, the activities of lysophospholipid acyltransferase, which recycles lysophospholipids into intact phospholipids, and glycerophosphocholine phosphodiesterase, which returns phospholipid catabolites to be used in phospholipid resynthesis, were increased by approximately 50-70% in the same brain areas. Brain activities of enzymes involved in de novo phospholipid synthesis (ethanolamine kinase, choline kinase, choline phosphotransferase, phosphoethanolamine cytidylyltransferase, and phosphocholine cytidylyltransferase) were either normal or only slightly altered. The activities of PLA2 and acyltransferase were normal in the degenerating cerebellum of patients with spinocerebellar atrophy type 1, whereas the activity of glycerophosphocholine phosphodiesterase was reduced, suggesting that the alterations in AD brain were not nonspecific consequences of neurodegeneration. Our data suggest that compensatory phospholipid metabolic changes are present in AD brain that reduce the rate of phospholipid loss via both decreased catabolism (PLA2) and increased phospholipid resynthesis (acyltransferase and glycerophosphocholine phosphodiesterase).
Acyltransferases↗
Familial respiratory distress syndrome in three consecutive full-term infants. Case reports and documentation of lung enzyme activities.
Familial respiratory distress syndrome in full-term newborn infants is a rare occurrence. Our patient delivered three consecutive full-term infants who developed findings consistent with respiratory distress syndrome. All three died from autopsy-proven hyaline membrane disease. Analysis of the activities of four enzymes that play an important role in the biosynthesis of lecithin (choline kinase, choline phosphotransferase, phospholipase A and lysolecithin acyltransferase) failed to disclose an abnormality in lung samples in our patient with familial respiratory distress syndrome.
1-Acylglycerophosphocholine O-Acyltransferase↗
Regulation of phosphatidylcholine biosynthesis in cultured chick embryonic muscle treated with phospholipase C.
Cultures of embryonic chick muscle cells grown in medium containing phospholipase C from Clostridium perfringens incorporated [3H]choline into lipid at a rate 3- to 5-fold higher than control cultures. To determine the mechanism by which stimulation of phosphatidylcholine synthesis occurred in phospholipase C-treated cells, activities of enzymes and levels of intermediates in the biosynthetic pathway for phosphatidylcholine were examined. Activities of choline kinase, choline phosphotransferase, glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate acyltransferase, acylglycerol-3-phosphate acyltransferase, and phosphatidic acid phosphatase in phospholipase C-treated cells were the same or only slightly higher than in control cells. CTP:phosphocholine cytidylyltransferase, on the other hand, was 3 times as active in homogenates from phospholipase C-treated cells. Levels of phosphocholine decreased and levels of CDP-choline increased in phospholipase C-treated cells, and a calculation of the disequilibrium ratio indicated that the cytidylyltransferase reaction was not at equilibrium. The cytidylyltransferase was, thus, identified as the regulatory enzyme for choline flux in these cells. The cytidylyltransferase was located in both the cytosolic and particulate fractions from cultured muscle cells and a much larger portion of enzyme activity was associated with the particulate fraction in cells treated with phospholipase C. Sonicated preparations of total chick lipids, phosphatidylethanolamine, and phosphatidylserine greatly stimulated the cytosolic cytidylyltransferase activity but had no effect on the particulate enzyme. Neither stimulation of incorporation of [3H]choline into lipid nor activation of the cytidylyltransferase was dependent on protein synthesis. A model for the mechanism of regulation of phosphatidylcholine synthesis in embryonic chick muscle is presented.
Animals↗
Evidence for the existence of multiple forms of choline (ethanolamine) kinase in rat tissues.
In order to characterize the form of choline kinase in rat tissues, both electrophoretic and gel chromatographic patterns of choline kinase activity were compared in the liver, kidney, lung, whole intestine and carbon tetrachloride-induced liver cytosols. Kinetic parameters of the reaction were also compared for the main forms of choline kinase protein from these tissues. The overall results suggested strongly that choline kinase does not exist in one particular active form but exists in multiple forms in rat tissues. In the study present here, the electrophoretic patterns of both choline kinase and ethanolamine kinase activities were compared in rat liver, kidney, lung and intestinal cytosols. The results strongly supported the view that both kinase activities are represented on the same enzyme protein(s) in each of the rat tissues examined.
Animals↗
Deletion and alanine mutation analyses for the formation of active homo- or hetero-dimer complexes of mouse choline kinase-alpha and -beta.
Choline kinase (CK) is the first-step regulatory enzyme for the biosynthesis of phosphatidylcholine in all mammalian cells. It exists as at least three isoforms (alpha1, alpha2 and beta) that are encoded by two separate genes termed ck-alpha and ck-beta. The active enzyme has been proposed to consist of either their homo- or hetero-dimeric forms. Here, we report on the identification of several essential domains and amino acid residues involved in their active dimer formation. Full-length cDNAs or their truncated or alanine-mutated versions for mouse CK-alpha1 and CK-beta tagged with either HA or Myc at their N-termini were expressed in COS-7 cells. Each dimer formation was analyzed by immuno-precipitation followed by Western blotting. Kinetic analysis for CK reaction was performed with different expression products. Both the N-terminal domain-1 and C-terminal portions (E424-K430 for CK-alpha1 and Q379-K385 for CK-beta) were shown to be critical for the formation of active homo- or hetero-dimer complex. Interestingly, D320 in the CK-motif of CK-alpha1 was found to be essential for alpha1/alpha1 homo-dimerization but not for alpha1/beta hetero-dimerization. A mutation of the corresponding D276 of CK-beta to A276 did not show any effect on either its homo- or hetero-dimerization but it caused a strong inhibition of CK activity in either case.
Alanine↗
Evidence for the existence of a single enzyme catalyzing the phosphorylation of choline and ethanolamine in primate lung.
Choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) has been isolated and purified 1000-fold from adult African Green monkey lung with a yield of 10%. The purified enzyme also phosphorylated ethanolamine (ratio of ethanolamine kinase to choline kinase = 0.30). This ratio remained constant throughout the purification procedure. The Km 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.
Animals↗
Purification and characterization of choline/ethanolamine kinase from rat liver.
Choline kinase, the first enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis, was purified 26,000-fold from rat liver to a specific activity of 143,000 nmol.min-1.mg-1 protein. The subunit molecular mass was 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the apparent native molecular mass was 160 kDa by size exclusion chromatography, suggesting a tetrameric structure. Two peaks of choline kinase activity were obtained by chromatofocusing. These isoforms eluted at pH 4.7 (CKI) and 4.5 (CKII). CKII appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Peptide mapping of two isoforms indicated a high degree of similarity, although there were peptides not common to both. Ethanolamine kinase activity copurified with both isoforms. The ratio of choline to ethanolamine kinase activity was 3.7 +/- 0.7 throughout the purification procedure. Choline and ethanolamine were mutually competitive inhibitors. The respective Km values, 0.013 and 1.2 mM, were similar to the Ki values of 0.014 and 2.2 mM. An antibody raised against CKII immunoprecipitated both choline and ethanolamine kinase activities from liver cytosol at the same titer. These data suggest that both activities reside on the same protein and occur at the same active site. Similarly, both activities were immunoprecipitated from brain, lung, and kidney cytosols. Western blot analysis showed both purified liver isoforms, as well as brain, lung and kidney enzymes, to have a molecular mass of 47 kDa.
Acetates↗
Choline pathways during normal and stimulated renal growth in rats.
Cellular membrane synthesis occurs during normal and stimulated renal growth. Choline in the kidney is utilized as a precursor for membrane synthesis via the choline kinase reaction. We investigated choline phosphorylation during normal and stimulated renal growth. Rapidly growing neonatal rat kidneys contained relatively high levels of choline kinase activity (61 pmol phosphorylcholine/min per mg protein). Choline kinase activity and phosphorylcholine production then fell gradually over the 1st mo of life; by 1 mo phosphorylcholine production was 34 pmol phosphorylcholine/min per mg protein. Choline kinase activity increased by 27% (P less than 0.001) in 28-day-old rats when renal growth was stimulated by contralateral nephrectomy; the increase occurred within 2 h after surgery. Thus, changes in the activity of this important enzyme in the initiation of membrane synthesis is associated both with normal renal development and with adaptation to nephron loss. The findings further suggest that the cell membrane may be involved in the initiation of compensatory renal growth.
Animals↗
Phosphatidylcholine biosynthesis and choline transport in the anaerobic protozoon Entodinium caudatum.
Choline accumulation and phosphatidylcholine biosynthesis were investigated in the choline-requiring anaerobic protozoon Entodinium caudatum by incubating whole cells or subcellular fractions with [14C] choline, phosphoryl [14C] choline and CDP-[14C] choline. 2. All membrane fractions contained choline kinase (EC 2.7.1.32) and CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2), although the specific activities were less in the cell-envelope fraction. Choline phosphate cytidylyltransferase (EC 2.7.7.15) was limited to the supernatant, and this enzyme was rate-limiting for phosphatidylcholine synthesis in the whole cell. 3. Synthesis of phosphatidylcholine from free choline by membranes was only possible in the presence of supernatant. Such reconstituted systems required ATP (2.5 mM), CTP (1 mM) and Mg2+ (5 mM) for maximum synthesis of the phospholipid. CTP and Mg2+ were absolute requirements. 4. Hemicholinium-3 prevented choline uptake by the cells and was strongly inhibitory towards choline kinase; the other enzymes involved in phosphatidylcholine synthesis were minimally affected. 5. Ca2+ ions (0.5 mM) substantially inhibited CDP-choline-1,2-diacylglycerol cholinephosphotransferase in the presence of 15 mM-Mg2+, but choline phosphate cytidylyltransferase and choline kinase were less affected. 6. No free choline could be detected intact cells even after short (10-180s) incubations or at temperatures down to 10 degrees C. The [14C] choline entering was mainly present as phosphorylcholine and to a lesser extent as phosphatidylcholine. 7. It is suggested that choline kinase effectively traps any choline within the cell, thus ensuring a supply of the base for future growth. At low choline concentrations the activity of choline kinase is rate-limiting for choline uptake, and the enzyme might possibly play an active role in the transport phenomenon. Thus the choline uptake by intact cells and choline kinase have similar Km values and show similar responses to temperature and hemicholinium-3.
Calcium↗
Extracellular calcium stimulates DNA synthesis in synergism with zinc, insulin and insulin-like growth factor I in fibroblasts.
In serum-starved mouse NIH 3T3 fibroblasts cultured in 1.8 mM Ca2+-containing medium, addition of 0.75-2 mM extra Ca2+ stimulated DNA synthesis in synergism with zinc (15-60 microM), insulin and insulin-like growth factor I. Extra Ca2+ stimulated phosphorylation/activation of p42/p44 mitogen-activated protein kinases by an initially (10 min) zinc-independent mechanism; however, insulin, and particularly zinc, significantly prolonged Ca2+-induced mitogen-activated protein kinase phosphorylation. In addition, extra Ca2+ activated p70 S6 kinase by a zinc-dependent mechanism and enhanced the stimulatory effect of zinc on choline kinase activity. Insulin and insulin-like growth factor I also commonly increased both p70 S6 kinase and choline kinase activities. In support of the role of the choline kinase product phosphocholine in the mediation of mitogenic Ca2+ effects, cotreatments with the choline kinase substrate choline (250 microM) and the choline kinase inhibitor hemicholinium-3 (2 mM) enhanced and inhibited, respectively, the combined stimulatory effect of extra Ca2+ (3.8 mM total) and zinc on DNA synthesis. In various human skin fibroblast lines, 1-2 mM extra Ca2+ also stimulated DNA synthesis in synergism with zinc and insulin. The results show that in various fibroblast cultures, high concentrations of extracellular Ca2+ can collaborate with zinc and certain growth factors to stimulate DNA synthesis. Considering the high concentration of extracellular Ca2+ in the dermal layer, Ca2+ may promote fibroblast growth during wound healing in concert with zinc, insulin growth factor-I insulin, and perhaps other growth factors.
3T3 Cells↗
Comparison of the enzyme activities of phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol synthesis in freshly isolated type II pneumocytes and whole lung from the adult rat.
We compared the activities of enzymes of phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol synthesis in whole lung tissue and freshly isolated type II pneumocytes from adult rats. The activities of 1-acylglycerophosphocholine acyltransferase and CDPdiacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase were 2.9- and 4.4-fold higher, respectively, in type II cell sonicates than in whole lung homogenates. There was little difference between the type II cells and whole lung in the activities of choline kinase, choline-phosphate cytidyltransferase, cholinephosphotransferase, phosphatidate phosphatase, phosphatidate cytidylytransferase or CDPdiacylglycerol-inositol 3-phosphatidyltransferase. Since the type II cell is the source of pulmonary surfactant, and disaturated phosphatidylcholine and phosphatidylglycerol are major components of surfactant, it is of interest that this cell is enriched in the activities of enzymes exclusively involved in the synthesis of these lipids. In view of possible proteolytic damage during isolation we compared freshly isolated type II cells with those cultured for 1 day. The rates of incorporation of [methyl-3H]choline and [2-3H]glycerol into phospholipids, L-[U-14C]phenylalanine into protein and [methyl-3H]thymidine into DNA were the same in the freshly isolated and cultured cells. The composition of the phospholipids synthesized from [2-3H]glycerol and sodium [1-14C]acetate were also the same. The freshly isolated cells were at least 90% pure and did not release significant amounts of lactate dehydrogenase. Since use of freshly isolated cells avoids cell loss during culture they provide an attractive alternative, particularly in studies requiring large amounts of material.
1-Acylglycerophosphocholine O-Acyltransferase↗
The cellular mechanism of glucocorticoid acceleration of fetal lung maturation. Fibroblast-pneumonocyte factor stimulates choline-phosphate cytidylyltransferase activity.
The cellular mechanism by which glucocorticoids stimulate phosphatidylcholine biosynthesis has been studied in the fetal rat lung in vivo and in cultured fetal rat lung cells of varying levels of complexity. Administration of dexamethasone to pregnant rats at 18 days gestation resulted in a significant increase in saturated phosphatidylcholine content in fetal lung 24 h after injection. Dexamethasone administration increased the activity of fetal lung choline-phosphate cytidylyltransferase by 34%. It had no effect on the activities of fetal lung choline kinase and choline phosphotransferase. Exposure of fetal lung type II cells in organotypic cultures (which contain both type II cells and fibroblasts) to cortisol resulted in a 1.6-fold increase in the incorporation of [Me-3H]choline into saturated phosphatidylcholine. The activities of the enzymes in the choline pathway for the de novo biosynthesis of phosphatidylcholine were not significantly altered except for a 105% increase in choline-phosphate cytidylyltransferase activity. Treatment of monolayer cultures of fetal type II cells with cortisol-conditioned medium from fetal lung fibroblasts resulted in a 1.5-fold increase in saturated phosphatidylcholine production. This effect correlated with a doubling of choline-phosphate cytidylyltransferase activity. Additional evidence that this stimulatory action is mediated by fibroblast-pneumonocyte factor, produced by fetal lung fibroblasts in response to cortisol, was obtained. The factor was partially purified from cortisol-conditioned medium of fetal lung fibroblasts by gel filtration and affinity chromatography. Based on biological activity, a 3000-fold purification was obtained. Stimulation of saturated phosphatidylcholine synthesis in type II cells by fibroblast-pneumonocyte factor was maximal within 60 min of incubation. Pulse-chase experiments indicated that the stimulatory effect was correlated with an increased conversion of choline phosphate into CDP choline. Moreover, the enhanced phosphatidylcholine formation by fetal type II cells in response to fibroblast-pneumonocyte factor was accompanied by decreased levels of cellular choline phosphate. These findings further support the concept that glucocorticoid action on surfactant-associated phosphatidylcholine synthesis occurs ultimately at the level of the alveolar type II cell and involves fibroblast-pneumonocyte factor which stimulates the activity of choline-phosphate cytidylyltransferase.
Animals↗
A novel high-molecular mass mammalian ethanolamine kinase.
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.
Animals↗
Sympathetic neurons expressing cholinergic properties are poised to allocate choline symmetrically between acetylcholine and the phosphatidylcholine-generating pathway in growing neurites.
We have examined the question of how regenerating sympathetic neurons that are concomitantly induced to become cholinergic regulate choline allocation between ACh and the phospholipid synthetic pathway. The allocation of choline into ACh increased parabolically with time in culture, and by 3 weeks, cultures with neurites of approximately 6 mm length were incorporating over 85% of the choline locally in the neurites into four major metabolites: ACh, phosphorylcholine, cytidine diphosphocholine, and phosphatidylcholine. The near-equivalent distribution of labeled choline between intracellular choline, ACh, and phosphorylcholine was independent of time (5 min to 6 hr) and choline concentration (0.125-30 microM), phosphatidylcholine being the sole metabolite whose level in the neurites increased steadily with incubation time. Relative choline distribution into ACh and phosphorylcholine was unaltered even after a brief depolarizing prepulse, which caused a two- to fourfold enhancement in the total choline incorporated. These observations, allied with the similar half-saturation constants and Vmax values of CAT and choline kinase for intracellular choline, suggest that growing sympathetic neurons are poised to allocate choline symmetrically between the synthesis of ACh and phosphatidylcholine in the neurites. When, however, the supply of choline was limited either by replacement of Na+ in the medium with N-methyl-D-glucamine, or by vesamicol, a 90-97% reduction in intracellular choline caused a similar decline in ACh levels but synthesis of metabolites of the phosphatidylcholine pathway was maintained unperturbed, as if no drug was present. We suggest that this can be accounted for by a 10-fold increase in choline kinase activity. Thus, growing sympathetic neurons that express cholinergic properties not only maintain their chief cellular phosphatidylcholine-synthesizing activity concomitantly with ACh synthesis in the neurites, but may also preserve phosphatidylcholine synthesis more effectively than ACh synthesis when the supply of choline is perturbed. Relinquishing ACh synthesis during growth may be one way of conserving and encouraging neurite regeneration.
Acetylcholine↗
Evolutionary comparison of enzyme activities of phosphatidylcholine metabolism in the nervous system of an invertebrate (Loligo pealei), lower vertebrate (Mustelus canis) and the rat.
While steady-state kinetic parameters (metabolite pools, Km and activation energies) are partially known for the enzymes involved in phosphatidylcholine synthesis and degradation in mammalian brain, they are not available for the nervous system of lower vertebrates or invertebrates. Since the extent of evolutionary development of an enzyme is not known a priori, we evaluated the kinetic and thermodynamic parameters of choline kinase, CTP:phosphocholine cytidylyltransferase, choline phosphotransferase and glycerophosphorylcholine phosphodiesterase in squid (Loligo pealei) optic lobe, dogfish (Mustelus canis) and rat brain. For all these enzyme activities, basic similarities in Km and inhibitor effect were found. The same was true for the activation energies Ea, with the exception of squid choline kinase and dogfish cytidylyltransferase. Treatment of microsomal membranes with phospholipase C sharply inhibited cytidylyltransferase activity in all three animal species. In dogfish brain, glycerophosphorylcholine phosphodiesterase activity was undetectable. Our results are consistent with the notion that the kinetic properties of the enzyme activities leading to the preservation of the phosphatidylcholine membranous pool may have appeared early in metazoan evolution and been fully conserved in mammals.
Animals↗