Choline kinase and ethanolamine kinase are separate, soluble enzymes in rat liver.

Choline kinase and ethanolamine kinase are located in the cytosol from rat liver and have been copurified more than 500-fold by affinity chromatography [P. J. Brophy and D. E. Vance (1976) FEBS Lett. 62, 123-125]. Kinetic properties of the two activities were determined. Choline kinase had a Km for choline of 0.033 mM and ethanolamine was a competitive inhibitor (Ki = 6.2 mM). Ethanolamine kinase had a Km for ethanolamine of 7.7 mM and choline was a 'mixed' type of inhibitor with a Ki of 0.037 mM. Both enzymes activities responded in a similar fashion to the adenylate energy charge. Betaine and choline phosphate partially inhibited both kinases with a 93% inhibition of the ethanolamine kinase by 5 mM choline phosphate. CTP and ethanolaminephosphate partially inhibited the ethanolamine kinase, but not the choline kinase. Other metabolites tested had negliglible effects on both kinases. The affinity-column-purified enzyme was analyzed by disc gel electrophoresis which resolved the two activities. Hence, although many of the properties of the two activities are similar, choline kinase and ethanolamine kinase must be separate enzymes. Analysis of rat liver cytosol by disc gel electrophoresis indicated four isoenzymes for choline kinase and ethanolamine kinase.     

Complete co-purification of choline kinase and ethanolamine kinase from rat kidney and immunological evidence for both kinase activities residing on the same enzyme protein(s) in rat tissues

Choline kinase and ethanolamine kinase were completely co-purified from rat kidney cytosol through acid treatment, ammonium sulfate fractionation, DEAE-cellulose column chromatography, Sephadex G-150 gel filtration followed by choline-Sepharose affinity chromatography. The final preparation appeared to be highly homogeneous with respect to both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Ishidate, K., Nakagomi, K. and Nakazawa, Y. (1984) J. Biol. Chem. 259, 14706-14710). Throughout the purification steps, the ratio of ethanolamine kinase activity to choline kinase activity was almost constant in a range of 0.3-0.4, which strongly indicated that, in rat kidney, both activities could reside on a single enzyme protein. The rabbit polyclonal antibody raised against highly purified rat kidney choline (ethanolamine) kinase protein inhibited both choline and ethanolamine kinase activities in a parallel manner in crude enzyme preparations not only from rat kidney, but also from rat liver, lung and intestinal cytosols. The results, together with our previous findings, suggested strongly that, in rat tissues, at least large portions of both kinase activities are present on the same enzyme protein(s). The kinetic properties of both kinase reactions with the highly purified kidney enzyme were compared in some detail and the overall result suggested that choline kinase and ethanolamine kinase activities may not have a common active site on a single enzyme protein.     

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.     

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.Abbreviations Etn ethanolamine - MeEtn N-monomethylethanolamine - Me₂Etn N, N-dimethylethanolamine     

Purification and properties of an ethanolamine-serine base exchange enzyme of rat brain microsomes

The activity of an ethanolamine and serine base exchange enzyme of rat brain microsomes was copurified to near homogeneity. The purification sequence involved detergent solubilization, Sepharose 4B column chromatography, phenyl-Sepharose 4B column chromatography, glycerol gradient sedimentation, and agarose-polyacrylamide gel electrophoresis under non-denaturing conditions. The ratio of the ethanolamine and serine base exchange activities remained almost constant during purification, and both enzyme activities were enriched 25-fold over the initial microsomal suspension. The final enzyme preparation which contained both enzyme activities showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel, having an apparent molecular mass of about 100 kDa. Serine inhibited the ethanolamine incorporation by this preparation and ethanolamine inhibited the serine incorporation. The competitive nature of this inhibition was apparent from Lineweaver-Burk plots, suggesting that the enzyme catalyzes the incorporation of both ethanolamine and serine into their corresponding phospholipids. The Km and Ki values for ethanolamine were quite similar, being 0.02 and 0.025 mM, respectively. The Km and Ki values for serine were also quite similar being 0.11 and 0.12 mM, respectively. The pH optimum was the same at 7.0 with both substrates. The optimum Ca2+ concentration was 8 mM for serine incorporation.     

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.     

Choline kinase and ethanolamine kinase activity in the cytosol of nerve endings from rat forebrain.

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.     

Effects of deoxycholate and phospholipase A2 on choline and ethanolamine phosphotransferases of chicken brain microsomes.

Ethanolamine phosphotransferase (EC 2.7.8.1) and choline phosphotransferase (EC 2.7.8.2) activities were assayed in fresh microsomes from adult chicken brains with either diacylglycerols or alkylacylglycerols. Pretreatment of microsomes with 1.25 mM sodium deoxycholate, a concentration less than the critical micelle concentration, produced a slight inhibition of choline phosphotransferase activity. A deoxycholate concentration (5.0 mM) greater than the critical micelle concentration (3.0 mM) decreased the choline phosphotransferase activity by more than 70% but had no effect on ethanolamine phosphotransferase activity. Inclusion of 1.25 mM deoxycholate in the assay medium decreased choline phosphotransferase activity 35% but increased ethanolamine phosphotransferase activity 50%. The deoxycholate appeared to inactive the choline phosphotransferase. Phospholipase A2 (Vipera russelli) treatments of microsomes removed phosphoglycerides and decreased both phosphotransferase activities to a similar extent. Decreased activities are probably due to disruption of the membrane structure. Choline and ethanolamine phosphotransferase activities are apparently in different enzymes which lack specificity for the type of diglyceride. Thus, the systematic names should include 1,2-diradyl-sn-glycerol instead of 1,2-diacyl-sn-glycerol.     

Lipid metabolism in germinating seeds. Purification of ethanolamine kinase from soya bean.

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.     

Multiple isoforms of choline kinase from Caenorhabditis elegans: cloning,expression, purification,and characterization

Choline 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 K(m) values for CKA-2 were 1.6 and 2.4 mM for choline and ATP, respectively, and k(cat) 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 K(m) values for choline and ATP of 13 and 0.7 mM, and k(cat) 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.     

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.     

Choline Synthesis in Spinach in Relation to Salt Stress

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.     

Partial purification and characterization of monomethylethanolamine kinase and dimethylethanolamine kinase activities from rat liver.

Monomethylethanolamine (MEA) kinase and dimethylethanolamine (DEA) kinase activities were purified 950 and 750 fold respectively from rat liver by conventional procedures. Certain properties of the partially purified enzyme preparation suggest that they are different from both choline kinase activity and ethanolamine kinase activity and differ from one another. This is based upon the following observations: 1. The heat stabilities of MEA kinase and DEA kinase activities are significantly different from one another and are different from the stability of choline kinase and ethanolamine kinase activities. 2. K+ in the presence of Mg2+ increases MEA kinase activity by 100% but has no effect on DEA kinase activity. 3. Different Ki values and the types of inhibition by several structurally related amino alcohols were found for MEA kinase and DEA kinase activities. 4. The purification fold of MEA kinase and DEA kinase are different from each other and from that of choline kinase and ethanolamine kinase.     

Influence of choline and ethanolamine administration on choline and ethanolamine phosphorylating activities of mouse liver and kidney.

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.     

Purification and characterization of methylmalonate-semialdehyde dehydrogenase from rat liver. Identity to malonate-semialdehyde dehydrogenase

Methylmalonate semialdehyde dehydrogenase was purified from rat liver in order to define the distal portion of valine catabolism and related pathways in mammals. The purified enzyme is active with malonate semialdehyde and consumes both stereoisomers of methylmalonate semialdehyde, implicating a single semialdehyde dehydrogenase in the catabolism of valine, thymine, and compounds catabolized by way of beta-alanine. The oxidation of malonate and methylmalonate semialdehydes by this enzyme is CoA-dependent, the products being acetyl-CoA and propionyl-CoA, respectively. Expected activity with ethylmalonate semialdehyde as substrate was not found. Methylmalonate semialdehyde dehydrogenase was separated on DEAE-Sephacel into two isoforms which differ in mobility during nondenaturing polyacrylamide gel electrophoresis. The two forms are immunologically cross-reactive and exhibit the same N-terminal sequence, suggesting that one form is the product of the other. The monomer molecular mass, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 58 kDa. The native molecular mass, estimated by gel filtration, was 250 kDa, suggesting a tetrameric structure.     

Casein kinase II stimulates rat liver mitochondrial glycerophosphate acyltransferase activity

Rat liver mitochondrial glycerophosphate acyltransferase (mtGAT) possesses 14 consensus sites for casein kinase II (CKII) phosphorylation. To study the functional relevance of phosphorylation to the activity of mtGAT, we treated isolated rat liver mitochondria with CKII and found that CKII stimulated mtGAT activity approximately 2-fold. Protein phosphatase-lambda treatment reversed the stimulation of mtGAT by CKII. Labeling of both solubilized and non-solubilized mitochondria with CKII and [gamma-32P]ATP resulted in a 32P-labeled protein of 85kDa, the molecular weight of mtGAT. Our findings suggest that CKII stimulates mtGAT activity by phosphorylation of the acyltransferase. The significance of this observation with respect to hormonal control of the enzyme is discussed.     

Several lines of evidence demonstrating that Plasmodium falciparum, a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine

In Plasmodium falciparum-infected erythrocyte homogenates, the specific activity of ethanolamine kinase (7.6 +/- 1.4 nmol phosphoethanolamine/10(7) infected cells per h) was higher than choline kinase specific activity (1.9 +/- 0.2 nmol phosphocholine/10(7) infected cells per h). The Km of choline kinase for choline was 79 +/- 20 microM, and ethanolamine was a weak competitive inhibitor of the reaction (Ki = 92 mM). Ethanolamine kinase had a Km for ethanolamine of 188 +/- 19 microM, and choline was a competitive inhibitor of ethanolamine kinase with a very high Ki of 268 mM. Hemicholinium 3 inhibited choline kinase activity, but had no effect on ethanolamine kinase activity. In contrast, D-2-amino-1-butanol selectively inhibited ethanolamine kinase activity. Furthermore, when the two enzymes were subjected to heat inactivation, 85% of the choline kinase activity was destroyed after 5 min at 50 degrees C, whereas ethanolamine kinase activity was not altered. Our results indicate that the phosphorylation of choline and ethanolamine was catalyzed by two distinct enzymes. The presence of a de novo phosphatidylethanolamine Kennedy pathway in P. falciparum contributes to the bewildering variety of phospholipid biosynthetic pathways in this parasitic organism.     

Protein kinase C located in rat liver nuclei. Partial purification and biochemical and immunochemical characterization

In the rat liver homogenate, maximal protein kinase C activity was found at two calcium concentrations (1.75 and 3.5 mM). Subcellular fractionation of the liver homogenate revealed that the protein kinase C activity requiring 1.75 mM calcium was present only in the cytosolic and particulate subcellular fractions. The protein kinase C activity requiring 3.5 mM calcium concentration was mainly located in the rat liver nuclei preparation. About 19% of the liver homogenate protein kinase C activity requiring 3.5 mM calcium was present in the nuclei. Goat anti-rat brain protein kinase C antibodies revealed a single immunoreactive band at 80-82 kDa in the rat liver nuclear, particulate, or cytosolic fractions. Based on the ratio of plasma membrane marker enzyme activity determined in the nuclear preparation, the purity of the isolated nuclei was ascertained. Rat liver nuclear protein kinase C activity has been partially purified. The purification steps sequentially employed were Triton X-100 extraction of isolated nuclei, DEAE-cellulose chromatography, Phenyl-Superose, and Mono Q (fast protein liquid) chromatography. The final purification step revealed, by silver nitrate staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two protein bands at 80 and 66 kDa, respectively. These findings provide definitive data regarding the nuclear location of protein kinase C. The nuclear location of protein kinase C may lead to an understanding of the molecular pathway involved in signal transduction from the plasma membrane to the nucleus.     

A comparison of 1-aminocyclopropane-1-carboxylate synthase in vitro translation product and in-vivo-labeled protein in ripening tomatoes

We determined the time course of increases in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in ripening tomato (Lycopersicon esculentum (L.) Mill.) pericarp discs following wounding and treatment with 75 mM LiCl. Over the course of 24 h, we detected oscillations in the amount of enzyme activity from an initial peak at 6 h to a subsequent, even higher level at 18 h. In-vitro translation products derived from poly(A)⁺ RNAs isolated at various times of treatment and in-vivo-labeled proteins were immunoprecipitated using antibodies specific for ACC synthase. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that wounding and treatment with LiCl induced an accumulation of translatable ACC-synthase-specific mRNAs. In addition, single, prominent bands were apparent for both in-vivo and in-vitro samples but their molecular masses differed. It appears that the in-vitro translation product is a polypeptide of 56 kDa while the in-vivo-labeled enzyme has a molecular mass of 47 kDa. The authors greatly appreciate the skilled technical assistance of Renate deZacks and Gail Robinson. This research was supported by the National Science Foundation through Grant No. DCB-8718873 and by the Department of Energy through Contract No. DE-AC02-76ER-01338.     

Characterization of soybean choline kinase cDNAs and their expression in yeast and Escherichia coli.

An expressed sequence tag from Arabidopsis that displayed sequence homology to mammalian and yeast choline kinases was used to isolate choline kinase-like cDNAs from soybean (Glycine max L.). Two distinct cDNAs, designated GmCK1 and GmCK2, were recovered that possessed full-length reading frames, each sharing approximately 32% identity at the predicted amino acid level with the rat choline kinase. A third unique choline kinase-like cDNA, GmCK3, was also identified but was not full length. Heterologous expression of GmCK1 in yeast (Saccharomyces cerevisiae) and GmCK2 in both yeast and Escherichia coli demonstrated that each encodes choline kinase activity. In addition to choline, other potential substrates for the choline kinase enzyme include ethanolamine, monomethylethanolamine (MME), and dimethylethanolamine (DME). Both soybean choline kinase isoforms demonstrated negligible ethanolamine kinase activity. Competitive inhibition assays, however, revealed very distinct differences in their responses to DME and MME. DME effectively inhibited only the GmCK2-encoded choline kinase activity. Although MME failed to effectively inhibit either reaction, an unexpected enhancement of choline kinase activity was observed specifically with the GmCK1-encoded enzyme. These results show that choline kinase is encoded by a small, multigene family in soybean comprising two or more distinct isoforms that exhibit both similarities and differences with regard to substrate specificity.