The enzymatic oxidation of choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions, as such it is of potential interest for the genetic engineering of crops that do not naturally possess efficient pathways for the synthesis of glycine betaine, and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, one of the best characterized enzymes belonging to this pathway is the flavin-dependent choline oxidase from Arthrobacter globiformis. In this enzyme, choline oxidation proceeds through two reductive half-reactions and two oxidative half-reactions. In each of the reductive half-reactions the FAD cofactor is reduced to the anionic hydroquinone form (2 e- reduced) which is followed by an oxidative half-reaction where the reduced FAD cofactor is reoxidized by molecular oxygen with formation and release of hydrogen peroxide. In this dissertation the roles of selected residues, namely histidine at position 310, valine at position 464 and serine at position 101, that do not directly participate in catalysis in the reaction catalyzed by choline oxidase have been elucidated. The effects on the overall reaction kinetics of these residues in the protein matrix were investigated by a combination of steady state kinetics, rapid kinetics, pH, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects as well as X-ray crystallography. A comparison of the kinetic data obtained for the variant enzymes to previous data obtained for wild-type choline oxidase are consistent with the valine residue at position 464 being important for the oxidative half-reaction as well as the positioning of the catalytic groups in the active site of the enzyme. The kinetic data obtained for the serine at position 101 shows that serine 101 is important for both the reductive and oxidative half-reactions. Finally, the kinetic data for histidine at position 310 suggest that this residue is essential for both the reductive and oxidative half-reactions.

Choline oxidase catalyzes the two-step oxidation of choline to glycine betaine, one of limited osmoprotectants, with the formation of betaine aldehyde as an enzyme bound intermediate. Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism to withstand hyperosmolarity and elevated temperatures. This makes the genetic engineering of relevant plants which lack the property of salt accumulation of economic interest, and the biosynthetic pathway of the osmolyte a potential drug target in microbial infections. The reaction of alcohol oxidation occurs via a hydride ion tunneling transfer from the substrate donor to a flavin acceptor within a highly preorganized active site environment in which choline and FAD are in a rigidly close proximity. In this dissertation, factors contributing to the enzyme-substrate preorganization which is required for the hydride ion tunneling reaction mechanism in choline oxidase have been investigated. Crystallographic studies of wild-type choline oxidase revealed a covalent linkage between C8M atom of the FAD isoalloxazine ring and the N(3) atom of the side chain of a histidine at position 99, and a solvent excluded cavity in the substrate binding domain containing glutamic acid at position 312 as the only negatively charged amino acid residue in the active site of the enzyme. The role of the histidine residue and the contribution of the 8á-N(3)-histidyl covalent linkage of the flavin cofactor to the reaction of alcohol oxidation was investigated in a variant form of choline oxidase in which the histidine residue was replaced with an asparagine. The role of the glutamate residue and the importance of the spatial location of the negative charge at position 312 was investigated in variant forms of choline oxidase in which the negatively charged residue was replaced with glutamine and aspartate. Mechanistic data obtained for the variant enzymes and their comparison to previous data obtained for wild-type choline oxidase are consistent with the residues at positions 99 and 312 being important for relative positioning of the hydride ion donor and acceptor. The residues are important for the enzyme-substrate preorganization that is required for the hydride tunneling reaction in choline oxidase.

Choline oxidase catalyzes the flavin-dependent, two-step oxidation of choline to glycine betaine via the formation of an aldehyde intermediate. The oxidation of choline includes two reductive half-reactions followed by oxidative half-reactions. In the first oxidation reaction, the alcohol substrate is activated to its alkoxide via proton abstraction and oxidized via transfer of a hydride from the alkoxide α-carbon to the N(5) atom of the enzyme-bound flavin. In the wild-type enzyme, proton and hydride transfers are mechanistically and kinetically uncoupled. The role of Ser101 was investigated in this dissertation. Replacement of Ser101 with threonine, alanine, cysteine, or valine demonstrated the importance of the hydroxyl group of Ser101 in proton abstraction and in hydride transfer. Moreover, the kinetic studies on the Ser101Ala variant have revealed the importance of a specific residue for the optimization of the overall turnover of choline oxidase. The UV-visbible absorbance of Ser101Cys suggests Cys101 can form an adduct with the C4a atom of the flavin. The mechanism of formation of the C4a-cysteinyl adduct has been elucidated. D-arginine dehydrogenase (DADH) catalyzes the oxidation of D-amino acids to the corresponding imino acids, which are non-enzymatically hydrolyzed to α-keto acids and ammonia. The enzyme is strick dehrogenase and deoesnot react with molecular oxygen. Steady state kinetic studies wirh D-arginine and D-histidine as a substrate and PMS as the electron acceptor has been investigated. The enzyme has broad substrate specificity for D-amino acids except aspartate, glutamate and glycine, with preference for arginine and lysine. Leucine is the slowest substrate in which steady state kinetic parameters can be obtained. The chemical mechanism of leucine dehydrogenation catalyzed by DADH was explored with a combination of pH, substrate and solvent kinetic isotope effects (KIE) and proton inventories by using rapid kinetics in a stopped-flow spectrophotometer. The data are discussed in the context of the crystallographic structures at high resolutions (<1.3 Å) of the enzyme in complex with iminoarginine or iminohistidine.

Young male rats fed a choline-deficient diet for three days accumulated triacylglycerol (TG) in the liver, and had reduced very low density lipoprotein (VLDL), but not high density lipoprotein (HDL), levels in the plasma. Cultured hepatocytes obtained from these rats were used as a model system to investigate how choline deficiency affected hepatic lipogenesis, apolipoprotein synthesis and lipoprotein secretion. When the cells were cultured in a medium free of choline and methionine, the secretion of TG and phosphatidylcholine (PC) was impaired. Supplementation of choline, methionine or lysophosphatidylcholine (lysoPC) to the culture medium increased the secretion of these lipids to normal levels, and stimulated PC biosynthesis. Fractionation of the secreted lipoproteins by ultracentrifugation revealed that the reduced secretion of TG and PC from choline-deficient cells was mainly due to impaired secretion of VLDL. The secretion of HDL and lipid-free proteins (for example albumin), however, was not affected by choline and methionine deficiency. Supplementation of betaine and homocysteine also stimulated PC biosynthesis and enhanced hepatic VLDL secretion. However, supplementation of ethanolamine, N-monomethylethanol-amine or N, N-dimethylethanolamine did not correct the impaired VLDL secretion from the hepatocytes, although an active synthesis of phosphatidylmonomethyl-ethanolamine (PMME) and phosphatidyldimethylethanolamine was observed. Choline deficiency had no effect on the rate of incorporation of [³H]leucine into cellular apolipoprotein B, E and C or on the rate of disappearance of radioactivity from the labeled apolipoproteins. These results suggest that biosynthesis of PC is specifically required for hepatic VLDL (but not HDL) secretion, and any one of the three synthetic pathways, the CDP-choline pathway, methylation of phospha-tidylethanolamine (PE) or reacylation of lysoPC, is sufficient to provide the required PC. The total activity of cytidylyltransferase in liver was unchanged in choline deficiency. However, choline deficiency caused an abnormal distribution of cytidylyltransferase activity between rat liver cytosol and microsomes (mainly endoplasmic reticulum), a decrease in the cytosolic enzyme activity and an increase in the microsomal enzyme activity. In cultured hepatocytes from the choline-deficient rat, the abnormally distributed cytidylyltransferase activity could be rapidly reversed by the addition of choline, but not lysoPC, to the culture medium. The stimulated microsomal activity of cytidylyltransferase during choline deficiency might be a mechanism whereby the cells could more effectively utilize phosphocholine to maintain a normal CDP-choline level in the choline-deficient liver. Rat liver PE N-methyltransferase catalyzes all three transmethylation reactions in the conversion of PE to PC. The in vitro activity of PE N-methyltransferase was increased in choline-deficient livers using endogenous PE as the methyl group acceptor. However, no significant changes were observed in the enzyme activity when exogenous PMME was used as the methyl group acceptor. Addition of methionine to the cultured hepatocytes obtained from choline-deficient rats resulted in a concomitant reduction in cellular PE levels and the specific activity of PE-dependent methyltransferase. However, the specific activity of PMME-dependent methyltransferase was not significantly altered upon the addition of methionine. No change in PE N-methyltransferase activity was observed in the cultured hepatocytes supplemented with choline. Immunoblotting of PE N-methyltransferase, in crude liver microsomes and in membrane fractions of cultured hepatocytes, revealed that the enzyme mass was not altered by choline and methionine deficiency. Thus, hepatic PE N-methyltransferase is preserved in choline deficiency, and its activity is probably dependent on the availability of metabolic substrates (i.e. methionine and PE). / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21806号 / 農博第2319号 / 新制||農||1065(附属図書館) / 学位論文||H31||N5178(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 松井 徹, 教授 久米 新一, 教授 廣岡 博之 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23055号 / 医博第4682号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 伊達 洋至, 教授 長船 健二 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Introduction [11C]choline is a very effective PET radiopharma-ceutical for the study of prostate cancer. To support the increasing demand for [11C]choline, several different synthetic approaches have been described in the literature, including different automated production methods using remote-controlled synthesis modules [1–4]. The most popular method uses a C18 Sep-Pak as solid support for methylation and, subsequently, a CM Sep-Pak for purification [2]. We report an optimized method for producing [11C]choline using only one CM Sep-Pak for both reaction and purification as was shown in the literature [4]. For synthesis of [11C]choline we used two modules Tracerlab FXC for preparation of methylation reagent [11C]CH3I and GPF-101 for [11C]choline synthesis. Material and Methods TracerlabFXC GE, GPF-101 Veenstra Instrument, 2-(dimethylamino)-ethanol (DMAE) ABX, Sep-Pak Light Accell Plus CM cation-exchange cartridges Waters used without conditioning, precursor 50 µL of DMAE dissolved in 25 µL of ethanol and loaded on a CM Sep-Pak. Schematic diagram of the automated system for the production of [11C]choline is given below. [11C]CH4 was produced in two standard Nitra target IBA irradiation of mixture 90 % N2/10 % H2 with 15 MeV protons using dual beam. Results and Conclusion [11C]CH4 was prepared in the targets and connected with Tracerlab FXC. [11C]CH3I was pre-pared in a loop in which allowed to react of elemental iodine at a temperature 720 oC. Con-version to [11C]CH3I usually is around 50% uncorrected activity. Activity is within the range 15–18 GBq of [11C]CH3I and time of production 10 min. Synthesis of [11C]choline is based on the reaction DMAE with [11C]CH3I on a Accell Plus CM cation-exchange column which serves both as a support for reaction and for separation of choline from DMAE by ethanol washing. The basic parameters are shown in TABLE 1. Beam current 2X 20 µA Irradiation time 30 min DMAE 50 µl Synthesis time from EOB 25 min Absolute yield without correction 6.6 GBq Radiochemical purity > 99 % Residual DMAE in product < 5 ppm Ethanol < 1000 mg/L pH 4.5–8.5 TABLE 1. Reaction parameters and result of production of [11C]choline syntheses Conclusion We have applied a simple synthesis method for [11C]choline preparation using automated commercial equipments with one column used both for reaction and separation purpose. The main advantage of using one column is lower contamination of the product [11C]choline with DMAE. When for synthesis of [11C]choline two columns C18 for synthesis and CM for separation is used, higher contamination of DMAE can be found in the product due to a release of DMAE from C18 column.

Background: One key component in the development of sustainable farming and foodstuffs is to increase the cultivation of legumes, due to their environmental and health benefits. Legumes contain several essential vitamins and minerals, protein and fiber, but cultivation can be problematic due to their weak stem strength which results in loss of crops as a result of crop lodging and susceptibility to weed invasion. One possible solution is co-cultivation with cereal crops such as oats as they provide support and outcompete weed growth, however, it is still unknown if co-cultivation will affect the nutrient content of lentils. Aim: The thesis has two aims: first, to compare choline, betaine, folate, resistant starch and dietary fiber content in two different types of lentils: Gotland lentils and Anicia lentils; and second to examine if cocultivation with oats will affect the nutrient content of the two lentil types. Methods: Betaine and choline were analyzed through high pressure liquid chromatography-mass spectrometry (HPLC-MS) and folate through high pressure liquid chromatography with fluorometric/UV detection (HPLC-FL/UV). Resistant starch, non-resistant starch and dietary fiber were analyzed according to enzymatic assay kits by Megazyme. Results: Significant differences in nutrient content between Anicia and Gotland lentils were seen regarding resistant starch content, with Gotland showing a 50 % higher content. A significantly greater choline content was found in Anicia lentil samples that were co-cultivated with oats, showing approx. a 15 % higher choline content. Gotland lentils co-cultivated with oats showed a significantly greater choline, resistant starch and dietary fiber content by 15 %, 70 % and 10 %, respectively. Conclusions: There was no reported significant difference in choline, betaine, folate and dietary fiber content, but in resistant starch between the two lentil types, with Gotland lentil showing a higher resistant starch content. There appeared to be a positive effect of co-cultivation with oats since a significantly higher choline content in both lentil types co-cultivated with oats and a significant increase resistant starch and dietary fiber in Gotland lentils co-cultivated with oats was reported. This suggests that cocultivation can lead to an increase in nutritional content for some nutrients in Gotland and Anicia lentils. The dietary fiber analysis confirmed that legumes are a great source of fiber by one portion providing approx. half the recommended daily amount. Cultivation and consumption of lentils can give both environmental and health benefits. Further studies are needed to explore other pulses and effect on other nutrients. / Svenska ekologiska linser

Cholinergic enzymes and muscarinic receptors in nuclei of rat medulla oblongata were examined after unilateral vagotomy to determine their association with efferent vagal neurons. Vagotomy caused an ipsilateral depletion of acetylcholinesterase from the dorsal motor nucleus of the vagus (DNV) and the nucleus ambiguus (NA). Choline acetyltransferase activity was reduced in ipsilateral DNV, nucleus tractus solitarius and rostral NA. Muscarinic receptor localization by autoradiography with [3H]quinuclidinyl benzilate (QNB) revealed marked intranuclear variations in receptor density. Vagotomy had no effect on the QNB binding pattern. Loss of cholinergic enzymes is a consistent response of motor and preganglionic autonomic neurons to axotomy. Depletion of muscarinic receptors is an additional component of axon reaction in brain stem motoneurons. Accordingly, previous studies have shown a decrease in neurotransmitter-related proteins after axotomy of motoneurons. In the present study, cholinergic enzymes were depleted from axotomized vagal neurons but receptors were not. It is concluded that muscarinic receptors in the DNV and NA are not associated with vagal efferent neurons.

Trimethyltin (TMT) destroys specific subfields of the hippocampus in the rat. TMT also increases choline acetyltransferase (ChAT) activity in CA1 of Ammon's horn and the outer molecular layer of the dentate gyrus. This observation suggests that axonal sprouting occurs in the cholinergic septohippocampal system in response to TMT. However, neither does-response nor time course data are available for the effects of TMT on this enzyme. The effects of three dose levels of TMT on ChAT activity in CA1 and the dentate gyrus were determined in Experiment 1 and ChAT activity in these two areas was measured at six time points following exposure to TMT in Experiment 2. Only the highest dose of TMT (6 mg/kg) significantly increased ChAT activity. ChAT activity in the dentate gyrus increased significantly by 3 d after administration and continued to increase until 21 d after exposure. A significant increase was not observed in CA1 until 7 d after exposure to TMT. Asymptotic levels were still reached at d 21. These results indicate a steep dose-response curve for TMT-induced changes in ChAT activity in the hippocampal formation and that this marker of cholinergic activity is more sensitive to perturbation by TMT in the dentate gyrus than Ammon's horn.