Hiroyasu Taguchi

Glyceryl-ether monooxygenase [EC 1.14.16.5]. A microsomal enzyme of ether lipid metabolism

The history, biological, and medical aspects of glyceryl ethers, as well as their chemical syntheses, biosynthesis, and their chemical and physical properties are briefly reviewed as background information for appreciating the importance of the enzyme glyceryl‐ether monooxygenase, and for embarking on new studies of this enzyme. The occurrence, isolation and general properties of the microsomal, membrane‐bound, glyceryl‐ether monooxygenase from rat liver are described. Radiometric, nonradiometric, and coupled and direct spectrophotometric assays for this enzyme are detailed. The effects of detergents on the kinetics of this enzyme are described together with the stoichiometry and the effects of inhibitors. The structure‐activity relationships of pterin cofactors and of ether lipid substrates, including their stereospecificities, have been summarized from enzyme kinetic data which are also tabulated. The mechanism of enzymic hydroxylation of glyceryl ethers and a model for the active site of glyceryl‐ether monooxygenase are proposed from these apparent kinetic data. Notes on useful future studies of this monooxygenase have been made.

Lipophilic 5,6,7,8-tetrahydropterin substrates for phenylalanine hydroxylase (monkey brain), tryptophan hydroxylase (rat brain) and tyrosine hydroxylase (rat brain)

A high yielding unambiguous synthesis of (±)-6-alkyl-5,6,7,8-tetrahydropterin 5a–f hydrochlorides starting from ethyl α-isocyanoacetate 1 and the respective alkanoic anhydrides or alkanoyl chlorides in four steps is described. All the six pterins 5a–f that have been synthesised are substrates for mammalian phenylalanine, tryptophan and tyrosine hydroxylases and their activities have been compared with those of natural 6R-tetrahydrobiopterin under similar conditions. The data allowed the choice of 6-n-propyl-5,6,7,8-tetrahydropterin 5c for further studies as a candidate for tetrahydrobiopterin drug therapy.

MR tracking of transplanted glial cells using poly-l-lysine-CF3

Magnetic resonance (MR) imaging using super-paramagnetic iron oxides (SPIOs) is a powerful tool to monitor transplanted cells in living animals. Since, however, SPIOs are negative contrast agents, positive agents have been explored. In this study, we examined the feasibility of FITC-labeled poly-L-lysine-CF3 (PLK-CF3) using glial cells. FITC-labeled PLK-CF3 was easily internalized by neuroblastoma cells and glia as adding it into culture medium. No toxicity was seen at the concentration of less than 80 microg/ml. MR images positively detected labeled cells transplanted in the brain of living mouse. The results indicate that FITC-labeled PLK-CF3 is a useful positive contrast agent for MR tracking.

Glyceryl-ether Monooxygenase [EC 1.14.16.5J Part V: Some Aspects of the Stoichiometry

Summary A stoichiometry of one is found for the reaction between RS-batyl alcohol and 6-methyl-5,6,7,8-tetrahydropterin catalysed by glyceryl-ether monooxygenase during the first two minutes. The apparent decoupling of these two substrates after the first two minutes of reaction is discussed. The stoichiometry of the oxidation of 6-methyl-5,6,7,8-tetrahydropterin [to quinonoid 6-methyl-7,8(6H)-dihydropterinJ to the conversion of NADH to NAD in the monooxygenase-dihydropteridine reductase coupled reaction is ca one at concentrations of 6-methyl-5,6,7,8-tetrahydropterin less than 100 μM. At higher concentrations of the tetrahydropterin the ratio decreases and it is shown to be due to product inhibition of the reductase. R( +)- and S( - )-6-Methyl- 5,6,7,8-tetrahydropterin are about equally effective substrates for glyceryl-ether monooxygenase. A new and convenient synthesis of 1-14C-batyl alcohol is reported.

Glyceryl ether monooxygenase [EC 1.14.16.5]: stoichiometry and inhibition.

Glyceryl ether monooxygenase is a microsomal enzyme which hydroxylates the α-carbon atom of the fatty side-chain of glyceryl ethers of long-chain fatty alcohols. The reaction requires oxygen and a tetrahydropterin cofactor. It is a mixed-function oxidase and by analogy with phenylalanine hydroxylase the reaction shown in Scheme 1 was postulated.1 We investigated aspects of the stoichiometry of the oxygenase because two earlier reports were not entirely satisfactory. Tietz, Lindberg and Kennedy1 had shown that the amount of fatty aldehyde produced, in a non regenerating system, was 40-50% of tetrahydropterin oxidised and attributed this to the fact that the pterin was a DL mixture. This statement is incorrect in view of later work2 and because we have found that the monooxygenase activity with the two separate enantiomers S(-) and R(+) 6-methyl-5,6,7,8-tetrahydropterin as cofactors is the same. The second report was by Kotting, Unger and Eibl3 who determined the stoichiometry of the reaction in a coupled reaction with DHPR and NADH, and showed that the amount of NADH oxidised after one minute was equivalent to the total amount of fatty aldehyde, alcohol and acid produced.3 Since the fatty aldehyde, alcohol and acid are produced by several reactions following enzymic hydroxylation of the ether substrate the conclusion was dubious.

Glyceryl-ether monooxygenase [EC 1.14.16.5]. Part 9. Stereospecificityof the oxygenase reaction

(2RS,1′R)-[1′-3H1 n]- and (2RS, n1′S)-[1′-3H1]-Hexade ncyloxypropane-1,2-diols (chimyl alcohols) have been prepared and their nstereochemistry has been confirmed by synthesizing the n[2H1]-analogues using similar procedures. nWhen they were used as substrates for glyceryl-ether monooxygenase from nrat liver in the presence of oxygen and n(RS)-6-methyl-5,6,7,8-tetrahydropterin as co-factor, the n1′S-isomer released 37% of its tritium into the aqueous nbuffer after 20 mins, whereas the 1′R-isomer released nonly 6.5% showing that the reaction was stereospecific for the npro-HS hydrogen atom of the glyceryl ether nsubstrate. This was in agreement with the kinetic parameters of nunlabelled-(2RS)-3-, (2RS, n1′R)-3-[1′-2H1]-, n(2RS, n1′S)-3-[1′-2H1]- and n(2RS)-3-[1′,1′-2H2]- nhexadecyloxypropane-1,2-diols where the apparent Km nvalues were about the same (49.4, 53.7, 49.3 and 54.0 µM nrespectively) but the apparent maximum velocities n(Vmax in nmol min-1 nmg-1 protein) of the first two substrates (37.5 and n37.5) were faster than for the latter two substrates (22.5 and 23.6), nconsistent with the pro-HS hydrogen atom nbeing replaced by the hydroxy group and a primary deuterium isotope neffect of ≈1.6.

Glyceryl-Ether Monooxygenase [EC 1.14.16.5J Part VIII. Probing the Nature of the Active Site

Summary The V /K kinetic parameters for glyceryl-ether monooxygenase with 3-1 -n-alkoxypropane-I,2-diols having alkyl sidechains varying from Cll to C22 (lb-k, 2a and b, and 3d) were determined. Maximum activity was found with the CI6 ethers, dropping almost to zero with ethers that have sidechains shorter than Cl2 and longer than C19. The thioether analogues with C16 and CI8 sidechains were poorer substrates than the respective oxygen ethers by one order of magnitude. The kinetic parameters for 2-1 -n-hexadecyloxy- (6a) and 2-1 -n-octadecyloxy- (6d) ethanol revealed that they were also better substrates than the respective thio-ethers by one order of magnitude and with these compounds those with C16 sidechains were better substrates than the C 18 analogues. L2-bis-3-1 -n-Hexadecyloxypropan-3-01 (3c) and 1,3-bis-3- 1-n-hexadecyloxypropan-3-01 and 2- 01 (4a) were inactive but 3-1-n-hexadecyloxy- 2-methoxypropan-I-ol (3b) and 3-1-n-hexadecyloxy-l-methoxypropan-2-01 (4b) which have one long fatty chain were viable substrates because they possessed a free hydroxy group, but the ethers (6b, c and e), (7c and d) and (5) which do not have a free hydroxy group were inactive. These data, taken with those reported previously, have allowed us to postulate a model for the active site of the alkyl-ether substrates of the monooxygenase and to define the contours of the hydrophobic and hydrophilic pockets.

Syntheses of 3-substituted 6-amino-2-methylpyrimido[4,5-e][1,2,4]triazine-8-ones (7-substituted 6-methyl-6-azapterins) as inhibitors of dihydropteridine reductase from human brain

Condensation of 5,5-dibromo-2-aminopyrimidine-4,6-dione with S-methyl-, S-benzyl- and S-p-carboxybenzyl- 2-methylisothiosemicarbazide hydrobromides provided 3-methylthio-, 3-benzylthio- and 3-p-carboxybenzylthio-6-amino-2-methylpyrimido[4,5-e][1,2,4]triazin-8-ones. Similar condensations with 1-amino-1-methylguanidinium bromide and 2,3-dimethylamidrazone hydrochloride gave 3-amino- and 3-methyl- 6-amino-2-methylpyrimido[4,5-e][1,2,4]triazin-8-one. The 3-thio derivatives hydrolyse in aqueous solution to the same 2-methylpyrimido[4,5-e][1,2,4]triazine-3,8-dione. The ionization and 13 C nmr spectra are consistent with the stated structures. These triazinones are not substrates for dihydropteridine reductase from human brain but are inhibitors of the reductase and their K i values are reported

New Inhibitors of Dihydropteridine Reductase (Human Brain)

The dihydropteridine reductase (DHPR) gene from rat liver has recently been cloned1 and the protein that was expressed from the cDNA was crystallised as the binary DHPR-NADH complex.2 The X-ray structure of the enzyme was determined and although the exact location of NADH in the enzyme was determined in the binary complex, the precise position of the pteridine cofactor was not obtained and had to be deduced from known kinetic data of various pteridine cofactor analogues.2 In order to obtain the precise position of the pteridine cofactor it is necessary to crystallise the ternary complex of DHPR, NADH and an inhibitor whose structure is so close to that of a viable cofactor (eg 1) that it would bind at the active site in the same manner as the pterin.

TAU IMAGING USING FLUORINE-19 MRI IN A MOUSE MODEL OF TAUOPATHY

of neuroticism, extroversion, openness, agreeableness, and conscientiousness and underwent [18F]-AV-1451 tau-PET and [18F]-AV-45 b-amyloid-PET imaging. Tau levels were assessed in four regions including the amygdala, entorhinal cortex, inferior temporal cortex, and lateral occipital cortex (Figure 1), which are known to display early tau accumulation in AD. b-amyloid was examined as a composite measure from previously well-defined AD-related regions. We utilized linear regression models, adjusting for age and sex, to evaluate the association between the each of the personality traits and regional tau accumulation. Secondary analyses additionally adjusted for b-amyloid deposition. Results: Elevated neuroticism scores were significantly associated with higher tau accumulation in the amygdala (p1⁄4.003), entorhinal cortex (p1⁄4.031), and inferior temporal cortex (p<.001) (Figure 2a). In contrast, extroversion, openness, agreeableness, and conscientiousness were not associated with tau deposition for any of these regions (Figure 2b-e). After additionally adjusting for b-amyloid, results remained essentially unchanged (Table 1). Conclusions: Our results indicate that