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  • In a study carried out by

    2020-10-22

    In a study carried out by Aunis et al. the properties of soluble DBH and membrane bound were examined [23]. The membrane bound enzyme was shown in glutamate transporter to the soluble form to have thermal denaturation at higher temperatures of 43.5–44°C. Similar discontinuities in the Arrhenius plots were obtained for the membrane bound DBH by the authors. Our findings demonstrate that the DBH–IAM and DBH–Glut-P interphases are representative of the membrane-bound and soluble enzyme.
    Acknowledgements
    Catecholamines play important roles in the central and peripheral nervous systems, where they are used as chemical messengers. The first step for their biosynthesis is the aromatic hydroxylation of amino-acid -tyrosine to -DOPA . -DOPA next undergoes a decarboxylation that produces dopamine. Dopamine is either used as such in the nervous system or transformed into norepinephrine and epinephrine. Conversion of dopamine into norepinephrine is catalyzed by dopamine β-hydroxylase (DbH), a copper-containing enzyme () , . The usual source for DbH is bovine chromaffin granule, from which it is extracted as a tetramer of 290kDa . Catalytic activity requires both molecular oxygen and ascorbate as cosubstrates . Ascorbate is oxidized into semidehydroascorbate, with a stoichiometry of two molecules per molecule of substrate transformed . EPR and EXAFS studies have shown that the active site of DbH contains two copper(II) ions displaying type II coordination in the resting state , , , . Still the global folding for DbH is unknown, which makes it impossible to determine how and at which sites the substrate and cosubstrates interact with the active site of the enzyme. Only hints about the interaction between the enzyme and its reduction cosubstrate have been obtained in solid state through a crystal structure of a neighboring enzyme, peptidylglycine α-hydroxylating monooxygenase (PHM), in the presence of reducer ascorbate . Various molecules have been assayed as reducing agents for DbH, such as ferrocyanide , ,-dimethyl--phenylenediamine , and thiophenyl derivatives . In a will to obtain data concerning putative interactions between the active site of the enzyme and its reducing cosubstrate, we studied a new family of molecules as cosubstrates for DbH. -Aryl--hydroxyguanidines () are well-known electron donors that act as substrates for heme-containing enzymes such as nitric oxide synthases (NOS) and peroxidases , . We were also interested in these compounds for their ability to release NO , inside the active site of the enzyme under oxidation, but were not able to obtain any conclusive result concerning that question. The present work uses for the first time these compounds with a copper-containing enzyme. It establishes that -aryl--hydroxyguanidines are reducing cosubstrates for DbH. Integrity of the hydroxyguanidine moiety is a prerequisite to activity. An influence of modifications of the aromatic ring substituents on catalytic activity was evidenced, and suggests that selective interactions between the active site of DbH and its reducing cosubstrate do occur during catalysis. Materials and methods Materials. Potassium phosphate salts, tyramine hydrochloride, sodium ascorbate, octopamine hydrochloride, catalase (from bovine liver), and bovine hemoglobin were purchased from Sigma. DEAE-cellulose and phenyl-Sepharose used for the purification of DbH were from Amersham–Pharmacia Biotech. Purification of dopamineβ-hydroxylase. Purification of soluble DbH from bovine adrenal medullae was performed following the procedure by Ljones [4], except for the hydrophobic interaction which was realized using a phenyl-Sepharose column. Purified DbH was more than 90% pure as judged by SDS–PAGE. Protein contents were measured using the Bradford assay from Bio-Rad and BSA as a standard. Syntheses.N-Aryl-N′-hydroxyguanidines 1 and 6–10 were obtained as well as small amounts of the corresponding N-arylureas, by the addition of hydroxylamine hydrochloride to intermediate cyanamides in anhydrous ethanol, following a previously described method [19]. N-Arylcyanamides were obtained by the addition of BrCN to the corresponding anilines following a general protocol [20]. N-(4-Methoxyphenyl)-N′-methoxyguanidine 2 was obtained by heating methoxyamine hydrochloride with 4-methoxyphenylcyanamide [16]. N-(4-Methoxyphenyl)acetamidoxime 3 was prepared by heating for 3 days under reflux 4-methoxyaniline in anhydrous ethanol in the presence of excess ethyl N-hydroxyacetimidate [21]. 4-Methoxyacetophenone oxime 4 and 4-methoxybenzamidoxime 5 were prepared by reacting hydroxylamine hydrochloride with 4-methoxyacetophenone and 4-methoxybenzonitrile, respectively, following conventional protocols [22], [23]. Complete physico-chemical characteristics of compounds 1–10 have been described elsewhere [16], [24].