Structural and Enzti1atic Studies of Dopamine-ß-Hydroxylase from Bovine Adrenal Chromaffin Granules
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Dopamine-ß-hydroxylase (DßH), an enzyme which catalyzes the conversion of dopamine to norepinephrine, is the only enzyme of the catecholamine biosynthetic pathway located in the chromaffin granules of adrenal medulla. Within the granules, two populations of DßH exist: a water-soluble fraction found within the granule matrix and a membrane-bound, amphiphilic fraction embedded in the surrounding bilayer. The amphiphilic form was purified to homogeneity following its extraction from the membrane with the non-ionic detergent BRIJ 58. Three steps were required to achieve complete purification: adsorption to ConA-Sepharose, adsorption to DEAE Sephadex A-25, and chromatography on Sephacryl S-200, Sepharose 6B, or Sepharose CL-4B. The presence of 0.1-0.2 mg/ml BRIJ 58 was essential for protein recovery. The enzymatic and structural characteristics of membrane-bound DßH were found to be similar to those of soluble DßH. Initial velocity data indicated a Ping-pong or double-displacement reaction with Km for the substrates, tyramine and ascorbate, of 2.13 mM and 0.88 mM, respectively. Dicarboxylic acids activated the enzyme but halides inhibited both forms of DßH at the optimal pH range of 5.0-5.5. The chromatographic elution profiles, fluorescence and circular dichroism spectra, and migration on sucrose density gradients were indistinguishable for the two forms of the enzyme. Although not identical, the amino acid compositions of the two forms also displayed considerable similarity. As determined by sedimentation equilibrium, molecular weight values of 295,000 ± 15,000 were obtained for both forms of DßH. Despite these similarities, membrane-bound DßH possesses amphiphilic properties lacking in the soluble form. Whereas purified amphiphilic DßH may be incorporated into egg phosphatidylcholine vesicles with a 75% efficiency, the soluble form does not interact with vesicles.
CitationAlbanesi, Joseph P. (1980). Structural and Enzti1atic Studies of Dopamine-ß-Hydroxylase from Bovine Adrenal Chromaffin Granules. Dissertation, Duke University. Retrieved from http://hdl.handle.net/10161/7389.
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