The Enzyme‐Bound Copper of Dopamine β‐Monooxygenase

Abstract

The enzyme‐bound copper of dopamine β‐monooxygenase reacted rapidly with the chelator bathocuproine disulfonate; the reaction in the presence of ascorbate was completed in 2 min at 25°C with 1 mM chelator. This reaction and also the reaction with EDTA could be used to prepare the apoenzyme, which in both cases was completely reactivated in less than 10 s. The reactivation data gave apparent Michaelis constants for copper of 0.03–0.2 μM. Trace amounts of copper in buffers and assay mixtures gave significant reactivation without added copper, unless they had been treated with a chelating resin. Titrations using the different chelation rates of free and enzyme‐bound copper indicated that four copper atoms are bound per enzyme molecule of four subunits. The native enzyme was more stable against thermal inactivation than the apoenzyme, but this stability was only partially restored by addition of copper to the apoenzyme.

Characterisation of N-terminal chromogranin A and chromogranin B in mammals by region-specific radioimmunoassays and chromatographic separation methods

“…The proteins were immunoblotted to nitrocellulose papers and the immunoreactive components were stained with the bCgA antibodies using the alkaline phosphatase reaction as previously described (Aardal et al 1993). Traces of the N-terminal sequence of the soluble DBH (Skotland & Ljones 1979) were not detected by these analyses and the protein failed to immunostain with a polyclonal antibody to bovine DBH (a kind gift from Dr D Aunis, Strasbourg, France).…”

Dopamine β‐Monooxygenase

“…The holoenzyme contained 4.2 copper atomsltetramer while the apoenzyme contained less than 0.04 copper atom/tetramer. Copper determination was performed by calculating the concentrations of the complex of Cu (1) and bathocuproine disulfonate from the absorbance at 483 nm [5,15]. The absorbance measurements were performed in a Cary 219 recording spectrophotometer.…”

“…The concentrations of holoenzyme and apoenzyme were estimated assuming an absorbance of 1.24 at 280nm for a solution of 1 mg/ml with a 10-mm light path [5,141. The holoenzyme contained 4.2 copper atomsltetramer while the apoenzyme contained less than 0.04 copper atom/tetramer.…”

“…The water-soluble form of dopamine fl-monooxygenase was purified from bovine adrenal medulla as described [6, 131; the inactive apoenzyme was prepared by dialysis against EDTA [5]. The concentrations of holoenzyme and apoenzyme were estimated assuming an absorbance of 1.24 at 280nm for a solution of 1 mg/ml with a 10-mm light path [5,141.…”

“…Although the copper of dopamine P-monooxygenase resembles type 2 copper in these respects, it is different by other criteria, especially by showing a high accessibility of the copper site. Thus, the copper atoms of dopamine P-monooxygenase can rapidly be removed at nondenaturing conditions by the chelators bathocuproine disulfonate or EDTA and the apoenzyme can be fully reactivated in less than two seconds by addition of CuSO, [5,6]. Moreover, the observation that enzyme preparations containing 4 copper atoms/tetramer gave only 80 of the maximal activity obtained after addition of an excess of copper ions, together with experiments with bathocuproine disulfonate [5,12], indicate that the specific binding of copper in this enzyme has a rather low association constant.…”