Properties of soluble and membrane bound dopamine-?-monooxygenase from bovine adrenal medulla cross-linked with dimethyl suberimidate

Abstract

Bovine dopamine-beta-monooxygenase from chromaffin granules in its soluble and membrane-bound forms was cross-linked with the bifunctional reagent dimethyl suberimidate, and its structural and kinetic properties were studied. 1. The cross-linking reaction does not affect the activity of soluble dopamine-beta-monooxygenase; it produces a ten percent inactivation in the membrane-bound enzyme, possibly because the linkage to other membrane proteins hinders its activity. 2. The soluble dopamine-beta-monooxygenase reaction mixture was analyzed by sodium dodecyl sulfate gel electrophoresis, showing appreciable amounts of dimer and tetramer, but only small amounts of trimer. In membrane-bound dopamine-beta-monooxygenase, subjected to the same treatment, appreciable amounts of dimer and higher aggregates were found. 3. The kinetic properties of soluble dopamine-beta-monooxygenase after the crosslinking reaction are the same as those of the native enzyme, with a ping-pong kinetic mechanism and the same real Michaelis constants for tyramine and ascorbate: KmT = 0.36 mM and KmA = 0.32 mM. Membrane-bound dopamine-beta-monooxygenase does not present a ping-pong mechanism before or after cross-linking; its real Michaelis constants are slightly modified by the cross-linking reaction: KmT = 0.4 mM and KMA = 0.4 mM.

Nucleotide Vesicular Transporter of Bovine Chromaffin Granules

“…Controls of chromaffin granule integrity were done by measuring the presence of catecholamines in the extragranular incubation media of transport experiments by electrochemical detection as described elsewhere (Castro et al, 1990). Dopamine ␤-monooxygenase activity was measured as already described (Miras-Portugal et al, 1980). Proteins were determined by the Bradford method.…”