Kinetic Evidence for Channeling of Dopamine between Monoamine Transporter and Membranous Dopamine-β-monooxygenase in Chromaffin Granule Ghosts

Abstract

The nature of coupling between the uptake and dopamine-p-monooxygenase (DβM) catalyzed hydroxylation of dopamine (DA) was studied in bovine chromaffin granule ghosts. Initial rate and transient kinetics of DA uptake and conversion were determined under a variety of conditions. The uptake kinetics of DA, norepinephrine (NE), and epinephrine demonstrate that DA is a better substrate than NE and epinephrine under optimal uptake conditions. The transient kinetics of DA accumulation and NE production under both optimal uptake and uptake and conversion conditions were zero-order with no detectable lag or burst periods. The mathematical analyses of the data show that a normal sequential uptake followed by the conversion process could not explain the observed kinetics, under any condition. On the other hand, all experimental data are in agreement with a mechanism in which DA is efficiently channeled from the vesicular monoamine transporter to membranous DβM for hydroxylation, prior to the release into the bulk medium of the ghost interior. The slow accumulation of DA under optimal conversion conditions appears to be caused by the slow leakage of DA from the channelling pathway to the ghost interior. Because DβM activity in intact granules is equally distributed between soluble and membranous forms of DβM, if an efficient channeling mechanism is operative in vivo, soluble DβM may not have access to the substrate, making the catalytic activity of soluble DβM physiologically insignificant, which is consistent with the increasing experimental evidence that membranous DβM may be the physiologically functional form.