Kinetic evidence for channeling of dopamine between monoamine transporter and membranous dopamine-beta-monooxygenase in chromaffin granule ghosts.
The nature of coupling between the uptake and dopamine-beta-monooxygenase (DbetaM) 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 DbetaM 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 channeling pathway to the ghost interior. Because DbetaM activity in intact granules is equally distributed between soluble and membranous forms of DbetaM, if an efficient channeling mechanism is operative in vivo, soluble DbetaM may not have access to the substrate, making the catalytic activity of soluble DbetaM physiologically insignificant, which is consistent with the increasing experimental evidence that membranous DbetaM may be the physiologically functional form.