Activation of dopamine beta-monooxygenase by external and internal electron donors in resealed chromaffin granule ghosts.
Membrane ghosts derived from chromaffin vesicles of bovine adrenal medullas have been used to examine the mechanism of reduction of dopamine beta-monooxygenase in its compartmentalized state. The rate of the dopamine beta-monooxygenase-catalyzed conversion of dopamine to norepinephrine is greatly stimulated by the presence of ATP, reflecting substrate hydroxylation on the ghost interior subsequent to the active transport of dopamine. We demonstrate a 2-3-fold increase in the turnover rate for ghosts resealed with 0.2-2 mM potassium ferrocyanide, conditions leading to a slight decrease in the rate of dopamine transport. These data provide the first evidence that an intravesicular pool of reductant can activate dopamine beta-monooxygenase, as required by models in which vesicular ascorbate behaves as enzyme reductant. Although there is sufficient catecholamine (endogenous plus substrate) to keep internal ferrocyanide reduced in these experiments, an additional 2-3-fold increase in turnover occurs in the presence of 0.2-2 mM ascorbate on the ghost exterior. The magnitude of this activation is found to be constant at all concentrations of internal ferrocyanide (both below and above saturation), implying that reductants on opposite sides of the membrane behave independently. Replacement of ascorbate by potassium ferrocyanide as external reductant leads to almost identical results, and we are able to rule out an inward transport of dehydroascorbate as the source of activation by external ascorbate. We conclude that external reductants are capable of reducing membrane-bound dopamine beta-monooxygenase from the exterior face of the vesicle, either by direct reduction or through a membrane-bound mediator. It appears that two viable modes for reduction of dopamine beta-monooxygenase may exist in vivo, involving the reduction of membrane-bound enzyme by cytosolic ascorbate as well as the reduction of soluble enzyme by the pool of intravesicular ascorbate present in chromaffin vesicles.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology*; D002838:Chromaffin System / Q000201:enzymology*; D003300:Copper / Q000494:pharmacology; D019327:Copper Sulfate; D004298:Dopamine / Q000378:metabolism; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism*; D004579:Electron Transport; D004789:Enzyme Activation; D005295:Ferrocyanides / Q000494:pharmacology; D006900:Hydroxylation; D007700:Kinetics; D008433:Mathematics
D005295:Ferrocyanides; D003300:Copper; D000255:Adenosine Triphosphate; D004299:Dopamine beta-Hydroxylase; C031835:potassium ferrocyanide; D019327:Copper Sulfate; D001205:Ascorbic Acid; D004298:Dopamine