Properties of soluble and membrane bound dopamine-beta-monooxygenase from bovine adrenal medulla cross-linked with dimethyl suberimidate.
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.
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002837:Chromaffin Granules / Q000201:enzymology; D003432:Cross-Linking Reagents; D004120:Dimethyl Suberimidate; D004299:Dopamine beta-Hydroxylase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins; D012995:Solubility; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D008565:Membrane Proteins; D004120:Dimethyl Suberimidate; D004299:Dopamine beta-Hydroxylase