Purification of membrane-bound dopamine beta-monooxygenase from chromaffin granules: relation to soluble dopamine beta-monooxygenase

Previous studies have indicated that α-d-1-fluoroglucose is a glycosyl donor for glucosyl transferases (5, 6) including dextransucrases formed by Leuconostoc and Streptococcus mutans. The present report confirms these observations with dextransucrase isolated from S. sanguis and conclusively establishes the details of this reaction as well as proving that mechanism of fluoroglucose transfer is comparable to that glucosyl transfer from sucrose. A new procedure for monitoring the reaction is reported, and is based on the measurement of proton formation using the pH indicator, bromcresol purple. Production of F^? was found to be stoichiometric with proton production. Rate studies with the substrate indicate that α-1-fluoroglucose undergoes spontaneous hydrolysis, which is greatly increased in the presence of nucleophilic buffers. When [¹⁴C]maltose and α-1-fluoroglucose or [¹⁴C]α-1-fluoroglucose and maltose were incubated with dextransucrase, a series of oligosaccharide products was observed. The results indicate that the glucosyl moiety of α-1-fluoroglucose transferred to the acceptor. The nature of formation of the products are consistent with a series of precursor-product reactions. Product analysis of the saccharides by borohydride reduction analysis demonstrated that the glucosyl unit was added to the nonreducing end of maltose. When either [¹⁴C]fructose or [¹⁴C]-α-1-fluoroglucose were incubated with enzyme, a reaction was observed which was analogous to the isotopic-exchange reaction catalyzed by the enzyme in the presence of [¹⁴C]fructose and sucrose.