The formation of dextran from sucrose can proceed by two reaction pathways, both of which are catalyzed by dextransucrase (EC 2.4.1.5). One pathway involves chain elongation via sequential transfers of D-glucosyl groups to nonreducing positions of acceptor saccharides' 4. The second pathway, which occurs in the absence of added acceptors, is considered to be an autopolymerization reaction, and may account for the de nova synthesis of dextran4-'.
Previous studies',' indicated that the dextranyl chains formed may be bound to the enzyme. The early experiments by Robyt et al.' provided evidence that supports the proposal that chain growth occurs at the reducing terminus. In their work, a crude enzyme preparation, immobilized by chemical cross-linking to Bio-Gel P-2, was treated with a low concentration of ['4C]sucrose for 10 h, during which time, the isotope became bound. Analyses showed that the bound isotope was a mixture of D-glucose and dextranyl chains. When the labeled enzyme was subsequently reacted with a high concentration of unlabeled sucrose, radioactivity at the reducing terminus diminished, and the conclusion was drawn that chain growth occurs at the reducing end.
Several criticisms have been made of these studies'. Firstly, the crudeness of the enzyme preparation, and the fact that the cells had been grown in the presence of sucrose, call into question the availability of endogenous, acceptor chains. Secondly, the cross-linking process yielded an enzyme that retained only 1% of the original activity. Finally, the extended reaction-time with [14C]sucrose cannot be considered to be representative of a catalytic process. Recently, we have obtained' highly purified enzyme preparation from S. sanguis ATCC-10558, and have developed a procedure for immobilization of the enzyme, such that a minimum of 80% of the akivity is expressed in the immobilized state'. With these developments, we considered it important to reexamine the issues raised by the work of Robyt et al.'. and now report the results of these studies.