On the Mechanism of Dextran Formation

Certain bacteria, e.g.Leuconostoc mesenteroides and related species, elaborate an enzyme, dextransucrase, which acts on sucrose to form dextrans, representing a-1,6-linked glucopyranose polymers, and free fructose.Hehre (1, 2) has demonstrated that this synthesis may also occur in cellfree systems.He postulated(3) that during the enzymatic reaction a glucosyl radical is split off from the substrate, sucrose, which can then attack the non-reducing end of a growing de&an chain.Although there is not yet direct evidence for such a "radical," glucose in trace amounts has been detected in the reaction system (4).The present experiments were undertaken in an attempt to obtain information on the mechanism of dextran synthesis with the aid of paper chromatography and CY4-labeled sugars. EXPERIMENTAL Materials and MethodsSubstrate-Stock solutions of sucrose were made up at an initial concentration of 10 per cent in 0.05 M acetate buffer, pH 5.0.When radioactive sucrose was employed, stable sucrose was added to adjust the total concentration to 10 per cent.The radioactive sucrose was uniformly labeled with Cl4 and had a specific activity of 0.6 PC. per mg.For most experiments 10 mg. of Cl4-sucrose were employed, since this activity was sufficient to give reproducible results both by means of direct counting and by autoradiography.Dextransucrase-All enzyme preparations were obtained from culture filtrates of L. mesenteroides, strain NRRL B-512, grown on a yeast or corn steep medium (5, 6).The enzyme was purified by ethanol precipitation and subsequent adsorption on calcium phosphate gel.The most active enzyme solutions showed an activity of 500 units per mg. of dry weight.