Uridine Diphosphate Galactose 4-Epimerase from Yeast

Uridine diphosphate galactose 4-epimerase (EC 5.1.3.2) from yeast was activated by cations, such as Na+, Mg2+, and a series of quaternary amines, among which spermine was the most effective. The activation by spermine could be explained by a decrease in the Km of the enzyme for uridine diphosphate galactose. Studies of sedimentation velocity in the analytical ultracentrifuge indicated that uridine diphosphate galactose 4-epimerase could assume three distinct states of aggregation, represented by averaged, unextrapolated sedimentation coefficients of approximately (a) 3.6 S, (b) 6.5 S, and (c) 10.9 S. These corresponded to (a) inactive enzyme, titrated with p-chloromercuribenzoate, (b) partially active enzyme, in solutions of low cation concentration, and (c) fully active enzyme, in the presence of relatively high concentrations of activating cations. Analysis of sedimentation equilibria together with previously published information on the molecular weight of the 6.5 S species (Darrow, R. A., and Rodstrom, R., Biochemistry, 7, 1645 (1968)) indicated that the three stages of aggregation are caused by a successive binary association of subunits, resulting in molecular weights of (a) 60,000, (b) 125,000, and (c) 250,000. Some preparations of uridine diphosphate galactose 4-epimerase contained a form of the protein which did not associate to a molecular weight of 250,000 from 125,000, regardless of the cation concentration. This nonassociating form, which still was partially activated by cations, could be separated from the associating form, to a limited extent, by chromatography on DEAE-cellulose.