CHARACTERIZATION OF ALPHA-GALACTOSIDASE FROM CORYNEBACTERIUM MURISEPTICUM AND MECHANISM OF ITS INDUCTION
1992 Volume 38 Issue 1 Pages 23-34
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1992 Volume 38 Issue 1 Pages 23-34
In C. murisepticum, alpha-galactosidase (EC 3.2.1.22) was induced during growth in the presence of melibiose or raffinose, and to a much lesser extent in the presence of galactose, as a single carbon source. Evidence was obtained to show that the enzymes induced by any of the sugars were identical with one another. The principal inducer of C. murisepticum alpha-galactosidase is melibiose. Raffinose elicits induction only indirectly by prior extracellular hydrolysis to melibiose. The enzyme was purified to homogeneity from melibiose-grown cells by column chromatographic fractionation of cell-free extract on Sephadex G-200, followed by that on QAE-Sephadex A-25, and finally by ultracentrifugation in a 5-20% sucrose density gradient. The purified C. murisepticum alpha-galactosidase is a homotetramer of 320 kDa molecular mass, with a Km for melibiose, 2mM; for p-nitrophenyl-alpha-D-galactopyranoside (PNPG), 0.17mM; and pH optimum of 7.5. Under optimal conditions of induction, 2% of total protein synthesized by C. murisepticum cells was alpha-galactosidase (as determined by selective immunoprecipitation of radiolabelled enzyme protein).
