Isolation, characterization, and sugar chain structure of endoPG Ia, Ib and Ic from Stereum purpureum.

Abstract

Three endopolygalacturonases (endoPG Ia, Ib, and Ic) were isolated from the culture filtrate of Stereum purpureum, the causative fungus of apple silver-leaf disease. Their properties, including specific activities, optimum pHs, thermal stabilities, and kinetic parameters (K(m) and Vmax) were compared. Their properties were very similar to one another except for the substrate specificity and relative molecular mass. The sugar chains of endoPG Is were released by hydrazinolysis, and one major sugar chain common to endoPG Is was isolated. The pyridylamino sugar was characterized by a two-dimensional mapping method using HPLC, and identified as a high mannose type N-linked sugar chain, Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3) Man beta 1-4 GlcNAc beta 1-4 GlcNAc (designated as M5.1). Observation of the course of Western blot analysis for the proteins from the culture filtrate with endoPG I antibodies showed that the fungus secreted three endoPG Is into the culture broth during the growing period.