INSITU LOCALIZATION OF THE SECRETION OF LIGNIN PEROXIDASES IN COLONIES OF PHANEROCHAETE-CHRYSOSPORIUM USING A SANDWICHED MODE OF CULTURE

Protein secretion and growth were investigated in Phanerochaete chryso sporium by using cultures sandwiched between perforated polycarbonate membranes. Labelling of colonies with radioactive N-acetylglucosamine and L-methionine indicated a close correlation between growth and gene ral protein secretion, even in a central area of the colony secreting the idiophase enzymes lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP). Comparison of the sites of release into the m edium of newly synthesized proteins and immuno-detected lignin peroxid ases suggested that diffusion of the enzymes from the walls was a limi ting step in the release of peroxidases into the medium. Microautoradi ography of colonies exposed to N-acetyl[H-3]glucosamine revealed the a pical growth of thin hyphae and branches (4 to 5 mum diameter on avera ge) in the central secreting area. These secondary hyphae showed perox idase activity and reacted with lignin peroxidase antibodies. Although it was not possible to directly visualize secretion at hyphal tips, t he results suggest that peroxidases (LiP and MnP) are initially secret ed at the apex of secondary growing hyphae and later slowly released i nto the surrounding medium.