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Immunolocalization of a proteinase produced by the sap-staining fungus, Ophiostome piceae

University of British Columbia

Ophiostoma piceae, the most frequently isolated sap-staining fungus in Canada, produces an extracellular subtilisin-like, serine proteinase. This enzyme is presumed to serve an important nutritive function for the fungus as it grows in wood where nitrogen is found primarily in the form of protein. To further characterize this proteinase, an attempt was made to detect and localize it in O. piceae cells. The fungus was grown in a minimal, liquid medium supplemented with either protein or inorganic nitrogen as nitrogen sources. The approaches taken to localize the proteinase in the cells included a) detection of cell-associated proteolytic activity and b) immunological detection of this proteinase in both whole cells and cell fractions. Proteolytic activity was assessed using both the general substrate azocoll and a subtilase specific substrate, succinyl- (Ala)2-Pro-Phe-p-nitroanilide. For immunological detection, polyclonal antibodies were prepared to localize the proteinase in O. piceae by immunoblotting methods and immunogold labelling with transmission electron microscopy. Polyclonal antibodies were first raised against proteinase K, an enzyme that is similar to the O. piceae proteinase, and then against the O. piceae extracellular proteinase purified by hydrophobic interaction chromatography. Characterization of these antibodies indicated that they recognized the O. piceae proteinase. In comparison to extracellular proteolytic activity, little activity was detected in washed cells and homogenized cells of O. piceae grown in soya milk medium. An even lower amount of activity was detected in cultures supplemented with inorganic nitrogen. By immunodetection, the enzyme was found to be associated with the O. piceae cells from both inorganic nitrogen cultures and protein provided cultures. Gold labelling was observed primarily in the cell wall and the extracellular sheath. The results suggested that the proteinase can be detected in the O. piceae cells whether or not they are secreting active proteinase. This was the first time the extracellular proteinase produced by O. piceae was studied with respect to the cells.

Thesis/Dissertation

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2009-01-21

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http://hdl.handle.net/2429/3836

Forestry

University of British Columbia

UBCV

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