Purification and characterisation of a novel 34,000-Mr cell-associated proteinase from the dermatophyte Trichophyton rubrum

Abstract

A novel cell-associated proteinase was purified to homogeneity from cytoplasmic antigen preparations of Trichophyton rubrum by sequential isoelectric focusing and gel filtration chromatography. The enzyme exhibited relative molecular masses of 34,000-Mr (non-reduced sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)), 15,000-Mr (reduced SDS-PAGE) and 37,000-Mr (substrate SDS-PAGE). It had a pH optimum of 7.5 and a pI of 4.5. The proteinase exhibited broad substrate specificity and it was strongly inhibited by the serine proteinase inhibitors phenylmethylsulfonyl fluoride and chymostatin. The N-terminal amino acid sequence of the 34,000-Mr proteinase shared 50% homology with the deduced amino acid sequence of a Coccidioides immitis wall-associated chymotrypsin-type serine proteinase. This is the first cell-associated proteinase to be purified and characterised from T. rubrum and it would appear to be related to the chymotrypsin-type serine proteinases, a class of enzymes that have rarely been isolated from fungi. The function of the proteinase remains speculative although it may play a role in the development and subsequent proliferation of the fungus in vivo.