Relationship of two ribonucleases with molecular masses of 45 kDa and 37 kDa from the culture medium Lentinus edodes

Lentinus edodes (shiitake) produces three base non-specific and acid ribonu cleases, RNase Le2, RNase Le37 and RNase Le45, The primary structures of th e former two RNases, having molecular masses about 24 and 37 kDa, respectiv ely, have been elucidated to be members of the RNase T2 family. The latter two are excreted from mycelia into the medium. In this report, we estimated the primary structure of RNase Le45 using the following experimental evide nce, (i) The partial amino acid sequence of RNase Le45 determined that up t o about 60% of total protein was identical with that of RNase Le37, (ii) Th e amino acid composition of RNase Le4 was identical to that of RNase Le37, (iii) The elution profiles on HPLC of lysylendopeptidase and Staphylococcus aureus V8 protease digests of RCM-RNase Le45 (reduced and S-carboxymethyla ted RNase Le45) were very similar to those of RNase Le37, except for the ab sence of C-terminus peptide which contained O-glycosylated peptides, Howeve r, RNase Le45 contained about 70 residues of mannose and 4 residues of hexo samine, These values were more than twice those of RNase Le37, (iv) RNase L e45 was immunologically indistinguishable from RNase Le37, (v) After treatm ent with both glycosidase EndoH and alpha-mannosidase, RNases Le37 and Le45 gave complex bands by slab-gel electrophoresis, However one of the major b ands with the highest mobility from RNase Le45 and Le37 showed the molecula r mass of 29 kDa in common, which is slightly larger than that of RNase Le2 containing no carbohydrate. These results indicated that RNase Le45 is an enzyme which is a heavily glycosylated species of RNase Le37.