Relationship of two ribonucleases with molecular masses of 45 kDa and 37 kDa from the culture medium of Lentinus edodes.
Lentinus edodes (shiitake) produces three base non- specific and acid ribonucleases, RNase Le2, RNase Le37 and RNase Le45. The primary structures of the former two RNases, having molecular masses about 24 and 37 kDa, respectively, 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 evidence. (i) The partial amino acid sequence of RNase Le45 determined that up to about 60% of total protein was identical with that of RNase Le37. (ii) The amino acid composition of RNase Le45 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-carboxymethylated RNase Le45) were very similar to those of RNase Le37, except for the absence of C-terminus peptide which contained O-glycosylated peptides. However, RNase Le45 contained about 70 residues of mannose and 4 residues of hexosamine. These values were more than twice those of RNase Le37. (iv) RNase Le45 was immunologically indistinguishable from RNase Le37. (v) After treatment with both glycosidase EndoH and alpha-mannosidase, RNases Le37 and Le45 gave complex bands by slab-gel electrophoresis. However, one of the major bands with the highest mobility from RNase Le45 and Le37 showed the molecular 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.
Kobayashi
H
H
Department of Microbiology, College of Pharmacy, Nihon University, Funabashi, Chiba, Japan.
Hara
J
J
Itagaki
T
T
Inokuchi
N
N
Koyama
T
T
Sanda
A
A
Iwama
M
M
Ohgi
K
K
Irie
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D003470:Culture Media / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D020942:Shiitake Mushrooms / Q000201:enzymology*
D000596:Amino Acids; D003470:Culture Media; D012260:Ribonucleases