Primary Structure of a Minor Ribonuclease from Aspergillus saitoi1

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Abstract
1. RNase Ms, a base non-specific RNase from Aspergillus saitoi was reduced and carboxymethylated (RCM-RNase Ms). RCM-RNase Ms was hydrolyzed with tryp-sin, and the trypsin digests were then treated with chymotrypsin. Trypsin digests were also treated with Staphylococcal protease and with chymotrypsin, separately. 2. By the analyses of the amino acid sequences of the peptides formed, the alignment of these peptides in RCM-RNase Ms was determined. 3. From the digest of heat-denatured RNase Ms with Bacillus subtilis protease, two peptides containing disulfide bridges were isolated. From the analyses of these two peptides, the locations of the bridges were determined. 4. The amino acid sequence of RNase M1 was compared with those of RNase T1 (Asp. oryiae, guanine specific), RNase U1 (Ustilago sphaerogena, guanine specific) and RNase U2 (Ustilago sphaerogena, purine specific). There are very similar sequences between these four RNases irrespective of their differences in base specificity. These were, in RNase Ms, tripeptide sequence containing His39 (Tyr-Pro-His), the tetrapeptide containing Glu67 (Glu-Tyr-Pro-Ile), the hexapeptide containing Arg76 (Asp-Arg-Val-Ile-Phe-Asp) and the hexapeptide containing His91 (Ile-Thr-His-Thr-Gly-Ala). The other sequences common for all four RNases are Tyr67, Phe100, and Cys103 in RNase Ms. Since among these peptides His39, Glu57, His91, and Arg76, in RNase Ms corresponded to His40, Glu68, His92, and Arg77 in RNase T1 which are known to be involved in the active site of RNase T1 the possible role of these amino acids in the active site of RNase Ms is discussed. 5. The sequence similarity of RNase Ms to that of RNase T1 was about 60% and to those of RNase U1 and RNase U2 was about 30%. 6. The details of the experimental evidence used to elucidate the amino acid sequence of RNase Ms are described in the supplemental miniprint.