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Primary structure of a minor ribonuclease from Aspergillus saitoi
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 trypsin, and the trypsin digests were then treated with chymotrypsin. Trypsin digests were also treated with Staphylococcus 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 analysis of these two peptides, the locations of the bridges were determined. 4. The amino acid sequence of RNase Ms was compared with those of RNase T1 (Asp. oryzae, guanine specific), RNase U1 (Ustilago sphaerogena, guanine specific) and RNase U2 (Ustilago sphaerogena, purine specific). There are very similar sequences between these for RNases irrespective of their differences in base specificity. These were, in RNase Ms, tripeptide sequence containing His39 (Tyr-Pro-His), the tetrapeptide containing Glu57 (Glu-Tyr-Pro-Ile), the hexapeptide containing Arg76 (Asp-Arg-Val-Ile-Phe-Asp) and the hexapeptide containing His 91 (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, Glu58, 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.
Insights
Researchers elucidated the amino acid sequence and disulfide bridges of Aspergillus saitoi ribonuclease Ms (RNase Ms). Sequence comparisons reveal similarities to other RNases, suggesting conserved active site residues despite differing base specificities.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Ribonucleases (RNases) are crucial enzymes involved in RNA metabolism.
- RNase Ms from Aspergillus saitoi is a base non-specific RNase.
- Understanding RNase structure-function relationships is key to their biological roles.
Purpose of the Study:
- To determine the complete amino acid sequence of RNase Ms.
- To identify the locations of disulfide bridges within RNase Ms.
- To compare the sequence of RNase Ms with other known RNases and infer functional insights.
Main Methods:
- Enzymatic hydrolysis of reduced and carboxymethylated RNase Ms (RCM-RNase Ms) using trypsin, chymotrypsin, and Staphylococcus protease.
- Peptide mapping and amino acid sequence analysis.
- Isolation and analysis of peptides containing disulfide bridges from heat-denatured RNase Ms digested with Bacillus subtilis protease.
Main Results:
- The complete amino acid sequence of RNase Ms was determined through peptide alignment.
- Two disulfide bridges were located by analyzing specific peptides.
- Significant sequence similarities were found between RNase Ms and other RNases (e.g., RNase T1), particularly in regions potentially involved in the active site.
- Sequence similarity to RNase T1 was approximately 60%, and to RNase U1 and U2 was about 30%.
Conclusions:
- The study successfully elucidated the primary structure and disulfide bridge locations of RNase Ms.
- Conserved amino acid sequences across different RNases suggest potential functional importance, even with varying base specificities.
- The findings provide a basis for understanding the catalytic mechanism and structural features of RNase Ms and related enzymes.