Limited proteolysis of RNAase-Aa1 (monodeamidated ribonuclease-A) by subtilisin results in the formation of an active RNAase-S type of derivative, namely RNAase-Aa1S. RNAase-Aa1S was chromatographically distinct from RNAase-S, but exhibited very nearly the same enzymic activity, antigenic conformation and susceptibility to trypsin as did RNAase-S. Fractionation of RNAase-Aa1S by trichloroacetic acid yielded RNAase-Aa1S-protein and RNAase-Aa1S-peptide, both of which are inactive by themselves, but regenerate active RNAase-Aa1S′ when mixed together. RNAase-Aa1S-peptide was identical with RNAase-S-peptide, whereas the protein part was distinct from that of RNAase-S-protein. Titration of RNAase-Aa1S-protein with S-peptide exhibited slight but noticeably weaker binding of the peptide to the deamidated S-protein as compared with that of native protein. Unlike the subtilisin digestion of RNAase-A, which gives nearly 100% conversion into RNAase-S, the digestion of RNAase-Aa1 gives only a 50% conversion. The resistance of RNAase-Aa1 to further subtilisin modification after 50% conversion is apparently due to the interaction of RNAase-Aa1 with its subtilisin-modified product. RNAase-S was also found to undergo activity and structural changes in acidic solutions, similar to those of RNAase-A. The initial reaction product (RNAase-Sa1) isolated by chromatography was not homogeneous. Unlike the acid treatment of RNAase-A, which affected only the S-protein part, the acid treatment of RNAase-S affected both the S-protein and the S-peptide region of the molecule.
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Research Article|
August 01 1977
Subtilisin modification of monodeamidated ribonuclease-A
B. N. Manjula;
B. N. Manjula
1Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
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A. Seetharama Acharya;
A. Seetharama Acharya
1Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
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Paul J. Vithayathil
Paul J. Vithayathil
1Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1977 London: The Biochemical Society
1977
Biochem J (1977) 165 (2): 337–345.
Citation
B. N. Manjula, A. Seetharama Acharya, Paul J. Vithayathil; Subtilisin modification of monodeamidated ribonuclease-A. Biochem J 1 August 1977; 165 (2): 337–345. doi: https://doi.org/10.1042/bj1650337
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