title: Subtilisin modification of monodeamidated ribonuclease-A.
abstract: Limited proteolysis of RNAase-Aa(1) (monodeamidated ribonuclease-A) by subtilisin results in the formation of an active RNAase-S type of derivative, namely RNAase-Aa(1)S. RNAase-Aa(1)S 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-Aa(1)S by trichloroacetic acid yielded RNAase-Aa(1)S-protein and RNAase-Aa(1)S-peptide, both of which are inactive by themselves, but regenerate active RNAase-Aa(1)S' when mixed together. RNAase-Aa(1)S-peptide was identical with RNAase-S-peptide, whereas the protein part was distinct from that of RNAase-S-protein. Titration of RNAase-Aa(1)S-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-Aa(1) gives only a 50% conversion. The resistance of RNAase-Aa(1) to further subtilisin modification after 50% conversion is apparently due to the interaction of RNAase-Aa(1) 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-Sa(1)) 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.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography; D006851:Hydrochloric Acid / Q000494:pharmacology; D011485:Protein Binding; D012260:Ribonucleases / Q000032:analysis / Q000037:antagonists & inhibitors; D013381:Subtilisins
chemical_list: D000596:Amino Acids; D012260:Ribonucleases; D013381:Subtilisins; D006851:Hydrochloric Acid