Manjula B N, Acharya A S, Vithayathil P J
Biochem J. 1977 Aug 1;165(2):337-45. doi: 10.1042/bj1650337.
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.
枯草杆菌蛋白酶对RNA酶-Aa(1)（单脱酰胺核糖核酸酶-A）进行有限的蛋白水解会导致形成一种具有活性的RNA酶-S型衍生物，即RNA酶-Aa(1)S。RNA酶-Aa(1)S在色谱上与RNA酶-S不同，但表现出与RNA酶-S非常接近的酶活性、抗原构象和对胰蛋白酶的敏感性。用三氯乙酸对RNA酶-Aa(1)S进行分级分离得到RNA酶-Aa(1)S-蛋白和RNA酶-Aa(1)S-肽，二者本身均无活性，但混合在一起时会再生出活性RNA酶-Aa(1)S'。RNA酶-Aa(1)S-肽与RNA酶-S-肽相同，而蛋白质部分与RNA酶-S-蛋白不同。用S-肽滴定RNA酶-Aa(1)S-蛋白时，与天然蛋白相比，该肽与脱酰胺化S-蛋白的结合略显微弱但明显较弱。与RNA酶-A经枯草杆菌蛋白酶消化后几乎100%转化为RNA酶-S不同，RNA酶-Aa(1)的消化仅产生50%的转化率。RNA酶-Aa(1)在50%转化后对进一步的枯草杆菌蛋白酶修饰具有抗性，这显然是由于RNA酶-Aa(1)与其枯草杆菌蛋白酶修饰产物之间的相互作用。还发现RNA酶-S在酸性溶液中会发生活性和结构变化，类似于RNA酶-A。通过色谱分离得到的初始反应产物（RNA酶-Sa(1)）不均匀。与仅影响S-蛋白部分的RNA酶-A的酸处理不同，RNA酶-S的酸处理会影响分子的S-蛋白和S-肽区域。
