The structure and function of ribonuclease T1. XXII. Tryptic cleavages of the single lysyl and arginyl bonds in ribonuclease T1.
1. When ribonuclease T1 [EC 3.1.4.8] was treated with trypsin [EC 3.4.21.4] at pH 7.5 and 37 degrees, activity was lost fairly slowly. At higher temperatures, however, the rate of inactivation was markedly accelerated. The half life of the activity was about 2.5 h at 50 degrees and 1 h at 60 degrees. 3'-GMP and guanosine protected the enzyme significantly from tryptic inactivation. 2. Upon tryptic digestion at 50 degrees, the Lys-Tyr (41-42) and Arg-Val (77-78) bonds were cleaved fairly specifically, yielding two peptide fragments. One was a 36 residue peptide comprizing residues 42 to 77. The other was a 68 residue peptide composed of two peptide chains cross-linked by a disulfide bond between half-cystines -6 and -103, comprizing residues 1 to 41 and 78 to 104. 3. When the trinitrophenylated enzyme, in which the alpha-amino group of alanine-1 and the episolone-amino group of lysine 41 were selectively modified, was treated with trypsin at 37 degrees, the activity was lost fairly rapidly with a half life of about 4 h. In this case, tryptic hydrolysis occurred fairly selectively at the single Arg-Val bond. Thus the enzyme could be inactivated by cleavage of a single peptide bond in the molecule, an indication of the importance of the peptide region involving the single arginine residue at position 77 in the activity of ribonuclease T1.
D000596:Amino Acids / Q000032:analysis; D001120:Arginine; D055598:Chemical Phenomena; D002621:Chemistry; D006152:Cyclic GMP / Q000494:pharmacology; D006151:Guanosine / Q000494:pharmacology; D008239:Lysine; D010446:Peptide Fragments; D006163:Ribonuclease T1 / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism*; D013696:Temperature; D014301:Trinitrobenzenes; D014357:Trypsin
D000596:Amino Acids; D010446:Peptide Fragments; D014301:Trinitrobenzenes; D006151:Guanosine; D001120:Arginine; D012260:Ribonucleases; D006163:Ribonuclease T1; D014357:Trypsin; D006152:Cyclic GMP; D008239:Lysine