Steps in the Formation of Active Derivatives of Staphylococcal Nuclease during Trypsin Digestion

Abstract

A staphylococcal nuclease (strain Foggi), very similar in chemical structure to the enzyme from strain V8, is cleaved in a limited way by trypsin in the presence of deoxythymidined/,5'-diphosphate and Ca++.Such treatment yields fragments PI (residues 1 to 5), PZ (residues 6 to 48), P3 (Psa, residues 49 to 149; and P3b, 50 to 149), and free lysine (residue 49).None of the products shows enzymatic activity.However, Fragments Pf and PB (either Paa or P& associate to form an active, noncovalently bonded derivative, nuclease-T.Evidence is presented to show that the cleavage of the peptide bonds in nuclease between residues 48 and 49 or between 49 and 50, but not the bond between residues 5 and 6, account for the decreased enzymatic activity and heat stability observed with nuclease-T. RESULTS Components of Nuclease-T-Whennuclease was digested with trypsin in the presence of pdTp and Ca++, less than 0.5 eq of 2 The abbreviations used are: DNP-, dinitrophenyl-; pdTp, deoxythymidine-3',5'-diphosphate.4778