STRUCTURE-FUNCTIONAL DOMAINS OF ELONGATIO N-FACTOR EF-2 - ANALYSIS OFFRAGMENTS OF THE EF-2 LIMITED HYDROLYSIS WITH TRYPSIN AND ELASTASE

Limited hydrolysis of EF-2 with trypsin in mild conditions leads to cl eavage at the N-terminal part of the protein, at the region of phospho rylation, at the Arg54 and Arg65 residues. The trypsinolysis product, fragment T1', containing Thr56 and Thr58, which are phosphorylated in EF-2, is also phosphorylated by EF-2-kinase at the same residues. In t he phosphorylated EF-2, digestion by trypsin takes place only at Arg65 , resulting in a reduction of the rate of hydrolysis in comparison wit h the native EF-2. Digestion of EF-2 with elastase results in the form ation of two fragments E1 and E2 (60 and 40 kDa, respectively). Fragme nt E1 represents the N-terminal part of EF-2. It is resistant to the f urther action of elastase, is not cleaved by trypsin, and loses its ca pability for phosphorylation. Fragment E2, and C-end part of the molec ule, is not resistant to the further action of elastase and retains it s capability for ADP-ribolyzation with the A fragment of diptheria tox in and NAD+. Electrophoretic analysis of EF-2 and its proteolytic frag ments according to O'Farrell showed that the modification, resulting i n the presence of two initial forms of EF-2, is located between the am ino acid residues 66 and 506 of the polypeptide chain. In conclusion a possibility of studying the formation of partial functional activitie s within the framework of individual structure-functional domains usin g a set of N-terminal fragments of various length is discussed.