In Vitro Adenylylation of Lysine-Sensitive Aspartylkinase from Escherichia coli TIR-8

Incubation of lysine sensitive aspartylkinase (AK III) from Escherichia coli Tir-8, with either [³H]ATP or [α-³²P]ATP, Mg²⁺ and E. coli extracts, results in a time-dependent incorporation of radioactive nucleotide (2-10%/subunit) into AK III. The nucleotide remains bound to the AK III after chromatography on Sephadex G-25, Sephadex G-200, DEAE-Sephadex, and polyacrylamide gel electrophoresis. Covalent attachment of the nucleotide is further demonstrated by isolation of labeled tryptic peptides by both Dowex 1-X2 chromatography and mapping of the peptides on paper by electrophoresis and chromatography. The bound nucleotide is sensitive to hydrolysis by snake venom phosphodiesterase indicating that the nucleotide is bound in a phosphodiester linkage.