Stimulation of Escherichia coli DNA photoreactivating enzyme activity by adenosine 5'-triphosphate.
A purification procedure consisting of Biorex-70, single-stranded DNA-agarose, and ultraviolet (UV) light irradiated DNA-cellulose chromatography has been adopted for the Escherichia coli photoreactivating enzyme. The purpose of this purification was to obtain enzyme preparations that are free of extraneous nucleic acid or nucleotides. The purification yields high specific activities (75 000 pmol h-1 mg-1) with a 50% recovery. Enzyme preparations have also been obtained from UV-irradiated DNA-cellulose by exposure to visible light. These enzyme preparations contain oligoribonucleotides, the largest found to be 26 nucleotides in length in relation to DNA size markers. However, the oligoribonucleotides associated with the enzyme are not essential for enzymatic activity. When the enzyme is preincubated with exogenous ATP for 4-10 h at 3 degrees C, a 10-fold stimulation in the enzyme activity has been observed. It has been determined by polyacrylamide gel electrophoresis and high-voltage diethylaminoethyl paper electrophoresis that the light-released enzyme samples from a preincubated and washed mixture of the enzyme, [gamma-32P]ATP, and UV-irradiated DNA-cellulose contained exogenous [gamma-32P]ATP. [gamma-32P]ATP eluted with the enzyme-containing fractions when subjected to Bio-Gel P-30 chromatography. GTP caused a slight enhancement of the enzyme activity while ADP strongly inhibited photoreactivation, at the same concentration and conditions as those for ATP. Higher (X5) concentrations of ADP and adenosine 5'-(beta, gamma-methylenetriphosphate) totally inhibited the enzyme activity. Dialysis of a photoreactivating enzyme preparation against a buffer solution containing 1 mM ATP caused a 9-fold stimulation of the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D012260:Ribonucleases
D000255:Adenosine Triphosphate; D012260:Ribonucleases; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase