Abstract
Flavokinase (ATP : riboflavin 5'-phosphotransferase, EC 2. 7. 1. 26, FK) was purified from chicken liver by gel filtration on Sephadex G-50 and affinity chromatography. The specific activity of the enzyme was 760 units/mg of protein. The enzyme was purified approximately 1,000-fold from the supernatant of the crude extract. The apparent molecular weight of the enzyme was calculated to be 14,000±1,000 by gel filtration on TSKgel G3000SW, and to be 13,500±1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal temperature of the reaction was lower than that of FK from rat liver and small intestine. The optimal pH was 9.0 for 10^<-4>M Zn(II) and 9.6〜for 10^<-4>M Mg(II). The purified FK showed the activity with ATP as the phosphate donor. No activity was observed with GTP, CTP, ADP, and AMP, as opposed to finding for the rat small intestine FK which was active with UTP as effective as ATP. The K_m value for ATP was calculated to be approximately 20 μM. The K_i value for ADP was calculated to be 200 μM against ATP. This K_i value was approximately 9 times as large as that of the rat liver FK reported by Yamada et al. The isoelectric point was determined to be approximately 5.7. The enhancement of FK activity attributable to addition of riboflavin binding protein was not confirmed.
