Purification, properties, and function of flavokinase from rat intestinal mucosa.
Flavokinase (ATP:riboflavin 5'-phosphotransferase) [EC 2.7.1.26] was purified to apparent homogeneity from rat intestinal mucosa by fractionation with ammonium sulfate, gel filtration, and flavin affinity chromatography. The addition of ATP to the enzyme solution was necessary for its binding to the affinity gel. The apparent molecular weight of the enzyme was estimated to be 13,500 by gel filtration on Sephadex G-100 and by SDS-PAGE. The properties of the enzyme, including its flavin specificity, were studied. Three types of riboflavin analogues were used for the flavin specificity study; namely, ones modified at the ribityl group, and at positions 3 and 8 of the isoalloxazine ring. Of the analogues modified at the ribityl group or position 3 of the isoalloxazine ring, only 2'-deoxyriboflavin was phosphorylated and then only weakly. On the other hand, most analogues modified at position 8 of the isoalloxazine ring were good substrates for the kinase, an appropriate increase in the substituent volume at position 8 of the isoalloxazine ring resulting in an increase in the Vmax value. In a previous paper on the mechanism of intestinal absorption of riboflavin, we proposed that one of the specific processes for the absorption of riboflavin is phosphorylation by flavokinase [Kasai, S. et al. (1988) J. Nutr. Sci. Vitaminol. 34, 265-280]. The present results support this conclusion because analogues that were absorbed at low concentrations through a process specific for riboflavin in our previous study were phosphorylated effectively by the enzyme, whereas those that were absorbed solely through simple diffusion at all concentrations were not phosphorylated or only phosphorylated weakly. The properties of the flavokinases from intestinal mucosa and liver were compared.
Kasai
S
S
Research Institute for Atomic Energy, Osaka City University.
Nakano
H
H
Maeda
K
K
Matsui
K
K
D000818:Animals; D002846:Chromatography, Affinity; D005415:Flavins / Q000378:metabolism; D005782:Gels; D007413:Intestinal Mucosa / Q000201:enzymology*; D007583:Jejunum / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008297:Male; D008970:Molecular Weight; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D005415:Flavins; D005782:Gels; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022532:riboflavin kinase