The Intracellular Localization, Partial Purification, and Properties of Flavokinase from Rat Liver

Flavin mononucleotide is an essential coenzyme for several known oxidative enzymes and is the precursor of flavin adenine dinucleotide (2). Some information is available concerning production of flavin mononucleotide from riboflavin and adenosine triphosphate in several bacteria (3-5)) yeast (6-S), higher plants (9, lo), and intestine (11) ; a reaction which is catalyzed by flavokinases. Despite the intrinsic importance of this enzyme, only the kinase from yeast has been studied in reasonable detail (6), especially with regard to substrate specificity (7). Indeed, the occurrence and function of flavokinase in the mammal remained uncertain until recently. It had been suggested that intestinal mucosa is the site of phosphorylation of riboflavin within the mammal (12), perhaps by a transphosphorylation catalyzed by nonspecific alkaline phosphomonoesterase (13). The recent demonstration that extracts from several rat tissues contain flavokinase activity which is largely masked by phosphatases (14), makes less tenable the suggestion of a primary role played by the small intestine in the production of flavin mononucleotide for use in other organs. Furthermore, with the partial purification and characterization of flavokinase from rat liver (l), it seems necessary to invoke the role of a kinase in the synthesis of flavin mononucleotide, rather than a transphosphorylation catalyzed by a phosphatase. Those acid phosphatases which most readily hydrolyzed flavin mononucleotide are widespread among animal tissues, but apparently do not catalyze transphosphorylation with usual phosphate esters and riboflavin (15). The present study describes the intracellular localization, partial purification, and properties of flavokinase from rat liver. The substrate specificity of the liver enzyme appears similar to that of the yeast kinase insofar as comparisons have been made. In addition to those structural analogues of riboflavin which were used to incompletely ascertain the specificity of the yeast kinase (7), several others have been included in the present work.