PURIFICATION AND CHARACTERIZATION OF A RIBOFLAVIN-BINDING PROTEIN FROM FLAGELLA OF EUGLENA-GRACILIS

Phototaxis of the flagellate Euglena gracilis has been thought to be m ediated by flavin photoreceptor molecules localized in the paraflagell ar body (PFB). From isolated flagella of Euglena a riboflavin (RF)-bin ding protein was solubilized and purified using nonionic detergents, h igh ionic strength, affinity chromatography and standard column separa tions. Sodium dodecyl sulfate gel electrophoresis showed an apparent m olecular weight of 68 kDa for the binding protein. Its hydrophobicity was confirmed by Triton X-114 phase partitioning. Binding affinity for tritiated RF was high in the oxidized state (K-D = 4 nM) as well as u nder reducing conditions in the presence of dithionite (K-D = 6 nM). A ffinities towards flavin mononucleotide and flavin adenine dinucleotid e were lower. Based on binding data and on estimates of the purified 6 8 kDa polypeptide, approximately 10(6) flavin-binding sites were deter mined per one flagellum. Evidence is discussed that the flavin-binding protein is part of the entire flagellar membrane and does not reside in the PFB. If not the photoreceptor, the flagellar RF-binding protein may have a functional role in the biochemical chain leading from the reception of the phototactic stimulus to the motile response.