Electron-transferring flavoprotein has an AMP-binding site in addition to the FAD-binding site.
Mammalian electron-transferring flavoprotein (ETF) has been reported to consist of two non-identical subunits and one FAD. The present paper shows that ETF purified from pig kidney contains one more molecule, an AMP. ETF was denatured by guanidine hydrochloride and ultrafiltered for the purpose of removing proteins. The filtrate was analyzed by reverse-phase chromatography. Two peaks appeared on the chromatogram: they were identified as FAD and AMP, and their molar amounts were identical, indicating that ETF contains one AMP molecule. ApoETF, which was prepared by KBr treatment of ETF, also contains one AMP molecule. ApoETF, which was prepared by KBr treatment of ETF, also contain one AMP molecule. These results clearly demonstrate that ETF has an AMP-binding site in addition to the FAD-binding site. AMP-free apoETF was prepared by guanidine treatment of ETF. Mixing AMP-free apoETF, FAD, and AMP produced reconstituted ETF, which showed the same properties as native ETF. Mixing AMP-free apoETF and FAD produced AMP-free ETF, regardless of the coexistence of ATP or ADP: the AMP-binding site cannot bind FAD, ADP, or ATP. The enzymatic activity of the AMP-free ETF for electron transfer from substrate-reduced medium-chain acyl-CoA dehydrogenase to 2,6-dichlorophenolindophenol was identical to that of native ETF. This indicates that the AMP contained in holoETF has no apparent influence on this enzymatic activity. A role of AMP recognized in this study is that AMP facilitates the formation of holoETF from AMP-free apoETF, FAD, and AMP.
Sato
K
K
Department of Physiology, Kumamoto University School of Medicine.
Nishina
Y
Y
Shiga
K
K
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D044927:Electron-Transferring Flavoproteins; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism; D008433:Mathematics; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate