ELECTRON-TRANSFERRING FLAVOPROTEIN HAS AN AMP-BINDING SITE IN ADDITION TO THE FAD-BINDING SITE

Mammalian electron-transferring flavoprotein (ETF) has been reported t o consist of two non-identical subunits and one FAD. The present paper shows that ETF purified from pig kidney contains one more molecule, a n AMP. ETF was denatured by guanidine hydrochloride and ultrafiltered for the purpose of removing proteins. The filtrate was analyzed by rev erse-phase chromatography. Two peaks appeared on the chromatogram: the y were identified as FAD and AMP, and their molar amounts were identic al, indicating that ETF contains one AMP molecule. ApoETF, which was p repared by KBr treatment of ETF, also contained one AMP molecule. Thes e results clearly demonstrate that ETF has an AMP-binding site in addi tion to the FAD-binding site. AMP-free apoETF was prepared by guanidin e treatment of ETF. Mixing AMP-free apoETF, FAD, and AMP produced reco nstituted ETF, which showed the same properties as native ETF. Mixing AMP-free apoETF and FAD produced AMP-free ETF, regardless of the coexi stence of ATP or ADP: the AMP-binding site cannot bind FAD, ADP, or AT P. The enzymatic activity of the AMP-free ETF for electron transfer fr om substrate-reduced medium-chain acyl-CoA dehydrogenase to 2,6-dichlo rophenolindophenol was identical to that of native ETF. This indicates that the AMP contained in holoETF has no apparent influence on this e nzymatic activity. A role of AMP recognized in this study is that AMP facilitates the formation of holoETF from AMP-free apoETF, FAD, and AM P.