8-N-3-3 '-biotnyl-ATP, a novel monofunctional reagent: Differences in the F-1- and V-1-ATPases by means of the ATP analogue

A novel photoaffinity label, 8-N-3-3'-biotinyl-ATP, has been synthesized. T he introduction of an additional biotin residue is advantageous for easy de tection of labeled proteins. This could be first tested by reaction with th e F-1-ATPase from the thermophilic bacterium PS3 (TF1). UV irradiation of T F1 in the presence of 8-N-3-3'-biotinyl-ATP results in a nucleotide-depende nt binding of the analogue in the noncatalytic a and the catalytic beta sub units of TF1, demonstrating the suitability of this analogue as a potential photoaffinity label. Reaction with the V-1-ATPase, however, led to labelin g of subunit E, which has been suggested as a structural and functional hom ologue of the gamma subunit of the F-ATPases. MALDI-TOF mass spectrometry h as been used to map the regions of subunit E involved in the binding of 8-N -3-3'-biotiinyl-ATP. (C) 2001 Academic Press.