0000000000294854
AUTHOR
Yasuo Kagawa
8-N(3)-3'-biotinyl-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. The introduction of an additional biotin residue is advantageous for easy detection of labeled proteins. This could be first tested by reaction with the F(1)-ATPase from the thermophilic bacterium PS3 (TF(1)). UV irradiation of TF(1) in the presence of 8-N(3)-3'-biotinyl-ATP results in a nucleotide-dependent binding of the analogue in the noncatalytic alpha and the catalytic beta subunits of TF(1), demonstrating the suitability of this analogue as a potential photoaffinity label. Reaction with the V(1)-ATPase, however, led to labeling of subunit E, which has been suggested as a structural and functional homologue of …
Photoaffinity cross-linking of F1ATPase from the thermophilic bacterium PS3 by 3′-arylazido-β-alanyl-2-azido ATP
AbstractThe photoactivatable bifunctional 3′-arylazido-β-alanyl-2-azido ATP (2,3′-DiN3ATP) has been applied to study the localization of the nucleotide-binding sites of coupling factor 1 (F1ATPase, TF1) from the thermophilic bacterium PS3 by photoaffinity cross-linking. UV irradiation of TF1 in the presence of 2,3′-DiN3ATP results in the nucleotide-dependent formation of various higher molecular mass cross-links formed by two, three or even four α- and/or β-subunits. The differences observed upon photoaffinity cross-linking by the bifunctional 2-azido ATP or 8-azido ATP analog are discussed. They are probably due to the varied maximal distance between both azido groups, or to the different …
Photoaffinity labeling of the coupling factor 1 from the thermophilic bacterum PS3 by 8-azido ATP
AbstractTo localize the nucleotide binding sites of the F1ATPase (TF1) from the thermophilic bacterium PS3 we have used 14C-labeled 8-azido ATP (8-N3ATP) as photoaffmity label. 8-N3ATP is hydrolyzed by the F,ATPase in the absence of ultraviolet light. Irradiation by ultraviolet light of the enzyme in the presence of 8-N3ATP results in reduction of ATPase activity and in preferential nucleotide specific labeling of the α subunits (0.8–0.9 mol 8-N3ATP/TF1,α:β = 4:1). Inactivation and labeling do not depend on the presence of Mg2+. Both effects decrease upon addition of various nucleotide di- or triphosphates.
2,8-Diazido-ATP — a short-length bifunctional photoaffinity label for photoaffinity cross-linking of a stable F1 in ATP synthase (from thermophilic bacteria PS3)
Abstract To demonstrate the direct interfacial position of nucleotide binding sites between subunits of proteins we have synthesized the bifunctional photoaffinity label 2,8-diazidoadenosine 5′-triphosphate (2,8-DiN3ATP). UV irradiation of the F1-ATPase (TF1) from the thermophilic bacterium PS3 in the presence of 2,8-DiN3ATP results in a nucleotide-dependent inactivation of the enzyme and in a nucleotide-dependent formation of α-β crosslinks. The results confirm an interfacial localization of all the nucleotide binding sites on TF1.