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 the gamma subunit of the F-ATPases. MALDI-TOF mass spectrometry has been used to map the regions of subunit E involved in the binding of 8-N(3)-3'-biotinyl-ATP.
Schäfer
H J
HJ
Institut für Biochemie, Johannes Gutenberg-Universität, Mainz, D-55099, Germany.
Coskun U
Eger
O
O
Godovac-Zimmermann
J
J
Wieczorek
H
H
Kagawa
Y
Y
Grüber
G
G
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000737:chemistry* / Q000494:pharmacology*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000737:chemistry* / Q000494:pharmacology; D002384:Catalysis; D002417:Cattle; D018402:Manduca; D008956:Models, Chemical; D008958:Models, Molecular; D019920:Photoaffinity Labels / Q000494:pharmacology; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013053:Spectrophotometry; D013997:Time Factors; D014466:Ultraviolet Rays; D025262:Vacuolar Proton-Translocating ATPases
C436521:8-N(3)-3'-biotinyl-ATP; D001426:Bacterial Proteins; D019920:Photoaffinity Labels; D001710:Biotin; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases