Gabriele Rathgeber

Photoaffinity cross‐linking of F1ATPase from the thermophilic bacterium PS3 by 3′‐arylazido‐β‐alanyl‐2‐azido ATP

The 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 conformations (anti/syn) of these analogs. The results confirm our suggestion that several (possibly all) nucleotide‐binding sites of F1ATPases are located at the interfaces between α‐ and β‐subunits.

3′-Arylazido-8-azido ATP—A cross-linking photoaffinity label for ATP binding proteins

Abstract The synthesis of 3′-O-{3-[N-(4-azido-2-nitrophenyl) amino] propionyl} 8-azido-adenosine 5′-triphosphate—a 3′-arylazido-8-azido ATP—is described. The ATP derivative is characterized by thin layer chromatography, infrared spectroscopy, and optical spectroscopy. Its photolysis upon irradiation with uv light and its stability in dependence on pH are tested. Its two photolabile azido groups allow the use of this ATP analog as a photoaffinity label for cross-linking the subunits of ATP binding proteins.

Photoaffinity cross-linking of oligomycin-sensitive ATPase from beef heart mitochondria by 3′-arylazido-8-azido ATP

Abstract Photoaffinity cross-linking of the oligomycin-sensitive ATPase from beef heart mitochondria by 3′-arylazido-8-azido ATP results in a nucleotide specific formation of a cross-link between α and/or β subunits. Moreover a nucleotide independent decrease of the heterogeneous 29–31 kd protein band is observed. This decrease can be reduced by addition of 2,4-dinitrophenol or 2-azido-4-nitrophenol.

Fluorescent photoaffinity labeling of F1 ATPase from Micrococcus luteus with 8-azido-1,N6-etheno-adenosine 5′-triphosphate

Abstract A fluorescent photoaffinity label—8-azido-1-N6-etheno-adenosine 5′-triphosphate (8-N3e ATP)—for ATP-binding proteins has been synthesized. The effectiveness of the label is demonstrated with F1ATPase from Micrococcus luteus. 8-N3e ATP is a substrate for the enzyme in the presence of bivalent cations. Ultraviolet irradiation of F1ATPase in the presence of the label and Mg2+ ions inhibits the enzyme irreversibly. The fluorescent label is bound preferentially to the β subunit of the enzyme. Labeling and inactivation are decreased by protection with ATP or ADP.

Photoaffinity labeling of the coupling factor 1 from the thermophilic bacterum PS3 by 8-azido ATP

To 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)

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.

3'-Arylazido-β-alanyl-2-azido ATP, a Cross-Linking Photoaffinity Label for F1ATPases

Abstract The synthesis of the 3′-arylazido-2-azido ATP derivative 3′-O-{3-[N-(4-azido-2-nitrophenyl)-amino]propionyl}2-azido-adenosine 5′-triphosphate (2,3′-DiN3ATP) is described. The bifunc tional photoreactive ATP analog is characterized spectroscopically. Photoaffinity labeling of F, ATPase from Micrococcus luteus by this analog results in the inactivation of the enzyme and in the formation of higher molecular weight cross-links,