Hans-Jochen Schäfer

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.

Identification of purine binding sites on torpedo acetylcholine receptor

Electrophysiological studies from this and other laboratories have suggested a direct action of ATP on nicotinic acetylcholine receptors (nAChR). To determine the site of binding of this purine derivative, we have covalently modified the nAChR from Torpedo marmorata electrocytes employing 2-[3H]-8-azido-ATP as a photoactivable affinity label. Covalently attached radioactivity was predominantly found in the beta-polypeptide of the receptor. Based on the results of protection studies with several nAChR ligands whose target sites at the receptor are known, we conclude that the purine site(s) differ from those of acetylcholine and of physostigmine, galanthamine and related ligands, and those of local anesthetics.

Conversion of the Ca2+-ATPase from Rhodospirillum rubrum into a Mg2+-dependent enzyme by 1,N6-etheno ATP

Nucleoside triphosphate hydrolysis of R.rubrum ATPase complexes can be changed from Ca2+-dependence to Mg2+-dependence by replacing ATP with 1,N6-etheno ATP. Four ATPase complexes which have been prepared by different procedures hydrolyze ATP and 1,N6-etheno ATP at different rates in dependence on the added metal ions. These differences allow an easy distinction of the various enzyme forms.

In human and rat lung membranes [35s]GTPγS binding is a tool for pharmacological characterization of G protein-coupled devucleotide receptors

The P2Y receptor family is activated by extracellular nucleotides such as ATP and UTP. P2Y receptors regulate physiological functions in numerous cell types. In lung, the P2Y2 receptor subtype plays a role in controlling Cl- and fluid transport. Besides ATP or UTP, also diadenosine tetraphosphate (Ap4A), a stable nucleotide, seems to be of physiological importance. In membrane preparations from human and rat lung we applied several diadenosine polyphosphates to investigate whether they act as agonists for G protein-coupled receptors. We assessed this by determining the stimulation of [35S]GTPgammaS binding. Stimulation of [35S]GTPgammaS binding to G proteins has already been successfully applied to elucidate agonist binding to various G protein-coupled receptors. Ap(n)A (n = 2 to 6) enhanced [35S]GTPgammaS binding similarly in human and rat lung membranes, an indication of the existence of G protein-coupled receptor binding sites specific for diadenosine polyphosphates. Moreover, in both human and rat lung membranes comparable pharmacological properties were found for a diadenosine polyphosphate ([3H]Ap4A) binding site. The affinity for Ap2A, Ap3A, Ap4A, Ap5A, and Ap6A was also comparable. 8-Diazido-Ap4A and ATP were less potent, whereas the pyrimidine nucleotide UTP showed hardly any affinity. Thus, we present evidence that different diadenosine polyphosphates bind to a common G protein-coupled receptor binding site in membranes derived either from human or rat lung.

Photoaffinity labelling of a low-affinity nucleotide binding site on the β-subunit of yeast mitochondrial F1-ATPase

Mitochondrial ATP synthase from yeast is an oligomeric enzyme consisting of a hydrophobic membrane part (Fe) and a hydrophilic part (Fr). Fr is composed of 5 different polypeptide chains (o to c) [Il. Lowand high-affinity nucleotide diand triphosphate binding sites have been characterized on the enzyme from various sources by different kinetic techniques [2,3]. Alternatively, binding sites have been located on specific subunits by photoaffinity labelling [4-81. Recently, the development of a fluorescent photoaffinity label (8-azido-I ,Ne-ethenoATP) was reported [9], which might allow the identification of a binding site with respect to the hydrolytic or synthetic function of Fr . We have therefore investigated the interaction of this compound with yeast Fr . It will be shown that the label interacts with a low-affinity nucleotide binding site, and that upon photolabelling it is bound to the P-subunit.