title: [(3)H]Adenine is a suitable radioligand for the labeling of G protein-coupled adenine receptors but shows high affinity to bacterial contaminations in buffer solutions.
abstract: [(3)H]Adenine has previously been used to label the newly discovered G protein-coupled murine adenine receptors. Recent reports have questioned the suitability of [(3)H]adenine for adenine receptor binding studies because of curious results, e.g. high specific binding even in the absence of mammalian protein. In this study, we showed that specific [(3)H]adenine binding to various mammalian membrane preparations increased linearly with protein concentration. Furthermore, we found that Tris-buffer solutions typically used for radioligand binding studies (50 mM, pH 7.4) that have not been freshly prepared but stored at 4 degrees C for some time may contain bacterial contaminations that exhibit high affinity binding for [(3)H]adenine. Specific binding is abolished by heating the contaminated buffer or filtering it through 0.2-mum filters. Three different, aerobic, gram-negative bacteria were isolated from a contaminated buffer solution and identified as Achromobacter xylosoxidans, A. denitrificans, and Acinetobacter lwoffii. A. xylosoxidans, a common bacterium that can cause nosocomial infections, showed a particularly high affinity for [(3)H]adenine in the low nanomolar range. Structure-activity relationships revealed that hypoxanthine also bound with high affinity to A. xylosoxidans, whereas other nucleobases (uracil, xanthine) and nucleosides (adenosine, uridine) did not. The nature of the labeled site in bacteria is not known, but preliminary results indicate that it may be a high-affinity purine transporter. We conclude that [(3)H]adenine is a well-suitable radioligand for adenine receptor binding studies but that bacterial contamination of the employed buffer solutions must be avoided.
authors.affiliation: University of Bonn, Pharmaceutical Institute, Pharmaceutical Sciences Bonn (PSB), Pharmaceutical Chemistry I, Bonn, Germany.
authors.forename: Anke C
authors.initials: AC
authors.lastname: Schiedel
authors.forename: Heiko
authors.initials: H
authors.lastname: Meyer
authors.forename: Bernt B A
authors.initials: BB
authors.lastname: Alsdorf
authors.forename: Simone
authors.initials: S
authors.lastname: Gorzalka
authors.forename: Hannelore
authors.initials: H
authors.lastname: Brüssel
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller