Properties of the Ribose‐Ring‐Opened Adenine Nucleotide, 2,2′[1‐(9‐adenyl)‐1′‐(tri‐,diphosphoryl‐oxymethyl)]‐dihydroxydiethylether in Mitochondrial Adenine‐Nucleotide Translocation

Abstract

(Adenine-'4C) or (y3'P)-labelled 2,2'[1-(9-adenyl)-I '-(tri-, diphosphoryl-oxymethy1)l-dihydroxydiethylether (rroANP) was obtained from ANP by cleavage of the C-2'-C-3' bond by sodium periodate oxidation and subsequent borohydride reduction.Binding of rroANP to rat liver mitochondria revealed carrier-linked (atractyloside-sensitive) and unspecific (atractyloside-insensitive) binding but no transfer across the inner mitochondrial membrane. Kinetic data indicate rroANP as acompetitive inhibitor for AhT uptake with Ki = 9.3 x M. Experimental rroANP confirmed that an intact adenine base and three anionic charges of the phosphate chain are essential for the recognition between AM'-carrier and nucleotide but insufficient for the induction of a transmembrane ANP exchange. In addition mobilisation of the carrier-nucleotide complex requires an intact ribofuranoside ring system. Mitochondria1 AM' translocation is a key step for energy supply processes of aerobic organisms. Much interest has been directed to the catalytic mechanism of this carrier-mediated adenine nucleotide transfer across the inner mitochondrial membrane in the last decade [1,2]. Up to now, the molecular nature of the membrane integral Am-carrier is still unknown, but lately isolation and characterisation techniques have been developed [3 -61. Studies of specific interactions between the carrier and its substrate are actually still confined to measurements in situ. Analogues of the adenine nucleotides, therefore, are very useful probes for studying structural and electronic factors involved in the catalytic mechanism of AAPdependent biological processes in general and, as shown in this paper, on mitochondrial ANP translocation [7,8, and cited references].This publication describes carrier-linked binding and exchange properties of the ribose-ring opened Am-analogue 2,2'[1-(9-adenyl)-l'-(tri-, diphosphoryl-oxymethyl)]-dihydroxydiethylether rro-AT(D)P. Our interest was directed to investigations of struc- tural requirements of the mitochondrial membrane integral Am-carrier for substrate binding and transfer across the inner mitochondrial membrane. EXPERIMENTAL PROCEDURE MATERIALS Preparation of rroATPThe ribose-ring opened ATP analogue was prepared by cleavage of the C-2' -C-3' bond in a modidure [9 -111 by sodium periodate oxidation and subsequent sodium borohydride reduction.8.25 pmol ATP disodium salt was dissolved in 0.22 ml 0.1 M phosphate buffer pH 7.0 and 0.44 ml 0.1 M sodium periodate were added. Excess periodate was destroyed by addition of 0.045 ml 1 M glucose solution. (Reaction time 45 min). After oxidation 5 m g sodium borohydride solved in 0.43ml double distilled water was added. The pH of the reaction mixture was adjusted to 7.8 by 2 M sodium acetate buffer pH 5.2. After a reaction period of 24 h at room temperature excess sodium borohydride was destroyed by adjusting the pH to 5.2. The reaction mixture was then applied to a DEAE-cellulose column (Whatman DEAE 32-cellulose, 1 x 5 cm). The modified ANP was eluted by a linear gradient of trieth...

Adenosine di- and triphosphate transport in mitochondria. Role of the amidine region for substrate binding and transport

“…Effect of Substrate Analogues on the Uptake of [U-UC\-ANP by Rat Liver Mitochondria. Sampling for rate measurements was performed within the first 4-15 s of [U-14C]-ANP uptake (Boos & Schlimme, 1979). Competition studies were carried out with analogues which were found to bind specifically to the carrier protein, i.e., 1, 3, 4, and 10, or those showing distinct unspecific binding, i.e., 9.…”

“…chemically phosphorylated to the corresponding S'-monophosphates by using [32P] phosphorus oxychloride in either acetonitrile (Sowa & Ouchi, 1975) or trimethyl phosphate (Yoshikawa et al, 1967), respectively. The a-32P-labeled nucleoside S'-triphosphates were prepared via the phosphorylimidazolide (Cramer et al, 1961) according to Hoard & Ott (1965) in a modified procedure (Boos & Schlimme, 1979). Separation of the products from the reaction mixture was achieved by DEAE 52-cellulose column chromatography with a linear triethylammonium hydrogen carbonate gradient (Boos & Schlimme, 1979).…”

“…The a-32P-labeled nucleoside S'-triphosphates were prepared via the phosphorylimidazolide (Cramer et al, 1961) according to Hoard & Ott (1965) in a modified procedure (Boos & Schlimme, 1979). Separation of the products from the reaction mixture was achieved by DEAE 52-cellulose column chromatography with a linear triethylammonium hydrogen carbonate gradient (Boos & Schlimme, 1979). iV'-Methyl-ANP (6) was prepared as an unlabeled compound and was purified by CM 52-cellulose column chromatography using ammonium formate (0-0.5 M, pH 2.6).…”

“…Mitochondrial adenine nucleotide translocation studies were carried out at 5 °C by using the inhibitor stop method described by Pfaff & Klingenberg (1968). For further details in assaying, see Boos & Schlimme (1979). In inhibition experiments, sampling for rate measurements was performed within the linear phase (4-15 s) of [14C]adenine nucleotide uptake at 5 °C under varying substrate concentrations i«Km) according to Boos et al (1975).…”

“…For further details in assaying, see Boos & Schlimme (1979). In inhibition experiments, sampling for rate measurements was performed within the linear phase (4-15 s) of [14C]adenine nucleotide uptake at 5 °C under varying substrate concentrations i«Km) according to Boos et al (1975).…”