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The conformationally restricted adenine nucleotide analogs 8,3'anhydro-8-oxy-9-(beta-D-xylofuranosyl)adenine-5'-O-tri(di)-phosphate (I), and 8,2'-anhydro-8-oxy-9-(beta-D-arabinofuranosyl) adenine-5'-O-tri(di)-phosphate (II), were prepared chemically as their alpha-32P-labelled compounds and compared with syn-structured 8-bromo AT(D)P in mitochondrial adenine nucleotide translocation. The experimental findings demonstrate that the heterocycle-ribose orientation affects the carrier mediated adenine nucleotide transport very strongly, i.e. a non fixed adenine heterocycle in the anti region is prerequisite for the bound nucleotide to induce the transfer action of the adenine nucleotide carrier.

1. P1, P5-Bis-(5'-adenosyl) pentaphosphate (AP5-A) inhibits "soluble" adenylate kinase even when this enzyme is an integral part of the complete mitochondrion. The Ki is 10(-5) M, i.e. about two orders of magnitude higher than the inhibitor contants determined for the purified adenylate kinase of rabbit muscle and an enzyme preparation separated from the mitochondrial intermembrane space. The weaker inhibitory effect is due to a lower accessibility of the enzyme. 2. as to be expected Ap5A which is of the "multisubstrate analogue"-type does not affect mitochondrial nucleoside diphosphate kinase. 3. Though Ap5A owns the structural elements of both ATP and ADP it is not a substrate of the adenine nucleotide carrier, i.e. neither it is exchanged across the inner mitochondrial membrane nor speicifically bound. 4. Ap5A is not metabolized by rat liver mitochondria.

Adenylate kinase activity of intact mitochondria is strongly inhibited by Ap5A, i.e. p1,p5-Di (adenosine-5') pentaphosphate, whereas oxidative phosphorylation is not affected. Therefore, Ap5A is a useful tool to distinguish between oxidative and non oxidative ATP generating reactions.

The ADP,ATP carrier of rat liver mitochondria is specifically inhibited by Meisenheimer-type trinitrophenyl (TNP) derivatives of ADP and ATP. Due to a systematic inhibition study we could show that the TNP-moiety itself, even in the 2', 3'-O-cyclic Meisenheimer complex, revealed no inhibition of mitochondrial ADP,ATP transport. Nucleosidic TNP-compounds are weak inhibitors. Introduction of a phosphate group at the 5'-position, however, enhances the inhibitory power markedly. Our findings point to a synergistic effect of the 5'-phosphate chain and the TNP-moiety. Irreversible inhibition by TNP-nucleotides can be ruled out to the fact that the inhibited ADP,ATP-transport system is fully reactivated by addition of albumin.

An ADP analogue, i.e. 3H- or 14C-labeled 5'-diphosphate of 6-(beta-D-ribofuranosyl-methyl)-4-pyrimidinamine, was synthesized, the structure of which was deduced from structure-activity studies on the substrate specificity of the nucleotide-binding center of the inner mitochondrial membrane integrated ADP/ATP carrier protein. Bearing only the minimal but substrate-essential recognition structures, minimum structured ADP (msADP) is bound to the cytosol-facing active center and transported by the highly specific carrier system across the inner mitochondrial membrane.

A variety of base-modified nucleotide analogues was prepared and characterized as their alpha-32P- or U-14C-labeled compounds. Carrier-linked nucleotide binding and carrier-catalyzed exchange across the inner membrane of rat liver mitochondria were measured by using an inhibitor (atractyloside) stop method. Kinetic data of carrier-specific bound analogues were evaluated from Dixon plots and indicate that these analogues are competitive inhibitors for mitochondrial [14C]ADP uptake. Km and Vmax values for carrier-mediated uptake of nucleotide analogues were calculated from Lineweaver-Burk plots. By means of the analogues, a systematic mapping of the essential chemical and steric interactions between the transporter protein and the heterocycle of its substrate in the course of the binding as well as transfer step was achieved. Prerequisites for carrier-specific binding (recognition) are (A) an anti- or syn-positioned beta-glycosyl-linked heterocycle, (B) a nitrogen ring atom in position 7 for syn-structured analogues, and (C) an electron-rich region at the N(1) position, i.e., a permanent dipole moment oriented toward N(1) for anti-structured analogues. Additional requirements for subsequent transport catalysis are (A) a non-fixed anti-positioned base moiety with a beta-glycosyl torsion angle of about -20 degrees, (B) a C(6)-positioned amino group, and (C) an unsubstituted C(2) atom. The complementary binding site at the carrier protein to the N(1)-C(6)(-NH2) amidine region is proposed to be represented by two juxtaposed and invariant bonding points, i.e., an asparagine or glutamine residue.

(Adenine-14C) or (gamma-32P)-labelled 2,2'[1-(9-adenyl)-1'-(tri-, diphosphoryl-oxymethyl]-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 a competitive inhibitor for ANP uptake with Ki = 9.3 X 10(-5) M. Experimental rroANP confirmed that an intact adenine base and three anio.nic charges of the phosphate chain are essential for the recognition between ANP-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.

1. Solubility of mitochondrial membranes in various solvent systems was determined quantitatively. The most effective agent was the anionic detergent, sodium dodecylsulphate, which solubilizes 90% of the protein at the concentration of 0.1% followed by Triton X-100 (70%), sodium deoxycholate (60%), Brij 56 (50%), and guanidine hydrochloride (40%) at a concentration of 2 M. 2. Affinity chromatography of a clear 0.1% sodium dodecylsulphate solution of digitonized mitochondria on Sepharose 4B containing carboxyatractylate always resulted in the separation of two fractions, one of which was not retained by the column and the other which could be obtained after elution with 2% sodium dodecylsulphate. 3. The retained protein showed a high binding specificity for ATP and [3H]atractylate when compared with the unretained fraction. The amount of bound [3H]atractylate or carboxyatractylate-sensitive binding of ATP was 10.5 +/- 4 nmol/mg protein, and 22 +/- 8 nmol/mg protein, respectively. 4. The major component within the retained fraction, comprising 85% of the total weight, was protein, followed by phospholipids (14%) and approximately 1% triglycerides. Sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed a major (95%) and a minor (5%) component with an apparent molecular weight of 26000 +/- 1000 and 8300 +/- 400, respectively. The gels did not stain for carbohydrates. Ultracentrifugal analysis showed a single, symmetrical boundry. 5. Double immunodiffusion analysis gave a single precipitin line with the corresponding antiserum. [14C]ADP exchange of digitonin particles was completely inhibited by an antiserum to the carboxyatractylate binding protein fraction, whereas the adenine nucleotide transport of intact mitochondria remained unaffected. In the presence of specific immunoglobulins state-3 respiration rate of digitonin particles was prolonged and reduced by approximately 25%. State-4 respiration rate was unaffected.