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

Abstract

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.

Substrate-induced modifications of the intrinsic fluorescence of the isolated adenine nucleotide carrier protein: demonstration of distinct conformational states

“…There are several lines of evidence indicating that the nucleotide-induced conformational changes of the isolated AdN carrier, as reflected by fluorescence changes, are similar to those occurring in the membrane-bound carrier during AdN transport: (1) Among natural nucleotides, only ADP and ATP are transported by mitochondria (Duée & Vignais, 1969) and are able to induce fluorescence changes in the isolated AdN carrier. (2) ADP/ATP transport in mitochondria is a two-step process; the first step corresponds to the binding of ADP or ATP to the membrane-bound carrier, the second step being the translocation of ADP or ATP across the membrane (Schlimme, 1980). Of the two steps, the second one that involves conformational changes of the carrier protein is probably the temperature-sensitive component of the transport process.…”

Chemical Probes of the Mitochondrial Atp Synthesis and Translocation

“…Lin-benzo ADP is a substrate for oxidative phosphorylation in submitochondrial particles, while lin-benzo ATP is hydrolyzed by soluble FI (61). To be transported by the AdN carrier, a nucleotide must have a non-fixed anti confonnation; additionally, transport requires a C6 positioned amino group and an unsubstituted C2 atom (36,63). For ADP or ATP analogs, this will depend on the substituted groups at the purine ring.…”

“…To be transported by the AdN carrier, a nucleotide must have a non-fixed anti confonnation; additionally, transport requires a C6 positioned amino group and an unsubstituted C2 atom (36,63). On the other hand, fonnycin di-and triphosphate (FDP and FTP) (63,71), tubercidin di-and triphosphate (TuDP and TuTP) (63,72), and I-N oxide-ADP and -ATP (32,73) that have the anti confonnation, and no steric restriction at difference with 2-azido ADP, bind to the AdN carrier and are transported. The 2-azido ADP analog presumably has the favorable anti conformation (67)(68)(69)(70).…”

Exploration of the nucleotide binding sites of the isolated ADP/ATP carrier protein from beef heart mitochondria. 2. Probing of the nucleotide sites by formycin triphosphate, a fluorescent transportable analog of ATP

“…The purpose of the present paper is to report on a similar study with formycin triphosphate (FTP), another fluorescent analogue of ATP (Ward et al, 1969a,b), which differs from N-ATP in that it is transported by the ADP/ATP carrier (Schlimme et al, 1980). The binding properties of FTP and the antagonistic effects of specific inhibitors were found to be significantly different from those described for N-ATP.…”