Exploration of nucleotide binding sites in the mitochondrial membrane by 2‐azido‐[α‐³²P]ADP

Abstract

The ADP/ATP carrier of beef heart mitochondria is able to bind 2-azido-[G2P]ADP in the dark with a Kd value of 'c 8 ,uM. 2-Azido ADP is not transported and it inhibits ADP transport and ADP binding. Photoirradiation of beef heart mitochondria with 2-azido-[a-32P]ADP results mainly in photolabeling of the ADP/ATP carrier protein; photolabeling is prevented by carboxyatractyloside, a specific inhibitor of ADP/ ATP transport. Upon photoirradiation of inside-out submitochondrial particles with 2-azido-[c+P]ADP, both the ADP/ATP carrier and the p subunit of the membrane-bound F,-ATPase are covalently labeled. The binding specificity of 2-azido-[cc-32P]ADP for the B subunit of F,-ATPase is ascertained by prevention of photolabeling of isolated F, by preincubation with an excess of ADP. Nucleotide binding Mitochondrial membrane 2-Azido-[a-32P]ATP

Labeling of two different regions of the nucleotide binding site of the uncoupling protein from brown adipose tissue mitochondria with two ATP-analogs

“…Nonspecific binding of 2-azido-ATP to mitochondrial membranes can also not be excluded. To determine the portion of 2-azido-ATP bound specifically to the UCP, binding of 2-azido-ATP was measured in the presence of GTP, because it is known that 2-azido-ATP but not GTP binds to the mitochondrial AAC (Dalbon et al, 1985;Weidemann et al, 1970). The GTPsensitive binding, corresponding to the specific binding of 2-azido-ATP to UCP, is much smaller and produces a linear relation in the mass action plot with a maximum of 1.0 nm.ol/g of protein, which is similar to the binding of GTP to mitochondria (Rafael et al, 1972;Nicholls, 1976), and gives a Kd value of 2.8 fiM (Figure 1A).…”

ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

“…2-Azido-ADP photolabels ␤ subunits exclusively upon photoirradiation, in contrast to 8-azido-ADP or -ATP, which modify both ␣ and ␤ subunits (86,89,419,421). 2-Azido-ADP binds to F 1 with an affinity similar to the affinity of ADP (45), and upon irradiation it modifies ␤Leu342, ␤Ile344, ␤Tyr345, ␤Pro346, or ␤Tyr368 (bovine heart MF 1 ) (111, 132).…”

8-N3-3′-Biotinyl-ATP, a Novel Monofunctional Reagent: Differences in the F1- and V1-ATPases by Means of the ATP Analogue

“…8-N 3 -ATP-analogues, on the other hand, most likely adopt a syn conformation in analogy to other nucleotides containing a bulky group at the five-member ring of the purine and thereby representing a conformational change inside the molecule (36,37). A selectivity of incorporation of a 2-N 3 -ATP-analogue in comparison with the 8-N 3 -ATP-analogue has been obtained by nucleotide binding studies with F 1 (38,39) or the fructose 1,6-bisphosphatase (40) with the latter showing a 1000-fold decreased affinity of fructose 1,6-bisphosphatase for the 8-N 3 -ATP-analogue (41) by comparison with the 2-N 3 -ATP-analogue (40). Photoirradiation of F 1 -ATPases with 2-azido-ADP resulted in the exclusive labeling of the ␤ subunit (38,39), thereby discriminating between the catalytic ␤ subunits, where the adenine is in contact with a hydrophobic interface (35), and the noncatalytic ␣ subunits, where binding of adenine involves several hydrogen bonds.…”

“…A selectivity of incorporation of a 2-N 3 -ATP-analogue in comparison with the 8-N 3 -ATP-analogue has been obtained by nucleotide binding studies with F 1 (38,39) or the fructose 1,6-bisphosphatase (40) with the latter showing a 1000-fold decreased affinity of fructose 1,6-bisphosphatase for the 8-N 3 -ATP-analogue (41) by comparison with the 2-N 3 -ATP-analogue (40). Photoirradiation of F 1 -ATPases with 2-azido-ADP resulted in the exclusive labeling of the ␤ subunit (38,39), thereby discriminating between the catalytic ␤ subunits, where the adenine is in contact with a hydrophobic interface (35), and the noncatalytic ␣ subunits, where binding of adenine involves several hydrogen bonds. This is in contrast to 8-N 3 -ATP or -ADP, which was found to photolabel both the ␣ and ␤ subunits of F 1 (42)(43)(44)(45).…”