(1)
a photoactivable derivative of 5-adenylyl imidodiphosphate (Ado
PP[NH]
P), was synthesized. (2) Binding of
3H]NAP
4-Ado
PP[NH]
P to soluble ATPase from beef heart mitochrondria (F
1) was studied in the absence of photoirradiation, and compared to that of
. The photoactivable derivative of Ado
PP[NH]
P was found to bind to F
1 with high affinity, like Ado
PP[NH]
P. Once
had bound to F
1 in the dark, it could be released by Ado
PP[NH]
P, ADP and ATP, but not at all by NAP
4 or AMP. Furthermore, preincubation of F
1 with unlabeled Ado
PP[NH]
P, ADP, or ATP prevented the covalent labeling of the enzyme by
upon photoirradiation. (3) Photoirradiation of F
1 by
resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol
.
Photolabeling by NAP
4-Ado
PP[NH]
P was much more efficient in the presence than in the absence of MgCl
2. (4) Bound
was localized on the α- and β-subunits of F
1. At low concentrations (less than 10 μM), bound
was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F
1. (6) The covalently photolabeled F
1 was able to form a complex with aurovertin, as does native F
1. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F
1 was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F
1.
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