Interactions between the mitochondrial adenosinetriphosphatase and periodate-oxidized adenosine 5'-triphosphate, an affinity label for adenosine 5'-triphosphate binding sites

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Abstract
Periodate-oxidized ATP (o-ATP) was prepared as an affinity label of nucleotide binding sites on the chloroform-released ox heart mitochondrial ATPase. In the presence of MgSO4, o-ATP is a substrate for the ATPase. It can act as a reversible, competitive inhibitor of ATPase activity and can also induce an irreversible inhibition of ATPase activity. In parallel with the irreversible inhibition, covalent incorporation of [3H]o-ATP occurs. ATPase has about 1.05 mol of o-ATP bound per mol of ATPase when the enzyme is 50% inhibited. Most of the covalently bound o-ATP is associated with the .alpha. and .beta. subunits and is equally distributed between them. The incorporation of o-ATP into the ATP is reduced, and the irrevesible inhibition induced by o-ATP can be prevented totally by MgADP, MgATP, EDTA/ATP, or EDTA. The location, number and the functional significance of the o-ATP binding sites are discussed. o-ATP can decompose to form an adenine-containing compound and the tripolyphosphate anion in a .beta.-elimination reaction mechanism. The structures of the adenine-containing compound and its borohydride reduction product were determined. The adenine-containing comopund elimination product inhibited the mitochondrial ATPase activity at a rate greater than that observed with o-ATP. The nature and mechanism of the inhibition of ATPase activity exerted by o-ATP and the elimination product were examined. The significance of the .beta.-elimination reaction to the use of periodate-oxidized nucleotides as affinity labels of nucleotide binding sites on other proteins is discussed.