Nucleotide interactions with the dicyclohexylcarbodiimide-sensitive adenosinetriphosphatase from spinach chloroplasts.
The intrinsic nucleotide content of the dicyclohexylcarbodiimide-sensitive ATPase (DSA) from spinach chloroplasts and its interactions with ADP have been studied. Both partially purified and sucrose gradient purified DSA contain at least 1 mol of ADP/mol of enzyme and 1 mol of ATP/mol of enzyme, although considerable variation exists between different preparations. Radioactively labeled ADP is incorporated into DSA in the presence of 5 mM MgCl2 and 10 mM octyl glucoside with a half-life of approximately 30 min. Incorporation of ADP into DSA reconstituted in phospholipid vesicles occurs at about twice this rate, and a slightly slower rate of uptake is observed with [3H]ADP and [3H]ATP in the presence of 2 mM ethylenediaminetetraacetic acid. The [3H]ATP always appears as bound [3H]ADP on the enzyme. Nucleotide analyses indicate that this incorporation represents an exchange with tightly bound ADP. The nucleotide exchange requires binding at another nucleotide site or sites on the enzyme and is essentially a one-turnover process. Even during ATP synthesis less than 20% of incorporated 3H-labeled nucleotide is removed. Binding studies with forced dialysis indicate the presence of a reversible binding site for ADP distinct from the nucleotide exchange. Similar binding isotherms are obtained for the partially purified enzyme stabilized with 10 mM octyl glucoside, the gradient-purified enzyme stabilized with 0.4% sodium cholate, and the reconstituted, partially purified enzyme. The binding stoichiometry is approximately 0.5 mol of ADP/mol of DSA and the dissociation constant is approximately 2 microM, which is similar to the Michaelis constant for ADP estimated from kinetic studies of ATP synthesis.
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001692:Biological Transport; D002234:Carbodiimides / Q000494:pharmacology*; D002736:Chloroplasts / Q000201:enzymology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D007700:Kinetics; D010944:Plants / Q000201:enzymology
D002234:Carbodiimides; D004024:Dicyclohexylcarbodiimide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases