Abstract
Purified chloroplast ATP synthase (CF1) contains 1.2-2 mol of tightly bound ADP/mol of enzyme that resists removal by gel filtration or dialysis. CF1 was depleted of its endogenous nucleotide by treatment with alkaline phosphatase. Tightly bound nucleotide was demonstrated not to have an essential structural role. CF1 depleted of endogenous nucleotide retains its ability to catalyze Ca2+- and Mg2+-dependent ATPase activity and is not more sensitive to cold inactivation than untreated CF1. 2'(3')-O-Trinitrophenyladenosine 5'-diphosphate (TNP-ADP) binds tightly to two sites on nucleotide-depleted CF1, binding to either site at a faster rate than that of exchange of bound nucleotide for medium nucleotide. The nucleotide-depleted enzyme binds about one additional mol of TNP-ADP/mol of CF1, indicating that there is a tight TNP-ADP binding site that does not exchange readily with medium nucleotide. It is MgADP in this nonexchanging site, not the easily exchanging ADP binding site, that is responsible for the MgADP-induced inhibition of the ATPase activity. The rate of exchange of tightly bound ADP from CF1 matches the rate at which the Mg2+ATPase activity of CF1 is activated but is not itself responsible for the activation.
Highlights
It is MgADP in this nonexchanging site, not the been observed and appears to be induced by nucleotide binding [5]
The mechanism of alkaline phosphatase-induced release of bound ADP has not been investigated in detail, results of preliminary experiments suggest that alkaline phosphatase attacks ADP that had dissociated from CF1
This method has been routinely effective in producing nucleotide-depleted CF1 with an ADP content determined by high pressure liquid chromatography (HPLC) (Fig. 1) over 11 trials to be 0.094 Ϯ 0.04 mol/mol of CF1-⑀ compared with an untreated control content of 1.2 Ϯ 0.2 mol of ADP/mol of CF1-⑀
Summary
Reduced CF1-⑀ was prepared from market spinach by the procedure described in Shapiro and McCarty [5], with modifications described by Soteropoulos et al [10] and Digel and McCarty [7]. Alkaline phosphatase activity and must be removed from solution by passage through two consecutive 3-ml Sephadex G-50 columns equilibrated with 50 mM Tris-HCl, pH 8.0, 50 mM NaCl (TN buffer). This and all subsequent buffers were passed through a column of Chelex 100 resin to remove residual Mg2ϩ and were stored in plastic. ATPase activity of CF1-⑀ in the presence of Ca2ϩ was assayed by the incubation of 10 g of CF1-⑀ for 3–5 min at 37 °C in 50 mM Tris-HCl, 5 mM ATP, and 5 mM CaCl2.
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