Effect of Covalent Binding of a Derivative of 2', 3'-O-(2, 4, 6-Trinitrophenyl)-ADP to the Tight Binding Site of CF₁ on the Enzyme Activity

Abstract

Irradiation of isolated chloroplast thylakoids with TNP-ADP results in non-covalent binding and covalent incorporation of a reaction product of TNP-ADP formed by photo-synthetic reduction into the so-called “tight” nucleotide binding site of CF₁ [Ponse et al. (1992) Z. Naturforsch. 47 c, 264-274]. CF₁ extracted from thus-loaded thylakoid membranes yielded maximal incorporation of 1 mol/mol of CF₁. Almost half had the covalent bond with CF₁. In experiments with TNP-[¹⁴C] ADP, radioactivity was detected almost equivalently on α and β subunits, suggesting that the binding site may be at the interface between a and β subunits. Enzyme activities of the thylakoid membrane-bound enzyme after covalent labeling were measured. Inhibition, ranging from 20 to 25%, was less than expected from the percentage of CF₁ molecules labeled (40-50%). It is suggested that only half of the labeled enzymes, probably those with the nucleotide analog linked to the β subunit, are inactive.