[46] Binding of Fluorescent Nucleotide Analogs to Chloroplast Coupling Factor

This chapter discusses the binding of fluorescent nucleotide analogs to chloroplast coupling factor. The binding of _ЄADP and _ЄATP to membrane-bound ATPase (chloroplast coupling factor one or CF₁) was investigated by measuring the decrease of the fluorescence intensity or the increase in the polarization of fluorescence of the ligand upon binding. A binding system amenable to fluorescence intensity measurements developed consists of the fluorescent nucleotide analog, formycin di- and triphosphate and CF₁. It was found that the binding of formycin diphosphates (FDP) or formycin triphosphates (FTP) to CF₁ is accompanied by a two- to three-fold enhancement in fluorescence intensity of the protein–ligand complex and a markedly increased polarization of the nucleotide fluorescence. Fluorescence intensity measurements made with FTP or FDP and a chloroplast coupling factor released from lettuce chloroplasts by chloroform treatment were used to determine the nucleotide binding properties of CF₁. This method can be used to study the binding of FTP to the activated ATPase enzyme by a stopped-flow fluorescence technique and could be further developed to measure the nucleotide binding characteristics of the membrane-bound ATPase.