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.
ASJC Scopus subject areas
- Molecular Biology