A Fluorescence Study of the Effects of pH and Mg2+ on the Conformation of Fructose 1, 6-Diphosphatase from Spinach Chloroplasts

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Abstract
2-p-Toluidino-naphthalene-6-sulfonate is a sensitive fluorescent reporter group which can be used for the detection of the conformation of fructose I ,6-diphosphatase from spinach chloroplasts. When fructose 1,6-diphosphatase was added to a dilute solution of 2-p-toluidino- naphthalene-6-sulfonate at pH 9·0, the fluorescence intensity gradually increased. At this pH, the enzyme activity decreased at the same rate. However, at neutral pH (7·5), this time-dependent fluorescence change was not observed. In the presence of Mg2+ which is an acti vator of the enzyme, the fluorescence intensity was increased instantly and did not change for 30 mm in the pH range 8·0–9·0. From the concentration dependence of the fluorescence intensity, the dissociation constant for Mg2+ was determined, Kdis=3mM. The effects of pH and Mg2+ on the conformation and activity of chioroplast fructose 1,6-diphosphatase are discussed.