Efficient Purification and Molecular Properties of Spinach Chloroplast Fructose 1,6‐Bisphosphatase

Abstract

A relatively straightforward procedure has been developed for the purification of chloroplast fructose bisphosphatase from spinach leaves to apparent homogeneity and with 80% yield. The molecular weight of the enzyme was about 160000. Chloroplast fructosebisphosphatase consists of four possibly identical subunits and, at pH 8.8, easily dissociates into equal halves with lowered activity. Sigmoid saturation curves with Hill coefficients between 3.0 and 3.7 were obtained for fructose 1,6-bisphosphate and Mg²⁺. Incubation of the enzyme with 20 mM dithiothreitol slowly altered the response to pH from no activity measured at pH 7.5 and full activity at pH 8.8 to equal activity at each of these pH values; at the same time the number of freely available sulphydryl groups increased from four to twelve per molecule. These properties are considered in the context of the observed activation of this enzyme following illumination of chloroplasts.