Separation and characterization of two inorganic pyrophosphatases from spinach leaves

Abstract

Two inorganic pyrophosphatases (pyrophosphate phosphohydrolase, E.C.3.6.1.1) have been identified in spinach (Spinacia oleracea L.) leaves. The two isoenzymes were readily separated by polyacrylamide gel electrophoresis and by isoelectric focusing between pH 4 and 6. One isoenzyme is located in the chloroplasts whereas the other form was isolated from the soluble “cytoplasmic” fraction. In addition, a third form appeared when the isolation procedure started from a crude extract from whole leaves. It is suggested that this form represents an aggregation between the two natural species. The three forms differ in their kinetic properties, such as substrate affinity and pH optima. The apparent K _m values were determined to be 10^-5M for the chloroplastic isoenzyme, 7×10^-5 M for the “cytoplasmic” isoenzyme and 3×10^-5 M for the third form. At limiting Mg²⁺ concentrations, the corresponding pH optima were found to be 8.55, 8.95 and 8.75, respectively.