A membrane-bound alkaline inorganic pyrophosphatase in isolated spinach chloroplasts

Abstract

An alkaline inorganic pyrophosphatase is found in association with isolated spinach chloroplast membranes. The enzyme is not removed from chloroplasts by repeated washings in an iso-osmotic medium. Suspension of the chloroplasts in hyper- or hypo-osmotic medium, however, results in the loss of pyrophosphatase activity in the chloroplasts. Fractionation of an isolated chloroplast suspension by differential centrifugation yields chloroplast fractions possessing high levels of alkaline pyrophosphatase activity but practically devoid of cytoplasmic acid pyrophosphatase. The alkaline pyrophosphatase exhibits a pH optimum of 8.2–8.5. In addition, there is an absolute requirement for Mg 2+, with maximal rates of pyrophosphate hydrolysis occurring at Mg 2+ PP i ratios greater than 2. From these findings the actual substrate for the enzyme is evidently Mg 2P 2O 7 0 with pyrophosphate (P 2O 7 4−) acting as a potent inhibitor. The enzyme is inhibited by high concentrations of ATP (>3 m m), but increasing the concentration of Mg 2+ effectively relieves this inhibition. At lower ATP concentrations, however, there is a stimulation of pyrophosphatase activity. The rate of hydrolysis of pyrophosphate by isolated chloroplasts is not affected by methylamine, 4′-deoxyphlorizin, and light. The possible role of this enzyme in photophosphorylation is discussed.