Purification of kinetic properties of human erythrocyte Mg 2+-dependent inorganic pyrophosphatase

Abstract

Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from human erythrocyte henolysates has been purified up to 10 000-fold. The purified enzyme is homogenous and has a specific activity of 79.75 μmol PP i hydrolysed · min −1 · mg −1 at pH 8 ad 37°C. It was confirmed that it is a dimer with a molecular weight of 42 000, composed of two identical protomers. From kinetic studies, it is proposed that human erythrocyte inorganic pyrophosphatase activity depends on free Mg 2+ concentration in different ways. This ion constitutes part of the substrate (the Mg · PP i complex; K m = 1.4 · 10 −4 M) and probably acts as an allosteric activator (kinetic activation constant: K a Mg 2+ = 7.5 · 10 −4 M). Equilibrium binding studies performed in the absence of PP i showed 4 binding sites for Mg 2+, all having the same high affinity (dissociation constant: K d Mg 2+ = 4 · 10 t-6 M). Since the concentration of free Mg 2+ in red blood cells is very low and may vary with the oxygenation state, it is likely that in vivo erythrocyte pyrophosphatase activity is regulated.