Distribution and Properties of Alkaline Pyrophosphatases of Rat Liver

Abstract

A study of the distribution of alkaline inorganic pyrophosphatase [alkaline PP1ase, EC 3.6.1.1] in the subcellular fractions of rat liver indicated that in addition to the PP1ase present in the cytosol, there were two isoenzymes in mitochondria (Mt. I and Mt. II) which were electrophoretically different from the cytosol enzyme. PP1ase activities found in purified nuclei and lysosomes can be accounted for by contaminating mitochondria. All three PP1ases which were partially purified had pH optima at 8 and estimated molecular weight of 68, 000. They were activated by Mg2+, and markedly inhibited by Zn2+, Mn2+Hg2+, Cd2+, Ca2+ and F-; they were rather unstable to air oxidation and stablized in the presence of cysteine. The three isoenzymes were different with regard to the effect of Mg2+, substrate specificity and inactivation by urea. Mg2+ which was essential for the activity of all three enzymes was inhibitory when present in excess. This inhibition was more marked with the cytosol and Mt. II enzymes and less so with Mt. I. The cytosol PP1ase was rather specific for PP1 hydrolysis, while mitochondrial enzymes also hydrolyzed nucleo-side polyphosphates at considerable rates. The urea concentrations required for 50% inactivation were 1.2M for the cytosol enzyme and 2M for mitochondrial PP1ases.