Purification and properties of adenosine 3′,5′-monophosphate phosphodiesterase from baker's yeast

Abstract

Cyclic AMP phosphodiesterase from Saccharomyces cerevisiae was purified about 20,000-fold to homogeneity. The purified enzyme had a molecular weight of about 60,000 as estimated by gel filtration. The enzyme activity was optimal at pH 8.5–9.0 and was not stimulated by imidazole. Among cyclic 3′,5′-nucleotides, cyclic AMP was the most active substrate for the purified enzyme ( K m = 0.25 mM), but it was inhibitory at concentrations above 4 m m. N 6,O 2′ -dibutyryl cyclic AMP was not hydrolyzed at all. Unlike other cyclic AMP phosphodiesterases from various sources, the purified yeast enzyme did not require divalent metal ions for maximal activity and was rather inhibited in various degrees by added metal ions. The enzyme was not very sensitive to thiol inhibitors. The purified yeast enzyme was strongly inhibited by theophylline and slightly by caffeine. In contrast to the enzyme from S. carlsbergensis, the enzyme from S. cerevisiae was not inhibited at all by ATP or PP i. The enzyme activity was not released into the growth medium, and the intracellular distribution studies indicated that the enzyme was located mainly in the cytosol fraction.