| Abstract: | The high Km cyclic nucleotide phosphodiesterase of Saccharomyces cerevisiae was purified by an improved procedure. Its amino acid composition is reported. Its pI is 5.85 +/- 0.1. Sedimentation equilibrium analysis of the native enzyme gave Mr = 88,000 +/- 6,000, whilst gel electrophoresis in the presence of dodecyl sulfate gave a molecular weight of 43,000, indicating that the enzyme is a dimer. Preparations of 94 +/- 4% purity contained about 2.4 atoms of zinc/43,000 daltons. Inactivation of the enzyme by 8-hydroxyquinoline was accompanied by removal of about 2 zinc atoms per monomer. Partially inactivated enzyme regained activity during dialysis against zinc, or, with less effect, cobalt salts. 8-Hydroxyquinoline (Ki = 1.1 mM) and 1,10-phenanthroline (Ki = 0.6 mM) were competitive inhibitors. The enzyme was also inhibited by the nonchelating 1,7-and 4,7-phenanthrolines and by thiols and KCN, but not by NaN3. These inhibitors probably act by binding to, but not chelating, enzyme-bound zinc. |
