Purification of intact and nicked forms of a zinc-containing, Mg2+-dependent, low Km cyclic AMP phosphodiesterase from bakers' yeast.
A low Km cyclic AMP phosphodiesterase was purified to homogeneity from microsomes of bakers' yeast. "Intact" enzyme, purified from microsomes prepared in the presence of the protease inhibitor phenylmethylsulfonyl fluoride, had a specific activity of 0.6 mumol/min/mg of protein (30 degrees C, pH 8.0, 1 microM cyclic AMP), a pI of 6.65 +/- 0.15, and a molecular weight of 61,000 determined by gel electrophoresis in the presence of sodium dodecyl sulfate. Gel filtration of native enzyme suggested it is a monomer. When phenylmethylsulfonyl fluoride was omitted, a product ("nicked" enzyme) was obtained with a specific activity of 1.2 mumol/min/mg of protein, the same pI, and a similar amino acid composition; but gel electrophoresis now showed two bands, with molecular weights of 45,000 and about 17,000, together with a small amount of the 61,000 band. Apart from the higher specific activity of the nicked enzyme, no difference was found between the catalytic properties of the two enzyme forms. Between 40 nM and 1 microM cyclic AMP, an apparent Km of 170 nM was observed at pH 8.0, but at higher cyclic AMP concentrations (2-30 microM), Hofstee plots curved upwards. Cyclic deoxy-AMP was a substrate, but cyclic GMP was not and did not affect the activity towards cyclic AMP. Both enzyme forms contained tightly bound zinc. The metal chelators, 8-hydroxyquinoline and orthophenanthroline , caused progressive partial inactivation of the enzyme and a decrease in its affinity for cyclic AMP. Dialysis against Zn2+, Cu2+, Co2+, or Mn2+ (but not Mg2+ or Ni2+) reversed these changes.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D000242:Cyclic AMP / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D015125:Oxyquinoline / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D015032:Zinc / Q000032:analysis*
D000596:Amino Acids; D010618:Phenanthrolines; D015125:Oxyquinoline; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D008274:Magnesium; D015032:Zinc; C025205:1,10-phenanthroline