Heat-stable, calcium-inhibited cyclic nucleotide phosphodiesterase from Neurospora crassa

A heat-stable, Ca 2+-inhibited cyclic nucleotide phosphodiesterase (cPDE) (EC 3.1.4.17) from Neurospora crassa was isolated and could be separated into three aggregation forms ( ca M r values of 83 000, 151 000 and 330 000–400 000) by gel-filtration at 25° in the presence of EGTA, which suggests that the enzyme exists in monomeric, dimeric and tetrameric forms. Aggregation was temperature dependent; under similar conditions at 6°, a single form of ca M r 400 000 was present. The enzyme was not activated by calmodulin but was inhibited by micromolar concentrations of Ca 2+ and could be activated by addition of excess EGTA. Both cyclic AMP and cyclic GMP were substrates for the enzyme, and kinetic studies indicated that both bound at the same catalytic site. The enzyme had complex kinetics with two K m values. The affinity of the high K m component for both substrates was the same, whereas the low K m component had a higher affinity for cyclic GMP than for cyclic AMP. Ca 2+ was a competitive inhibitor of the high K m component, whereas inhibition of the low K m component was of the mixed type; Ca 2+ caused an increase in both K m values. The specific activity of the enzyme when cyclic AMP was used as a substrate was always higher than that with cyclic GMP, under all experimental conditions studied. A comparison of the heat-stable cPDE and cPDE activity in unheated extracts was also carried out using gel-filtration and ion-exchange chromatography. A working hypothesis is presented to account for the results.