Activity of cyclic-AMP phosphodiesterase in permeabilised cells of Bakers' yeast

Yeast cyclic-AMP phosphodiesterases were assayed in situ, using cells permeabilised with cytochrome c, to get information about the kinetics of these enzymes at the high concentrations of macromolecules occurring in vivo. Protamine treatment was not suitable, because it perturbed the intracellular localisations of both the (Mg-dependent) low-Km enzyme and the (EDTA-insensitive) high-Km enzyme. The pH-dependence of Km and V for EDTA-insensitive activity in situ agreed well with the behaviour of pure high-Km enzyme, except that near pH 8 Hofstee plots were bent slightly upwards both for activity in situ and with crude broken-cell preparations. Hofstee plots of Mg-dependent activity in situ were distinctly concave, and could be resolved mathematically into two activities, one (accounting for about 30% of the Mg-dependent V) with a Km close to the value in vitro of 0.2 microM, and the other with an apparent Km of 3 microM. The 3 microM Km activity probably represents a fraction of the low-Km enzyme that is particle-bound at the high protein concentrations occurring in situ.