UBC Theses and Dissertations
Studies on nucleoside 3', 5'-cyclic monophosphate 3'-phosphohydrolase from brain Wickson, Robert Douglas
A nucleoside 3',5'-cyclic monophosphate 3'-phosphohydrolase was partially purified from bovine cerebral cortex. The enzyme isolated hydrolyzed both cyclic AMP (K[sub m] = 30 uM) and cyclic GMP (K[sub m] = 3 uM). In the absence of metal ions, no activity was observed. Divalent cations (Mg⁺⁺ ≃ Mn⁺⁺ > Co⁺⁺ > Ni⁺⁺) stimulated activity with the activator constants being very low (e.g., 4.5 uM for Mg⁺⁺). Cyclic GMP inhibited cyclic AMP hydrolysis and vice versa. Cyclic AMP inhibition of cyclic GMP hydrolysis was competitive with a K[sub i] of 25 uM (cf., K[sub M] = 30 uM) indicating the presence of a single enzyme. 3-Isobutyl-l-methylxanthine inhibited cyclic AMP hydrolysis competitively with a of 1.5 uM. Gel filtration of the partially purified enzyme showed a decrease in the apparent molecular weight in the presence of Mg⁺⁺ and a further decrease in the presence of Mg⁺⁺ plus Ca⁺⁺. The phosphodiesterase present in the crude homogenate was inhibited by EGTA and activated by Ca⁺⁺. During DEAE-cellulose chromatography, a heat-stable protein factor was separated from the enzyme. In the absence of this factor, no stimulation by Ca⁺⁺ was observed. Phosphodiesterase was shown to require both this protein activator and Ca⁺⁺ in order to be stimulated. A model for the activation was proposed based upon the data obtained from kinetic experiments. Increasing concentrations of Ca⁺⁺ decreased the apparent activator constant for protein activator while increasing protein activator concentrations decreased the apparent activator constant for Ca⁺⁺. The observed activation by Ca⁺⁺ and protein activator suggested a means by which downward sloping Lineweaver-Burk plots could be obtained. In the case of phosphodiesterase, downward sloping Lineweaver-Burk plots have been interpreted by other workers as negative cooperativity. In the crude homogenate, there appeared to be a low K[sub m] phosphodiesterase specific for cyclic AMP in addition to the high K[sub m] enzyme partially purified. Calculations suggested that the high K[sub m] enzyme was at least as important as the low K[sub m] enzyme in hydrolyzing cyclic AMP. In addition, these calculations suggested that phosphodiesterase activity was not in a 10 to 100-fold excess over adenyl cyclase (or guanyl cyclase) activity. Phosphodiesterase and adenyl cyclase activities are probably delicately balanced, with any change in either of their activities capable of effecting changes in cyclic AMP concentrations.
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