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Cyclic nucleotide phosphodiesterases of the superior cervical ganglion

University of British Columbia

A modification of the assay of cyclic nucleotide phosphodiesterase involving batch use of Dowex 1 anion exchange resin was developed. This assay produced quantitative recoveries of adenosine, guanosine, and their metabolites from the resin slurry. The assay was suitable for use in crude preparations containing purine catabolizing enzymes. Cyclic nucleotide phosphodiesterase was examined in canine and bovine superior cervical ganglia. Activity in crude supernatant fractions was only slightly stimulated by Ca⁺⁺ despite the presence of protein activating factor. Three forms of phosphodiesterase were resolved from bovine ganglia supernatant extracts by chromatography on DEAE-cellulose. The first enzyme eluted (D[sub I]) was completely specific for cyclic GMP, while the other two (D[sub II]and D[sub III]) hydrolyzed both cyclic AMP and cyclic GMP ; all were free of heat stable protein activator. Each enzyme was inhibited by low concentrations of Ca⁺⁺ in the assay medium. Inhibition by Ca⁺⁺ was reversed by the addition of protein activator but activity did not increase above the control level. Cyclic AMP hydrolysis by D[sub II] was stimulated by micromolar concentrations of cyclic GMP. This stimulation was reduced by Ca⁺⁺ unless protein activator was present. The kinetics of cyclic nucleotide hydrolysis by each of the three enzymes were examined. All enzyme species displayed nonlinear double reciprocal plots of substrate hydrolysis. Through the use of reversible inhibition kinetics and nonlinear least squares fitting, rate equations to describe the data were evaluated. Apparent negatively cooperative behaviour was observed for the hydrolysis of cyclic GMP by the first peak eluted (K₁ < K₂ and V₁ < V₂). The two cyclic AMP-cyclic GMP hydrolyzing enzymes displayed apparent positive cooperativity for cyclic GMP hydrolysis (K₁ > K₂ and V₁ > V₂), and negative. cooperativity for cyclic AMP hydrolysis (K₁ < K₂ and V₁ < V₂). The kinetic behaviour displayed by all three enzyme species could be explained using variations of a model consisting of an enzyme with two initially equivalent interacting sites.

Text

2010-02-05

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http://hdl.handle.net/2429/19611

Pharmacology

University of British Columbia

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