A peripheral and an intrinsic enzyme constitute the cyclic AMP phosphodiesterase activity of rat liver plasma membranes

Abstract

1. Approx. 10% of the rat liver cellular cyclic AMP phosphodiesterase activity was associated with a plasma-membrane fraction. 2. Lineweaver-Burk plots of this activity were clearly non-linear, yielding extrapolated Km values of 0.7 and 60.6 microns. 3. Treatment of these membranes with high-ionic-strength NaCl solutions apparently released 80% of this activity assayed at 0.4 micron-cyclic AMP, and 15% of the activity assayed at 1 mM-cyclic AMP. 4. The high-salt-solubilized enzyme gave a non-linear Lineweaver-Burk plot. 5. The cyclic AMP phosphodiesterase activity of the washed high-salt-treated membranes exhibited a linear Lineweaver-Burk plot, yielding a Km of 60 microns. 6. The high-salt-solubilized enzyme exhibited a single peak of activity upon polyacrylamide-gel electrophoresis, a single peak upon sucrose-density-gradient centrifugation (3.9 S) and decayed as a single exponential upon heat-treatment (half-life 1 min at 55 degrees C). 7. The activity of washed high-salt-treated membranes decayed as a single exponential upon heat-treatment (half-life 42 min at 55 degrees C), and was solubilized in the detergent Triton X-100. 8. Cytosol-derived cyclic AMP phosphodiesterase activity could bind to washed high-salt-treated plasma membranes, but was totally eluted by washing with 1 mM-KHCO3, unlike the high-salt-solubilized enzyme, which required high salt concentrations to elute it. 9. We suggest that the cyclic AMP phosphodiesterase activity of rat liver plasma membranes can be resolved into two components: a single peripheral protein exhibiting apparent negative co-operativity, that is distinct from cytosol forms, and an intrinsic protein exhibiting normal Michaelis kinetics.

Insulin controls the cyclic AMP‐dependent phosphorylation of integral and peripheral proteins associated with the rat liver plasma membrane

“…This is a low Km cyclic AMP phosphodiesterase [25], which has been shown to be a peripheral enzyme [ 151 and is activated some 2-fold only when insulin, cyclic AMP and ATP are added together to a plasma membrane preparation [25]. The K, for activation by insulin and cyclic AMP, at saturating concentrations of the other ligand, reflects that obtained in this study for the phosphorylation of the peripheral species [25].…”

“…This is a low Km cyclic AMP phosphodiesterase [25], which has been shown to be a peripheral enzyme [ 151 and is activated some 2-fold only when insulin, cyclic AMP and ATP are added together to a plasma membrane preparation [25]. The K, for activation by insulin and cyclic AMP, at saturating concentrations of the other ligand, reflects that obtained in this study for the phosphorylation of the peripheral species [25]. Presumably the activation of this enzyme, which can only occur after intracellular cyclic AMP levels have been elevated above their basal levels, explains why insulin can depress those elevated cyclic AMP levels achieved by glucagon, but does not affect basal levels [21].…”

ERK2 Mitogen-activated Protein Kinase Binding, Phosphorylation, and Regulation of the PDE4D cAMP-specific Phosphodiesterases

“…Assay of cAMP PDE Activity-PDE activity was determined by a modification of the two-step procedure of Thompson and Appleman (31) as described previously by us (32). All assays were conducted at 30°C with initial rates taken from linear time courses.…”

The Unique Amino-terminal Region of the PDE4D5 cAMP Phosphodiesterase Isoform Confers Preferential Interaction with β-Arrestins

“…PDE Assays-PDE activity was assayed as described previously (38,39). The assays were performed at 30°C, and a freshly prepared slurry of Dowex/H 2 O/ethanol (1:1:1, v/v/v) was used.…”