A peripheral and an intrinsic enzyme constitute the cyclic AMP phosphodiesterase activity of rat liver plasma membranes.
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.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D006358:Hot Temperature; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D011485:Protein Binding; D051381:Rats; D012965:Sodium Chloride / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology
D012965:Sodium Chloride; D015105:3',5'-Cyclic-AMP Phosphodiesterases