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

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Abstract
Of the rat liver cellular cyclic[c]AMP phosphodiesterase activity, .apprx. 10% was associated with a plasma-membrane fraction. Lineweaver-Burk plots of this activity were clearly non-linear, yielding extrapolated Km values of 0.7 and 60.6 .mu.M. Treatment of these membranes with high-ionic-strength NaCl solutions apparently released 80% of this activity assayed at 0.4 .mu.M-cAMP and 15% of the activity assayed at 1 mM-cAMP. The high-salt-solubilized enzyme gave a non-linear Lineweaver-Burk plot. The cAMP phosphodiesterase activity of the washed high-salt-treated membranes exhibited a linear Lineweaver-Burk plot, yielding a Km of 60 .mu.M. 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.degree. C). The activity of washed high-salt-treated membranes decayed as a single exponential upon heat-treatment (half-life 42 min at 55.degree. C) and was solubilized in the detergent Triton X-100. Cytosol-derived cAMP 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. The cAMP phosphodiesterase activity of rat liver plasma membranes apparently can be resolved into 2 components: a single peripheral protein exhibiting apparent negative co-operativity, that is distinct from cytosol forms, and an intrinsic protein exhibiting normal Michaelis kinetics.