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Characterization of the phosphorylated form of the insulin-stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes.

ABSTRACT: Incubation of intact purified rat liver plasma membranes with insulin, cyclic AMP and ATP led to the activation of the peripheral "low-Km" cyclic AMP phosphodiesterase. When (gamma-32P]ATP was included in the incubation mixture, after purification of this enzyme to homogeneity it was found to contain 1 mol of alkali-labile 32P/mol of enzyme. Treatment of the homogeneous phosphorylated enzyme with alkaline phosphatase released all of the 32P from the protein while restoring its activity to the native state. The reversibility of the activation that is achieved by the phosphorylation of this enzyme could also be demonstrated with a high-speed supernatant from rat liver. This restored the activity of the activated membrane-bound enzyme to its native state. The Ka for the cyclic AMP-dependence of this process (1.6 micrometer) was unaffected by a range of ATP concentrations (1-10 mM) and by a range of membrane protein concentrations (0.2-2 mg/ml). Adenylyl imidodiphosphate could not substitute for ATP, and concanavalin A could not substitute for insulin, as essential ligands in the activation process. The purified activated enzyme exhibited Km 0.6 microM, Vmax 10.9 units/mg of protein and Hill coefficient (h) 0.47. The Vmax. for this activated enzyme was much higher than that of the native enzyme, yet h was much lower.

SUBMITTER: Marchmont RJ

PROVIDER: S-EPMC1162937 | biostudies-other | 1981 Jun

REPOSITORIES: biostudies-other

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Purification and properties of the insulin-stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes.

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Identification and characterization of both the cytosolic and particulate forms of cyclic GMP-stimulated cyclic AMP phosphodiesterase from rat liver.

Project description:Two enzymes displaying cyclic GMP-stimulated cyclic AMP phosphodiesterase activity were purified from rat liver to apparent homogeneity: a 'particulate enzyme' found as an integral membrane protein associated with the plasma membrane, and a 'soluble' enzyme found in the cytosol. The physical properties of these enzymes were very similar, being dimers of Mr 134,000, composed in each instance of two subunits of Mr = 66,000-67,000. Both enzymes showed similar kinetics for cyclic AMP hydrolysis. They are both high-affinity enzymes, with kinetic constants for the particulate enzyme of Km = 34 microM and Vmax. = 4.0 units/mg of protein and for the cytosolic enzyme Km = 40 microM and Vmax. = 4.8 units/mg of protein. In both instances hydrolysis of cyclic AMP appeared to show apparent positive co-operativity, with Hill coefficients (happ.) of 1.5 and 1.6 for the particulate and cytosolic enzymes respectively. However, in the presence of 2 microM-cyclic GMP, the hydrolysis of cyclic AMP obeyed Michaelis kinetics (happ. = 1) for both enzymes. The addition of micromolar concentrations of cyclic GMP had little effect on the Vmax. for cyclic AMP hydrolysis, but lowered the Km for cyclic AMP hydrolysis to around 20 microM in both cases. However, at low cyclic AMP substrate concentrations, cyclic GMP was a more potent activator of the particulate enzyme than was the soluble enzyme. The activity of these enzymes could be selectively inhibited by cis-16-palmitoleic acid and by arachidonic acid. In each instance, however, the hydrolysis of cyclic AMP became markedly more sensitive to such inhibition when low concentrations of cyclic GMP were present. Tryptic peptide maps of iodinated preparations of these two purified enzyme species showed that there was considerable homology between these two enzyme forms.

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