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

Biochem J. 1981 Jun 1;195(3):645-52. doi: 10.1042/bj1950645.

Abstract

The peripheral high-affinity cyclic AMP phosphodiesterase from rat liver plasma membranes was purified to apparent homogeneity. The procedure used involved the initial purification of liver plasma membranes and the solubilization of the enzyme by using a high-ionic-strength medium. This was followed by chromatography of the enzyme on DEAE-cellulose, Affi-Gel Blue, a novel affinity column and Sephadex G-100. A 9500-fold purification of the enzyme with a 24% yield was achieved by this procedure. The purified enzyme was apparently monomeric (Mr 52000) as it exhibited identical molecular weights on analysis by gel filtration, sedimentation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It is suggested that the non-Michaelis kinetics exhibited by the enzyme are due to it obeying a mnemonical mechanism, where it displays Km 0.7 micrometer, Vmax. 9.1 units/mg of protein and Hill coefficient (h) 0.62. Cyclic GMP acts as a poor substrate for the enzyme, with Km 120 micrometer and Vmax. 0.4 unit/mg of protein, and also as an inhibitor of the enzyme, with I50 (concentration giving 50% inhibition) 150 micrometer when assayed at 0.4 micrometer-cyclic AMP. Inhibition by 5'-AMP is unlikely to be of physiological importance, as it is only a weak inhibitor of the enzyme (I50 47 mM assayed at 0.4 micrometer-cyclic AMP).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3',5'-Cyclic-AMP Phosphodiesterases* / antagonists & inhibitors
  • 3',5'-Cyclic-AMP Phosphodiesterases* / isolation & purification
  • Animals
  • Cell Membrane / enzymology
  • Chromatography, Affinity
  • Cyclic GMP / pharmacology
  • Electrophoresis, Polyacrylamide Gel
  • Insulin / pharmacology
  • Kinetics
  • Liver / enzymology*
  • Molecular Weight
  • Rats

Substances

  • Insulin
  • 3',5'-Cyclic-AMP Phosphodiesterases
  • Cyclic GMP