Open AccessPurification of an insulin‐sensitive cyclic AMP phosphodiesterase from rat liver
Author(s) -
BOYES Stewart,
LOTEN Ernest G.
Publication year - 1988
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1988.tb14098.x
Subject(s) - phosphodiesterase , biochemistry , cyclic nucleotide phosphodiesterase , enzyme , antiserum , proteolysis , chemistry , affinity chromatography , chromatography , molecular mass , gel electrophoresis , size exclusion chromatography , microbiology and biotechnology , biology , antibody , immunology
A low‐ K m cyclic nucleotide phosphodiesterase solubilised from rat liver membranes by mild proteolysis with chymotrypsin has been purified to apparent homogeneity. The purification included chromatography on cellulose phosphate, Ecteola‐cellulose, hydroxyapatite, a theophylline affinity matrix and HPLC on a DEAE‐substituted column. The purified enzyme has linear kinetic plots with a K m of 0.24 μM and a V max of 6.2 μmol mg −1 min −l with cyclic AMP as a substrate. It also hydrolyses cyclic GMP with a K m of 0.17 μM and a V max which is about a third of that with cyclic AMP. Cyclic GMP is also a competitive inhibitor of cyclic AMP hydrolysis with a K i of 0.18 μM. The proteolytically solubilised enzyme has a subunit molecular mass of 73 kDa by SDS gel electrophoresis and of 130 kDa by HPLC size‐exclusion chromatography, suggesting that it exists as a dimer. A partially purified preparation of this enzyme was used to raise antiserum in a sheep. The antiserum immunoprecipitated activity from liver and adipose tissue of rat and mouse. It had little activity against phosphodiesterase from other rat tissues or other species. Insulin‐activated phosphodiesterase from both adipocytes and hepatocytes was immunoprecipitated by the antiserum suggesting that the purified enzyme was an insulin‐sensitive phosphodiesterase.