Purification of an insulin-sensitive cyclic AMP phosphodiesterase from rat liver.
A low-Km 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 Km of 0.24 microM and a Vmax of 6.2 mumol mg-1 min-1 with cyclic AMP as a substrate. It also hydrolyses cyclic GMP with a Km of 0.17 microM and a Vmax which is about a third of that with cyclic AMP. Cyclic GMP is also a competitive inhibitor of cyclic AMP hydrolysis with a Ki of 0.18 microM. 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.
Boyes
S
S
Department of Clinical Biochemistry, University of Otago Medical School, Dunedin, New Zealand.
Loten
E G
EG
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification*; D000818:Animals; D002845:Chromatography / Q000379:methods; D002918:Chymotrypsin; D004789:Enzyme Activation / Q000187:drug effects; D007120:Immunochemistry; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000201:enzymology*; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology
D007328:Insulin; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D002918:Chymotrypsin