Characterization of a rat liver cyclic GMP-activated phosphodiesterase by chromatography on hexyl-agarose. Inhibition of phosphodiesterase activity by hexyl-agarose.
Chromatography on hexyl-agarose resolved a partially purified cyclic GMP-activated phosphodiesterase from rat liver into two peaks of activity: the first was eluted with 0.5 M-KCl and was cyclic AMP-specific. The second was tightly bound to hexyl-agarose and was not eluted with KCl (0--2.0 M), which enhanced the hydrophobic interactions of this form with the matrix. It was eluted with 0.5 M-Tris, hydrolysed cyclic AMP and cyclic GMP and was specifically activated by cyclic GMP. The cyclic GMP-activated phosphodiesterase was immobilized on hexyl-agarose. Enzyme activity, quantitatively bound to hexyl-agarose, was not released from the hydrophobic matrix in the presence of cyclic AMP or cyclic GMP, under our assay conditions. The immobilized form of the enzyme retained catalytic activity, was inhibited by 0.1 mM-cyclic AMP and was activated by micromolar concentrations of cyclic GMP to a lesser extent (7-fold) than the control, i.e. the enzyme mixed with unsubstituted agarose (15-fold). When the enzyme was immobilized, inhibition of cyclic AMP phosphodiesterase activity was only observed in the presence of cyclic GMP (at 3 microM); in its absence, activity remained unchanged. The kinetic behaviour of the immobilized enzyme is consistent with the hypothesis of a binding site distinct from the hydrolytic and activating sites.
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012685:Sepharose / Q000031:analogs & derivatives
D004800:Enzymes, Immobilized; D007527:Isoenzymes; C024903:aminohexyl-sepharose; D012685:Sepharose; D000242:Cyclic AMP; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP