Selective inhibition of two soluble adenosine cyclic 3',5'-phosphate phosphodiesterases partially purified from calf liver

Publisher Website
Google Scholar
Abstract
Low Km cAMP phosphodiesterase and cGMP-stimulated cyclic nucleotide phosphodiesterase activities were partially purified from calf liver supernatant by chromatography on DEAE-cellulose and DEAE-Sepharose and ammonium sulfate precipitation. The low Km phosphodiesterase was not retained on N6-H2N(CH2)2-cAMP-agarose and could be separated from the cGMP-stimulated phosphodiesterase which was absorbed by this matrix. From the proteins that did not bind, 2 distinct low Km cAMP phosphodiesterases were separated on Ultrogel AcA 34. One form (fraction C) hydrolyzed cAMP with an apparent Km of .apprx. 0.5 .mu.M and was very sensitive to inhibition by cGMP. Lineweaver-Burk plots of cAMP hydrolysis by a 2nd form (fraction B) were nonlinear, with an apparent low Km component of .apprx. 2 .mu.M. This form was rather insensitive to inhibition by cGMP. With both fractions, hydrolysis of cAMP relative to cGMP was much greater at low (.apprx. 1 .mu.M) than at high (.apprx. 100 .mu.M) substrate concentrations. Vmax for cAMP and cGMP were similar. From sedimentation equilibrium, the apparent weight-average MW of fraction B was estimated as 174,000, and that of fraction C was 85,000. Another fraction (A) of cAMP phosphodiesterase eluted at the void volume of the AcA 34 column. On the basis of the relative affinities for cAMP and cGMP and inhibition by cGMP, fraction A is most likely an aggregated form of fraction B. No apparent interconversion of fractions A, B or C was observed on high-performance liquid chromatography. Fractions B and C differed in their sensitivity to phosphodiesterase inhibitors as well as in other characteristics. The order of potency for inhibition of fraction B was RO 20-1724 [4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone] (IC50 [median inhibitory concentration], 2.2 .mu.M) > papaverine > isobutylmethylxanthine (IBMX) > cilostamide > theophylline > cGMP. The order for fraction C was cilostamide (IC50, 0.03 .mu.M) > cGMP (IC50, 0.75 .mu.M) > papaverine > IBMX > theophylline > RO 20-1724. The use of specific inhibitors may facilitate understanding the role of specific phosphodiesterases in the regulation of intracellular cAMP content.

This publication has 2 references indexed in Scilit: