par Erneux, Christophe ;Couchie, D;Dumont, Jacques Emile
Référence European journal of biochemistry / FEBS, 104, 1, page (297-304)
Publication Publié, 1980-02
Article révisé par les pairs
Résumé : Cyclic nucleotide phosphodiesterases have been characterized in crude soluble extract (100000 ×g supernatant) of horse thyroid. DEAE‐cellulose ion‐exchange chromatography at pH 7.4 followed by ultracentrifugation on sucrose‐density gradients separated the phosphodiesterases into three enzymatic activities: (a) a phosphodiesterase hydrolyzing both cyclic GMP (guanosine 3′,5′‐mono‐phosphate) and cyclic AMP (adenosine 3′,5′‐monophosphate), more efficient for cyclic GMP at micromolar levels, activated by the calcium‐dependent regulator or calmodulin; it is called the calmodulin phosphodiesterase; (b) a phosphodiesterase hydrolyzing cyclic GMP more efficiently than cyclic AMP, presenting positive cooperativity for both substrates and cyclic GMP activation of cyclic AMP hydrolysis; it is called the cyclic‐GMP‐activated phosphodiesterase; (c) a cyclic‐AMP‐specific phosphodiesterase not activated by cyclic GMP or calmodulin. Ultracentrifugation of crude horse thyroid preparation (100000 ×g supernatant) on linear sucrose density gradients (5 to 20%) resolved two peaks of activity. The first one (3.6 S) was cyclic‐AMP‐specific. The second one (6.6 S) hydrolyzed both substrates but was 2.5‐times more active against cyclic GMP than against cyclic AMP. Ultracentrifugation of separated DEAE‐cellulose peaks of activity purified the enzymes from contaminating activities: (a) a high molecular weight form (6.6 S) was specifically activated by calmodulin and was separated from the cyclic AMP enzyme (3.6 S); (b) the cyclic‐GMP‐activated phosphodiesterase prepared by anion‐exchange chromatography at pH 7.4 or 6 was still contaminated by the cyclic AMP enzyme (3.6 S) as shown by ultracentrifugation or gel filtration. After separation of the latter enzyme, cyclic GMP specifically activated one single non‐specific enzyme that sedimented at 6.2 S and whose stimulation factor was increased. The data are consistent with the presence in the thyroid of a mixture of three different phosphodiesterases; fractions containing two or three of these enzymes exhibited the properties of the sum of these activities; the enzymes were not interconvertible. Copyright © 1980, Wiley Blackwell. All rights reserved