Adenosine 3′,5′-cyclic phosphate phosphodiesterase from bovine thyroid: Isolation and properties of a partially purified, soluble fraction

Abstract

1. 1.|Adenosine 3′,5′-cyclic phosphate (cyclic AMP) phosphodiesterase was partially purified from a 10 000 × g supernate of bovine thyroid; the enzyme preparation so obtained was free of 5′-nucleotidase and deaminase activity and yielded [ 3H]-adenosine 5′-phosphate (AMP) as the end product of enzymatic hydrolysis of cyclic [ 3H]AMP. 2. 2.|Using an assay procedure that relied on physiologic substrate concentrations ( i.e confirming to known intracellular levels of cyclic AMP in thyroid), the partially purified thyroid phosphodiesterase was kinetically characterized and compared with a highly purified beef heart phosphodiesterase preparation. Double reciprocal plots of cyclic AMP hydrolysis yielded two apparent Michaelis constants: the lower in the 10 −6 M and the higher in the 10 −5 M range. Two apparent K m values were also obtained for Mg 2+ which was required for expression of maximal enzymatic activity. Other bivalent cations (Mn 2+, Co 2+, Ca 2+) activated the enzyme to a significantly lesser extent; Zn 2+ was stimulatory at 10 −4 M and inhibitory at ≥ 10 −3 M. 3. 3.|A number of cyclic nucleotides and acylated derivatives thereof interfered with the enzymatic hydrolysis of cyclic [ 3H]AMP to varying degrees; of these the mono- and dibutyryl derivatives of cyclic AMP were the most potent inhibitors. The inhibition was competitive in nature with K i values for dibutyryl cyclic AMP, N 6-monobutyryl cyclic AMP, and O 2′-monobutyryl cyclic AMP of 1.6·10 −4 M, 1.1·10 −4 M and 2.6·10 −5 M, respectively. 4. 4.|Methylxanthines and quazodine strongly inhibited thyroid phosphodiesterase whereas sulfonylureas were weakly inhibitory. 5. 5.|High concentrations of pituitary thyrotropin in vitro enhanced thyroid phosphodiesterase activity slightly while heat-inactivated thyrotropin, long-acting thyroid stimulator immunoglobulin G, and the prostaglandins E 2 and F 1 a were ineffective; prostaglandin E 1 was weakly inhibitory. The prostaglandin antagonists 7-oxa-13-prostynoic acid and polyphloretin phosphate effected significant inhibition of thyroid phosphodiesterase, possibly accounting for the agonist properties of these agents occasionally observed in vitro. 6. 6.|Upon complexing with a cyclic AMP-binding protein prepared from beef kidney, cyclic [ 3H]AMP became completely resistant to enzymatic breakdown by thyroidal phosphodiesterase.