The effect of slow freezing and hypertonic NaCl on the hydrolytic activity of rat erythrocyte cyclic AMP phosphodiesterase and its sensitivity to the inhibitor, d,l-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone

Abstract

Slow, in contrast to rapid, freezing of a rat erythrocyte hemolysate irreversibly activated the cyclic AMP phosphodiesterase (3′:5′-cyclic-AMP 5′-nucleotidohydrolase, EC 3.1.4.17) and reduced its sensitivity to the inhibitor, BMI ( d,l-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone) regardless of the presence of dithiothreitol. These effects could be mimicked by a 24-h exposure to 2 M NaCl at 4–5°C. Brief exposures (< 1 min) to this concentration of salt resulted in reversible activation of the enzyme and increased sensitivity to the inhibitor. Kinetic analyses demonstrated two K m values for the enzyme of approximately 4.3 μM and 5.0 mM. The 2-M NaCl had no effect on either K m but increased the V in the low substrate range analogous to the removal of a non-competitive inhibitor. The irreversibility of the salt effect seemed to be correlated with the reduction in potency of BMI rather than the kinetic aspects of the activation. It is proposed that the high salt concentration dissociates an enzyme aggregate and/or exposes certain groups to hydrolytic cleavage.