Regulation of Nucleoside Cyclic 3′:5′-Monophosphate Phosphodiesterase Activity from Rat Brain by a Modulator and Ca ²⁺

Abstract

Gel filtration of the 40,000 rpm supernatant fraction of a homogenate of rat cerebral cortex on a Sepharose 6B column yielded two fractions: fraction II with the “Ca 2+ plus Mg 2+ -dependent” phosphodiesterase activity and fraction III containing its modulator. The activity of fraction II was stimulated by micromolar concentrations of Ca 2+ and the modulator when present together; the modulator stimulated the activity of fraction II only when the Ca 2+ concentration was above a threshold value (about 2 μM with 0.4-1 μM substrate), and the stimulatory effect of Ca 2+ was dependent upon the presence of the modulator. A possibility is discussed that the modulator may reversibly bind to the enzyme, which by itself is inactive, to form an active enzyme-modulator complex and that Ca 2+ stimulates the activity of phosphodiesterase by shifting the equilibrium between these three species towards the formation of the active enzyme-modulator complex. Although fraction II hydrolyzed both cyclic AMP and cyclic GMP, hydrolysis of the latter was more significantly influenced by Ca 2+ and the modulator than that of the former, and the “Ca 2+ plus Mg 2+ -dependent” phosphodiesterase is likely to be a cyclic GMP enzyme. This conclusion is based on the following evidence: ( a ) Ca 2+ stimulated hydrolysis of cyclic GMP by fraction II more than that of cyclic AMP. ( b ) In the presence of Ca 2+ and the modulator, fraction II hydrolyzed cyclic GMP about 8 times faster than cyclic AMP when incubated with 0.4 μM substrate. ( c ) Half-maximal stimulation of hydrolysis of cyclic GMP was attained at a lower concentration of Ca 2+ (4 μM) than that of cAMP (8 μM). ( d ) Increase in the concentration of Ca 2+ from 0.06 μM to 12 μM in the presence of the modulator caused a decrease in the K m value of cyclic GMP hydrolysis by fraction II from 20 μM to 2 μM accompanied by 4-fold increase in the V max value. Under similar conditions, there was only a slight decrease in the K m value of cylic AMP hydrolysis (90 μM → 50 μM), although the V max value increased 7-fold. The anomalous shape of the kinetic plot of cyclic GMP hydrolysis became linear when the Ca 2+ concentration was increased in the presence of the modulator. The modulator seems to be a protein, but it is heat stable. It is probably identical to the protein activator of phosphodiesterase first described by Cheung.