Ca-2+/Mg-2+-dependent cyclic nucleotide phosphodiesterase and its activator protein

Abstract

(1) Ca-2+/Mg-2+-dependent cyclic nucleotide phosphodiesterase was found in the supernatant fluids of a variety of tissues, including cerebral cortex, cerebellum, kidney, liver, and heart. (2) this enzyme required Ca-2+, Mg-2+, and an activator protein (PAF) for the activity. In the presence of these ingredients the enzyme hydrolyzed cyclic GMP preferentially when incubated with a low concentration (0.4 muM) of substrate. (3) The enzyme devoid of PAF was eluted in fraction II with a molecular weight of approximately 150,000 after Sephadex G-200 gel filtration of the supernatant fluids using medium containing EGTA. PAF thus separated from the enzyme was eluted in a fraction corresponding to a molecular weight of approximately 28,000 by gel filtration. Stimulation of the activity of fraction II by Ca-2+ was completely dependent on the addition of PAF. (4) Formation of an active enzyme-PAF complex with an estimated molecular weight of 200,000 was demonstrated by gel filtration of a mixture of the enzyme and PAF in medium containing Ca-2+. It is likely that the activity of the Ca-2+/Mg-2+ in a concentration range of approximately 1 to 10 muM, as shown in the following equation: [Enzyme] inactive + PAF + Ca-2+ in equilibrium [enzyme - PAF - Ca-2+] active. More than one PAF protein may bind to one molecule of enzyme to form an active complex. Equilibrium of the above equation is probably determined mainly by the intracellular concentration of Ca-2+ in vivo. (5) The enzyme-PAF complex was more labile after heat treatment than the free form of enzyme. (6) PAF was isolated from all tissues listed in (1). The levels of PAF and of fraction II did not correspond exactly.