Calcium-dependent cyclic nucleotide phosphodiesterase from glial tumor cells

Abstract

A Ca 2+-dependent cyclic nucleotide phosphodiesterase has been identified in homogenates of C-6 glial tumor cells. The Ca 2+-dependent phosphodiesterase was resolved by ECTEOLA-cellulose chromatography into two fractions. One fraction contained a protein regulator of the enzyme which was identical to a homogeneous Ca 2+-binding protein (CDR) from porcine brain by the criteria of electrophoretic migration, biological activity, heat stability, and behavior in diverse chromatographic systems. The second fraction contained deactivated enzyme (CDR-dependent phosphodiesterase) which regained full activity upon the readdition of both Ca 2+ and CDR. In subcellular fractionation experiments both the CDR and the Ca 2+-dependent phosphodiesterase were predominantly located in the 100,000 g supernatant fraction. The apparent K m values of the phosphodiesterase for cyclic AMP (cAMP) and cyclic GMP (cGMP) were 10 and 1.2 μ m, respectively, when CDR was not rate limiting. Minor increases in the apparent K m for cAMP were observed at rate-limiting concentrations of CDR. At the ratio of CDR to CDR-dependent enzyme present in the C-6 cell homogenate, half-maximal activation was conferred by 4 μ m Ca 2+ for the hydrolysis of 25 μ m cGMP and by 8 μ m Ca 2+ for the hydrolysis of 25 μ m cAMP. Increased ratios of CDR to CDR-dependent phosphodiesterase increased the sensitivity of the enzyme to Ca 2+. The enzyme was more sensitive to CDR with cGMP as substrate than with cAMP, and more sensitive at high than at low cyclic nucleotide substrate concentrations. The quantity of enzyme in the assay also influenced the amount of CDR required for half-maximal activation.