Purification to homogeneity and general properties of a novel phosphodiesterase hydrolyzing cyclic CMP and cyclic AMP.

A cytidine 3'5'-monophosphate (cyclic CMP) phosphodiesterase was purified more than 10,000-fold to apparent homogeneity from the pig liver extract by the steps of pH and ammonium sulfate fractionations, and DEAE-cellulose, Sephadex G-100, and 8-HzN(CH&NHcyclic AMP Sepharose 4B affinity chromatographies.The preparation appeared as a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a M, of 33,000 & 400.The enzyme was an acidic protein, as indicated by an isoelectric point of about pH 4.6 and a high content of acidic amino acids.It had a sedimentation coefficient of 3.7 S and a Stokes radius of 24.0 f 1.3 A. It had a frictional ratio (f/f,,) of 1.1, indicating the enzyme is of a globular nature.The M, of the enzyme was also determined to be 31,000 2 3,200 by gel filtration and 36,500 by calculation from sedimentation coefficient and Stokes radius.The purified enzyme lacked absolute substrate specificity, hydrolyzing both cyclic CMP and cyclic AMP to a comparable degree and, to a lesser extent, cyclic GMP.The apparent K, for cyclic CMP (182 f 8 pM) was higher than the apparent K,,, for cyclic AMP (25 & 3 pM).The V,, (micromoles hydrolyzed/min/mg of enzyme) of the enzyme for cyclic CMP (4.1 -+ 0.2) was