Characterization of a calcium-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain

Abstract

A calmodulin-stimulated form of cyclic nucleotide phosphodiesterase from bovine brain was extensively purified (1000-fold). Its specific activity is .apprx. 4 .mu.mol min-1 (mg of protein)-1 when 1 .mu.M cGMP is used as the substrate. This form of calmodulin-sensitive phosphodiesterase activity differs from those purified previously by showing a very low maximum hydrolytic rate for cAMP vs. cGMP. The purification procedure utilizing ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephacryl S-300, isoelectric focusing and affinity chromatography on calmodulin-Sepharose and Cibacron blue-agarose results in a protein with greater than 80% purity with 1% yield. Kinetics of cGMP and cAMP hydrolysis are linear with Km values of 5 and 15 .mu.M, respectively. Addition of Ca and calmodulin reduces the apparent Km for cGMP to 2-3 .mu.M and increases the Vmax by 10-fold. cAMP hydrolysis shows a similar increase in Vmax with an apparent doubling of Km. Both substrates show competitive inhibition with Ki close to their relative Km values. Highly purified preparations of the enzymne contain a major protein band of MW 74,000 that best correlates with enzyme activity. Proteins of MW 59,000 and MW 46,000 contaminate some preparations to varying degrees. An apparent MW of 150,000 by gel filtration suggests that the enzyme exists as a dimer of MW 74,000 subunits. Phosphorylation of the enyzme preparation by cAMP-dependent protein kinase did not alter the kinetic or calmodulin binding properties of the enzyme. Western immunoblot analysis indicated no cross-reactivity between the bovine brain calmodulin-stimulated cGMP phosphodiesterase and the MW 60,000 high-affinity cAMP phosphodiesterase present in most mammalian tissues.