Cyclic CMP phosphodiesterase: isolation, specificity and kinetic properties.
Cyclic CMP phosphodiesterase activity was demonstrated in rat liver, heart, brain, kidney, intestine, skeletal muscle, blood, testes, ovaries, spleen and lung; that present in the liver was purified to homogeneity by a sequential process of ammonium sulphate fractionation, gel filtration, two ion-exchange chromatographic steps, preparative electrophoresis and two affinity chromatographic stages with selection at each stage for maximum specificity. The final enzyme preparation was confirmed as a single protein by HPLC and isoelectric focussing; the total yield obtained was 1.5% and the final specific activity of 48.6 mumol cyclic CMP hydrolysed/min/mg reflected a 88,000 fold purification. The phosphodiesterase had a Mr of 2.8 X 10(4), pH optimum 7.2-7.4, isoelectric point between 4.2 and 4.4 and a Km of 9.0 mM cyclic CMP. This enzyme differs from a previously isolated cyclic CMP phosphodiesterase in its amino acid composition and specificity. The absolute specificity for 3',5'-cyclic CMP as substrate distinguishes this cyclic CMP phosphodiesterase from all other reported phosphodiesterases and shows it to be a novel enzyme. Its potential as a research tool and the significance of its occurrence are discussed.
D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005260:Female; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014018:Tissue Distribution
D000596:Amino Acids; D010727:Phosphoric Diester Hydrolases; D063309:2',3'-Cyclic Nucleotide 3'-Phosphodiesterase; C571339:Cnp protein, rat