Purification of a cyclic nucleotide phosphodiesterase from bovine brain using blue dextran-Sepharose chromatography.
The soluble high Km form of cyclic nucleotide phosphodiesterase (EC 3.4.1.17) was purified over 2000-fold from bovine brain homogenates principally using blue dextran-Sepharose chromatography. The purified protein has a specific enzymic activity of 167 units/mg and appears homogeneous when examined by polyacrylamide gel electrophoresis. The enzyme has a molecular weight of 1.26 +/- 0.05 x 10(5) consisting of two apparently identical polypeptide chains. Kinetic measurements indicate that the substrates cyclic GMP and cyclic AMP each have a single Km value, 9 +/- 1 micron and 150 +/- 50 micron, respectively, that the two cyclic nucleotides compete for the same catalytic site, that the blue dye of blue dextran-Sepharose is a competitive inhibitor for the cyclic nucleotides, and that the Vmax with cyclic AMP as substrate is about an order of magnitude larger than that for cyclic GMP. Bovine brain calmodulin stimulates the catalytic rate of the purified enzyme in the presence of Ca2+ by increasing the Vmax associated with each cyclic nucleotide substrate.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification* / Q000378:metabolism; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D004396:Coloring Agents; D003911:Dextrans; D007700:Kinetics; D012685:Sepharose
D004396:Coloring Agents; D003911:Dextrans; D012685:Sepharose; C017080:blue dextran; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases