Purification and partial characterization of membrane-associated type II (cGMP-activatable) cyclic nucleotide phosphodiesterase from rabbit brain.
Membrane-associated, Type II (cGMP-activatable) cyclic nucleotide phosphodiesterase (PDE) from rabbit brain, representing 75% of the total homogenate Type II PDE activity, was purified to apparent homogeneity. The enzyme was released from 13,000 x g particulate fractions by limited proteolysis with trypsin and fractionated using DE-52 anion-exchange, cGMP-Sepharose affinity and hydroxylapatite chromatographies. The enzyme showed 105 kDa subunits by SDS-PAGE and had a Stokes radius of 62.70 A as determined by gel filtration chromatography. Hydrolysis of cAMP or cGMP showed positive cooperativity, with cAMP kinetic behavior linearized in the presence of 2 microM cGMP. Substrate concentrations required for half maximum velocity were 28 microM for cAMP and 16 microM for cGMP. Maximum velocities were approx. 160 mumol/min per mg for both nucleotides. The apparent Kact for cGMP stimulation of cAMP hydrolysis at 5 microM substrate was 0.35 microM and maximal stimulation (3-5-fold) was achieved with 2 microM cGMP. Cyclic nucleotide hydrolysis was not enhanced by calcium/calmodulin. The purified enzyme can be labeled by cAMP-dependent protein kinase as demonstrated by the incorporation of 32P from [gamma-32P]ATP into the 105 kDa enzyme subunit. Initial experiments showed that phosphorylation of the enzyme did not significantly alter enzyme activity measured at 5 microM [3H]cAMP in the absence or presence of 2 microM cGMP or at 40 microM [3H]cGMP. Monoclonal antibodies produced against Type II PDE immunoprecipitate enzyme activity, 105 kDa protein and 32P-labeled enzyme. The 105 kDa protein was also photoaffinity labeled with [32P]cGMP. The purified Type II PDE described here is physicochemically very similar to the isozyme purified from the cytosolic fraction of several bovine tissues with the exception that it is predominantly a particulate enzyme. This difference may reflect an important regulatory mechanism governing the metabolism of cyclic nucleotides in the central nervous system.
Whalin
M E
ME
University of South Alabama, College of Medicine, Department of Pharmacology, Mobile 36688.
Strada
S J
SJ
Thompson
W J
WJ
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D006152:Cyclic GMP / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D011233:Precipitin Tests; D011817:Rabbits; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology
D000911:Antibodies, Monoclonal; D010726:Phosphodiesterase Inhibitors; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP