Purification and partial characterization of membrane-associated type II (cGMP-activatable) cyclic nucleotide phosphodiesterase from rabbit brain

Biochim Biophys Acta. 1988 Oct 28;972(1):79-94. doi: 10.1016/0167-4889(88)90105-x.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3',5'-Cyclic-AMP Phosphodiesterases / immunology
  • 3',5'-Cyclic-AMP Phosphodiesterases / isolation & purification*
  • 3',5'-Cyclic-AMP Phosphodiesterases / metabolism
  • Animals
  • Antibodies, Monoclonal / immunology
  • Brain / enzymology*
  • Cell Membrane / enzymology
  • Chromatography, Affinity
  • Cyclic GMP / metabolism
  • Kinetics
  • Molecular Weight
  • Phosphodiesterase Inhibitors / pharmacology
  • Phosphorylation
  • Precipitin Tests
  • Rabbits
  • Solubility
  • Subcellular Fractions / enzymology

Substances

  • Antibodies, Monoclonal
  • Phosphodiesterase Inhibitors
  • 3',5'-Cyclic-AMP Phosphodiesterases
  • Cyclic GMP