Cyclic nucleotide phosphodiesterase and 5′-nucleotidase: A coupled system

Evidence is presented that multiple forms of cyclic nucleotide phophodiesterase (PDE) activity chromatographically separated from the soluble fraction of bovine hypothalamus are co-eluted with multiple forms of 5'-nucleotidase (5'N) activity. The enzymes could not be resolved from each other by anion-exchange chromatography on DEAE-TSK; by affinity chromatography on phenyl-, blue-, concanavalin A-, 5' AMP-sepharose, cAMP-silica gel; or by gel filtration on sephacryl S-200. The catalytic activities were found to be associated with the tetrameric, dimeric, and monomeric forms of the enzymes. The molecular weights determined by gel filtration or by SDS-gel electrophoresis were 220, 114, and 57 kDa, respectively. Kinetic analysis revealed that the first-order rate constant for 5' AMP hydrolysis measured in the reactions: cAMP?5'AMP?adenosine was 100 times higher than that in the reaction: 5'AMP?adenosine. Thus, functional interrelation between PDE and 5'N was expressed in drastic acceleration of the consecutive reactions: cAMP ?5'AMP?adenosine. The results confirm the conclusion about the existence of a multienzyme system involving PDE and 5'N or of a single bifunctional enzyme in brain tissue.