Several steps of purification (octyl-Sepharose chromatography,Blue Sepharose 6B Chromatography and sucrose density gradient centrifugation) led to a highly purified aggregate of the enzymes, 3′, 5′-cyclic-nucleotide phosphodiesterase (PDE) and nucleotidase. The purfied enzyme aggregate showed an S value of 7.3 (SE±0.3, n = 10). Further analysis by SDS-polyacrylamide gel electrophoresis (PAGE) reavealed two proteins near 67 and 60 kDa. Dissocation of the 7.3 S enzyme aggregate showed a 3.6 S PDE form and a nucleotidase form at 4.2 S. Additionally, higher S value forms of the necleotidase up to 17 S have been observed. Apparently, they had formed by self-association. SDS-PAGE of the 17 S nucleotidase form showed only one hand at 67 kDa. This was taken as evidence for the homogenity of the 17 S nucleotidase form and the self-association of the nuleotidase after dissociation from the 7.3 S enzyme aggregate. Furthermore, from this it could be concluded that the 67 kDa protin of the 7.3 S enzyme aggregate should be identified with the nucleotidase, and thus the 60 kDa band represents the PDE.
