Purification of an enzyme aggregate containing 3',5'-cyclic-nucleotide phosphodiesterase and nucleotidase.
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 purified enzyme aggregate showed an S value of 7.3 (SE +/- 0.3, n = 10). Further analysis by SDS-polyacrylamide gel electrophoresis (PAGE) revealed two proteins near 67 and 60 kDa. Dissociation 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 nucleotidase 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 band at 67 kDa. This was taken as evidence for the homogeneity of the 17 S nucleotidase form and the self-association of the nucleotidase after dissociation from the 7.3 S enzyme aggregate. Furthermore, from this it could be concluded that the 67 kDa protein of the 7.3 S enzyme aggregate should be identified with the nucleotidase, and thus the 60 kDa band represents the PDE.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification*; D015720:5'-Nucleotidase; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D009708:Nucleotidases / Q000302:isolation & purification*
D009097:Multienzyme Complexes; D009708:Nucleotidases; D015720:5'-Nucleotidase; D015105:3',5'-Cyclic-AMP Phosphodiesterases