Premium
Purification of an enzyme aggregate containing 3′, 5′‐cyclic‐nucleotide phosphodiesterase and nucleotidase
Author(s) -
Böldicke Thomas W.,
Schubert-Rehberg kristiane,
Wombacher Helmut
Publication year - 1986
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(86)81399-0
Subject(s) - 5' nucleotidase , chemistry , enzyme , phosphodiesterase , polyacrylamide gel electrophoresis , biochemistry , nucleotidase , chromatography , nucleotide , sepharose , gel electrophoresis , microbiology and biotechnology , biology , gene
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.