Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii
A partially purified preparation (200-fold) of cAMP phosphodiesterase (PDE) was obtained from Mucor rouxii grown and extracted under conditions minimizing endogenous proteolysis. Four purification steps were applied: batch DEAE-Sepharose, DEAE-Sepharose chromatography, Sephadex G-150 superfine gel f...
Guardado en:
| Autores principales: | , , |
|---|---|
| Publicado: |
1988
|
| Materias: | |
| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_08957479_v12_n5-6_p289_Tomes http://hdl.handle.net/20.500.12110/paper_08957479_v12_n5-6_p289_Tomes |
| Aporte de: |
| Sumario: | A partially purified preparation (200-fold) of cAMP phosphodiesterase (PDE) was obtained from Mucor rouxii grown and extracted under conditions minimizing endogenous proteolysis. Four purification steps were applied: batch DEAE-Sepharose, DEAE-Sepharose chromatography, Sephadex G-150 superfine gel filtration and sucrose gradient centrifugation. The final PDE preparation was activatable by cAMP-dependent phosphorylation and controlled trypsin treatment. A careful correlation of protein patterns with PDE activity was done throughout the whole procedure by analyzing the active fractions of each step by mini-polyacrylamide non-denaturing cell electrophoresis. The final preparation displayed four major protein bands, none of which corresponded to PDE, although PDE activity comigrated with two of them. Some properties of this preparation were studied. V(max) increased around 10-15 fold by activation of PDE by phosphorylation or proteolysis; K(m) values were unaffected. PDE had Stokes radius of 3.5 nm, sedimentation coefficient of 4.3 S and molecular weight of 70,000 daltons. The treatment of sucrose gradient fractions with [γ-32P] ATP and cAMP-dependent protein kinase catalytic subunit and further analysis through minigels showed that none of the visible bands was phosphorylated, and that among the four phosphorylated bands there was one that cosedimented and comigrated with PDE activity. Trypsin treatment of the phosphorylated samples removed the label but did not modify the staining pattern. |
|---|