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...

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Autores principales:	Tomes, Claudia Nora, Kerner, Néstor Alberto, Moreno, Silvia
Publicado:	1988
Materias:	cyclic amp phosphodiesterase article enzyme phosphorylation enzyme purification fungus mucor rouxii nonhuman priority journal 3',5'-Cyclic-Nucleotide Phosphodiesterase Enzyme Activation Mucor Phosphorylation Support, Non-U.S. Gov't Amylomyces rouxii Fungi
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:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	paper:paper_08957479_v12_n5-6_p289_Tomes
record_format	dspace
spelling	paper:paper_08957479_v12_n5-6_p289_Tomes2023-06-08T15:48:06Z Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii Tomes, Claudia Nora Kerner, Néstor Alberto Moreno, Silvia cyclic amp phosphodiesterase article enzyme phosphorylation enzyme purification fungus mucor rouxii nonhuman priority journal 3',5'-Cyclic-Nucleotide Phosphodiesterase Enzyme Activation Mucor Phosphorylation Support, Non-U.S. Gov't Amylomyces rouxii Fungi Mucor 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. Fil:Tomes, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Kerner, N. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Moreno, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 1988 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
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	cyclic amp phosphodiesterase article enzyme phosphorylation enzyme purification fungus mucor rouxii nonhuman priority journal 3',5'-Cyclic-Nucleotide Phosphodiesterase Enzyme Activation Mucor Phosphorylation Support, Non-U.S. Gov't Amylomyces rouxii Fungi Mucor
spellingShingle	cyclic amp phosphodiesterase article enzyme phosphorylation enzyme purification fungus mucor rouxii nonhuman priority journal 3',5'-Cyclic-Nucleotide Phosphodiesterase Enzyme Activation Mucor Phosphorylation Support, Non-U.S. Gov't Amylomyces rouxii Fungi Mucor Tomes, Claudia Nora Kerner, Néstor Alberto Moreno, Silvia Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii
topic_facet	cyclic amp phosphodiesterase article enzyme phosphorylation enzyme purification fungus mucor rouxii nonhuman priority journal 3',5'-Cyclic-Nucleotide Phosphodiesterase Enzyme Activation Mucor Phosphorylation Support, Non-U.S. Gov't Amylomyces rouxii Fungi Mucor
description	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.
author	Tomes, Claudia Nora Kerner, Néstor Alberto Moreno, Silvia
author_facet	Tomes, Claudia Nora Kerner, Néstor Alberto Moreno, Silvia
author_sort	Tomes, Claudia Nora
title	Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii
title_short	Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii
title_full	Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii
title_fullStr	Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii
title_full_unstemmed	Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii
title_sort	further studies on the phosphorylation-regulated camp-phosphodiesterase from the dimorphic fungus mucor rouxii
publishDate	1988
url	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
work_keys_str_mv	AT tomesclaudianora furtherstudiesonthephosphorylationregulatedcampphosphodiesterasefromthedimorphicfungusmucorrouxii AT kernernestoralberto furtherstudiesonthephosphorylationregulatedcampphosphodiesterasefromthedimorphicfungusmucorrouxii AT morenosilvia furtherstudiesonthephosphorylationregulatedcampphosphodiesterasefromthedimorphicfungusmucorrouxii
_version_	1768544918015311872

Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii

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