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Polymeric structure of the cyclic AMP-dependent protein kinase from the dimorphic fungus Mucor rouxii and purification of its catalytic subunit

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Summary

The polymeric structure of the cyclic AMP-dependent protein kinase (E.C.2.7.1.37) from the dimorphic fungus Mucor rouxii was analyzed through studies of gel filtration and sucrose gradient centrifugation of the holoenzyme and its subunits and by photoaffinity labeling of the regulatory subunit. It was demonstrated that it is a tetramer composed by two regulatory subunits (R) of mol. wt. 75 000 and two catalytic subunits (C) of mol. wt. 41 000 forming a holoenzyme R2C2 of mol. wt. 242 000. Frictional coefficients of 1.55 and 1.62 for the holoenzyme and for the regulatory dimer, respectively, indicate a significant degree of dimensional asymmetry in both molecules. A procedure for the purification of the catalytic subunit of the kinase is presented. The holoenzyme could be bound to a cyclic AMP-agarose column and the catalytic subunit could be eluted by 0.5 M NaCl, well resolved from the bulk of protein. This particular behaviour of the holoenzyme in cyclic AMP-agarose chromatography allowed the inclusion of this step in the purification of the catalytic subunit and corroborated that the holoenzyme was not dissociated by cyclic AMP alone. The isolated catalytic subunit displays Michaelis-Menten behaviour towards kemptide, protamine and histone and is inhibited by sulfhydryl reagents, indicating that the molecule has at least one cysteine residue essential for enzyme activity. The catalytic activity of the isolated C subunit is inactivated by the mammalian protein kinase inhibitor, and is inhibited by the regulatory subunit from homologous and heterologous sources. In general, the properties of the catalytic subunit suggest a structural similarity between Mucor and mammalian C subunits.

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Abbreviations

C:

catalytic subunit monomer of protein kinase

R:

regulatory subunit monomer of protein kinase

8-N3-cyclic AMP:

8-azido-cylic AMP

SDS:

sodium dodecyl sulfate

Pipes:

piperazine-N,N′-bis(2-ethanesulfonic acid)

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Authors and Affiliations

  1. Departamento de Química Biológica, Programa de Regulación Hormonal y Metabólica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428, uenos Aires, Argentina

    Ricardo Pastori, Silvia Moreno & Susana Passeron

Authors
  1. Ricardo Pastori
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  2. Silvia Moreno
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  3. Susana Passeron
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Career Investigators from the CONICET

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Pastori, R., Moreno, S. & Passeron, S. Polymeric structure of the cyclic AMP-dependent protein kinase from the dimorphic fungus Mucor rouxii and purification of its catalytic subunit. Mol Cell Biochem 69, 55–66 (1985). https://doi.org/10.1007/BF00225927

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