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Purification and characteristics of an inducible by polycyclic aromatic hydrocarbons NADP(+)-dependent naphthalenediol dehydrogenase (NDD) in Mucor circinelloides YR-1.
Camacho-Morales, Reyna Lucero; Zazueta-Novoa, Vanesa; Casillas, Juana Lizbeth González; Ballesteros, Elizabeth Aranda; Bote, Juan Antonio Ocampo; Zazueta-Sandoval, Roberto.
Afiliación
  • Camacho-Morales RL; Biology Department, Campus Noria Alta, University of Guanajuato, Noria Alta s/n, C.P. 36050 Guanajuato, Gto., Mexico. Electronic address: Lucy-2000@hotmail.com.
  • Zazueta-Novoa V; Biology Department, Campus Noria Alta, University of Guanajuato, Noria Alta s/n, C.P. 36050 Guanajuato, Gto., Mexico. Electronic address: vanesanovoa@yahoo.com.mx.
  • Casillas JL; Biology Department, Campus Noria Alta, University of Guanajuato, Noria Alta s/n, C.P. 36050 Guanajuato, Gto., Mexico. Electronic address: lizbeth_g_c_@hotmail.com.
  • Ballesteros EA; Departamento de Microbiología y Sistemas Simbióticos, Estación Experimental del Zaidín, Granada, Spain. Electronic address: elizabet.aranda@eez.csic.es.
  • Bote JA; Departamento de Microbiología y Sistemas Simbióticos, Estación Experimental del Zaidín, Granada, Spain. Electronic address: juanantonio.ocampo@eez.csic.es.
  • Zazueta-Sandoval R; Biology Department, Campus Noria Alta, University of Guanajuato, Noria Alta s/n, C.P. 36050 Guanajuato, Gto., Mexico. Electronic address: drbioqu1993@gmail.com.
Protein Expr Purif ; 97: 1-8, 2014 May.
Article en En | MEDLINE | ID: mdl-24530565
We detected NADP(+)-dependent dihydrodiol dehydrogenase (DD) activity in a cell-free extract from Mucor circinelloides YR-1, after high-speed centrifugation. We analyzed the enzymatic activity in the cytosolic fraction by zymograms, as described previously, and eight different DD activity bands were revealed. Five constitutive DD activities (DD1-5) were present when glucose was used as carbon source and three inducible activities (NDD, PDD1 and PDD2) when aromatic hydrocarbon compounds were used. NDD activity was induced all of the aromatic hydrocarbon compounds. The highest DD activity inducer was naphthalene and the lowest was pyrene. One of the enzymes showed higher activity with cis-naphthalene-diol rather than with trans-nahthalenediol as a substrate. We purified this particular enzyme to homogeneity and found that it had an isoelectric point of 4.6. The molecular weight for the native protein was 197.4kDa and 49.03±0.5kDa for the monomer that conforms it, suggesting a homotetrameric structure for the complete enzyme. Polyclonal antibodies were raised against it and obtained. NDD activity was almost totally inhibited when antibodies were used at low concentrations, and in native immunoblots only one band, which corresponds to the activity band detected in the zymograms, could be detected. In denaturing PAGE immunoblots only one band was detected. This band corresponds to the purified protein band of 49kDa detected in SDS-PAGE gels. The other two inducible enzymes PDD1 and PDD2 were present only when phenanthrene was used as sole carbon source in the culture media.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Hidrocarburos Policíclicos Aromáticos / Proteínas Fúngicas / Mucor / NADPH Deshidrogenasa Idioma: En Revista: Protein Expr Purif Asunto de la revista: BIOLOGIA MOLECULAR Año: 2014 Tipo del documento: Article Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Hidrocarburos Policíclicos Aromáticos / Proteínas Fúngicas / Mucor / NADPH Deshidrogenasa Idioma: En Revista: Protein Expr Purif Asunto de la revista: BIOLOGIA MOLECULAR Año: 2014 Tipo del documento: Article Pais de publicación: Estados Unidos