A New Real-Time Simple Method to Measure the Endogenous Nitrate Reductase Activity (Nar) in Paracoccus denitrificans and Other Denitrifying Bacteria
Por:
García-Trejo J.J., Rojas-Alcantar S., Alonso-Vargas M., Ortega R., Benítez-Guzmán A., Ramírez-Silva L., Pavón N., Peña-Segura C., Méndez-Romero O., Uribe-Carvajal S., Cadena-Ramírez A.
Publicada:
1 ene 2024
Resumen:
The transmembrane nitrate reductase (Nar) is the first enzyme in the dissimilatory alternate anaerobic nitrate respiratory chain in denitrifying bacteria. To date, there has been no real-time method to determine its specific activity embedded in its native membrane; here, we describe such a new method, which is useful with the inside-out membranes of Paracoccus denitrificans and other denitrifying bacteria. This new method takes advantage of the native coupling of the endogenous NADH dehydrogenase or Complex I with the reduction of nitrate by Nar through the quinone pool of the inner membranes of P. denitrificans. This is achieved under previously reached anaerobic conditions. Inner controls confirming the specific Nar activity determined by this new method were made by the total inhibition of the Nar enzyme by sodium azide and cyanide, well-known Nar inhibitors. The estimation of the Michaelis–Menten affinity of Nar for NO3- using this so-called Nar-JJ assay gave a Km of 70.4 µM, similar to previously determined values. This new Nar-JJ assay is a suitable, low-cost, and reproducible method to determine in real-time the endogenous Nar activity not only in P. denitrificans, but in other denitrifying bacteria such as Brucella canis, and potentially in other entero-pathogenic bacteria. © 2024 by the authors.
Filiaciones:
García-Trejo J.J.:
Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México (U.N.A.M.), Ciudad de México, 04510, Mexico
Rojas-Alcantar S.:
Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México (U.N.A.M.), Ciudad de México, 04510, Mexico
Alonso-Vargas M.:
Laboratorio de Bioprocesos Ambientales, Universidad Politécnica de Pachuca (U.P.P.), Pachuca, Zempoala, 43830, Mexico
Ortega R.:
Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México (U.N.A.M.), Ciudad de México, 04510, Mexico
Benítez-Guzmán A.:
Departamento de Microbiología e Inmunología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México (U.N.A.M.), Ciudad de México, 04510, Mexico
Ramírez-Silva L.:
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México (U.N.A.M.), Ciudad de México, 04510, Mexico
Pavón N.:
Departamento de Farmacología, Instituto Nacional de Cardiología “Ignacio Chávez”, Ciudad de México 14080, Mexico
Peña-Segura C.:
Departamento de Bioterio, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (U.N.A.M.), Ciudad de México, 04510, Mexico
Méndez-Romero O.:
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (U.N.A.M.), Ciudad de México, 04510, Mexico
Uribe-Carvajal S.:
Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (U.N.A.M.), Ciudad de México, 04510, Mexico
Cadena-Ramírez A.:
Laboratorio de Bioprocesos Ambientales, Universidad Politécnica de Pachuca (U.P.P.), Pachuca, Zempoala, 43830, Mexico
gold, All Open Access; Gold Open Access
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