• A New Real-Time Simple Method...

*Result*: A New Real-Time Simple Method to Measure the Endogenous Nitrate Reductase Activity (Nar) in Paracoccus denitrificans and Other Denitrifying Bacteria.

Title:
A New Real-Time Simple Method to Measure the Endogenous Nitrate Reductase Activity (Nar) in Paracoccus denitrificans and Other Denitrifying Bacteria.
Authors:
García-Trejo JJ; 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.), Zempoala, Pachuca 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.), Zempoala, Pachuca 43830, Mexico.
Source:
International journal of molecular sciences [Int J Mol Sci] 2024 Sep 10; Vol. 25 (18). Date of Electronic Publication: 2024 Sep 10.
Publication Type:
Journal Article
Language:
English
Journal Info:
Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE
Imprint Name(s):
Original Publication: Basel, Switzerland : MDPI, [2000-
MeSH Terms:
Nitrate Reductase*/metabolism , Paracoccus denitrificans*/enzymology , Enzyme Assays*/methods , Denitrification* , Computer Systems*, NADH Dehydrogenase/metabolism ; Electron Transport Complex I/metabolism ; Nitrates/metabolism ; Oxygen/metabolism ; Anaerobiosis ; Kinetics ; Time Factors ; Nitrogen
References:
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Contributed Indexing:
Keywords: Brucella; Nar; Paracoccus denitrificans; activity; denitrifiers; enteropathogenic; method; new; nitrate reductase; real-time
Substance Nomenclature:
EC 1.7.99.4 (Nitrate Reductase)
EC 1.6.99.3 (NADH Dehydrogenase)
EC 7.1.1.2 (Electron Transport Complex I)
0 (Nitrates)
S88TT14065 (Oxygen)
12136-83-3 (sodium nitride)
N762921K75 (Nitrogen)
Entry Date(s):
Date Created: 20240928 Date Completed: 20250206 Latest Revision: 20260127
Update Code:
20260130
PubMed Central ID:
PMC11431489
DOI:
10.3390/ijms25189770
PMID:
39337258
Database:
MEDLINE

*Further Information*

*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 NO<subscript>3</subscript><sup>-</sup> using this so-called Nar-JJ assay gave a K<subscript>m</subscript> 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.*