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In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis
- Source :
- Archives of Microbiology. 168:215-224
- Publication Year :
- 1997
- Publisher :
- Springer Science and Business Media LLC, 1997.
-
Abstract
- The nitrate assimilation pathway in Candida utilis, as in other assimilatory organisms, is mediated by two enzymes: nitrate reductase and nitrite reductase. Purified nitrite reductase has been shown to be a heterodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and NADPH as electron donors. FAD, which is an essential coenzyme, stabilised the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thiol reagents. One cysteine was demonstrated to be essential for the enzymatic activity. In vitro, the enzyme was inactivated by ammonium salts, the end product of the path way, proving that the enzyme is assimilatory in function. In vivo, the enzyme was induced by nitrate and repressed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated together, which indicated that the primary level of regulation of this enzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells grown with the same salts as the source of nitrogen, the residual enzymatic activities were similar. Thus, a study of the in vitro inactivation can give a clue to understanding the mechanism of in vivo regulation of nitrite reductase in Candida utilis.
- Subjects :
- 7-Dehydrocholesterol reductase
Nitrite Reductases
Nitrogen assimilation
p-Chloromercuribenzoic Acid
Biology
Reductase
Nitrate reductase
Biochemistry
Microbiology
Ammonium Chloride
Gene Expression Regulation, Enzymologic
Genetics
Cysteine
Molecular Biology
Candida
Microbiology & Cell Biology
Nitrite Reductase (NAD(P)H)
chemistry.chemical_classification
General Medicine
NAD
Nitrite reductase
Enzyme assay
Kinetics
Enzyme
chemistry
Enzyme Induction
Flavin-Adenine Dinucleotide
biology.protein
Enzyme Repression
Chloromercuribenzoates
NADP
Subjects
Details
- ISSN :
- 1432072X and 03028933
- Volume :
- 168
- Database :
- OpenAIRE
- Journal :
- Archives of Microbiology
- Accession number :
- edsair.doi.dedup.....6dc21729f2e4bf6c19b61d377aad2e82