Abstract
Nitrate reductase A has been solubilized from purified cytoplasmic membranes by extraction with terl-amyl alcohol. The resulting aqueous solution contained monomeric reductase which polymerized slowly to dimers and tetramers with sedimentation coefficients of respectively 10.5, 16 and 23 Svedbergunits. The polymerization could be stopped to some extent by addition of a small amount of Triton X-100. These distinct entities of nitrate reductase A were separable on electro-focusing, DEAE-column chromatography and polyacrylamide gel electrophoresis, and have been proved to consist of similar subunits with molecular weights of 104000, 63000, and 56000 daltons. The molecular weights of monomeric nitrate reductase A was found to be about 240000 daltons.
Chlorate reductase C has been solubilized by a similar procedure, resulting in only monomeric enzyme. Chlorate reductase C exhibited a sedimentation coefficient of 7.7 Svedbergunits, an isoelectric point of pH=4.55 and a molecular weight of approx. 180000 daltons. It was found to consist of three subunits with molecular weights of 75000, 63000 and 56000 daltons. The latter two subunits are most probably common in nitrate reductase A and chlorate reductase C.
Similar content being viewed by others
References
Chance, B., Maehly, A. C.: Assay of catalases and peroxidases. In: Methods in enzymology, Vol. II (S. Colowick, N. Kaplan, eds.), p. 764. New York: Academic Press 1955
Clegg, R. A.: Purification and some properties of nitrate reductase (EC 1.7.99.4) from Escherichia coli K12. Biochem. J. 153, 533–541 (1976)
Determann, H., Michel, W.: The correlation between molecular weight and elution behaviour in the gel chromatography of proteins. J. Chromatogr. 25, 303–313 (1966)
Enoch, H. G., Lester, R. L.: The role of a novel cytochrome b-containing nitrate reductase and quinone in the in vitro reconstitution of formate-nitrate reductase activity of E. coli. Biochem. biophys. Res. Commun. 61, 1234–1240 (1974)
De Groot, G. N., Stouthamer, A. H.: Regulation of reductase formation in Proteus mirabilis. I. Formation of reductases and enzymes of the formic hydrogenlyase complex in the wild type and in chlorate-resistant mutants. Arch. Mikrobiol. 66, 220–233 (1969)
De Groot, G. N., Stouthamer, A. H.: Regulation of reductase formation in Proteus mirabilis. III. Influence of oxygen, nitrate and azide on thiosulfate reductase and tetrathionate redutase formation. Arch. Mikrobiol. 74, 326–339 (1970)
Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265–275 (1951)
Lund, K., DeMoss, J. A.: Association-dissociation behaviour and subunit structure of heat-released nitrate reductase from Escherichia coli. J. biol. Chem. 251, 2207–2216 (1976)
MacGregor, C. H.: Solubilization of Escherichia coli nitrate reductase by a membrane-bound protease. J. Bact. 121, 1102–1110 (1975a)
MacGregor, C. H.: Anaerobic cytochrome b, in Escherichia coli: association with and regulation of nitrate reductase. J. Bact. 121, 1111–1116 (1975b)
MacGregor, C. H.: Biosynthesis of membrane-bound nitrate reductase in Escherichia coli: Evidence for a soluble precursor. J. Bact. 126, 122–131 (1976)
MacGregor, C. H., Schnaitman, C. A., Normansell, D. E.: Purification and properties of nitrate reductase from Escherichia coli K12. J. biol. Chem. 249, 5321–5327 (1974)
Martin, R. G., Ames, B. N.: A method for determining the sedimentation behaviour of enzymes: Application to protein mixtures. J. biol. Chem. 236, 1372–1379 (1961)
Maurer, H. R.: Disk-Electrophorese. Berlin: W. de Gruyter 1968
Nachbar, M. S., Winkler, W. J., Salton, M. R. J.: The effect of aliphatic alcohols upon the dissociation of Micrococcus lysodeikticus membrane lipids and proteins. Biochim. biophys. Acta (Amst.) 274, 83–94 (1972)
Oltmann, L. F., Reijnders, W. N. M., Stouthamer, A. H.: The correlation between the protein composition of cytoplasmic membranes and the formation of nitrate reductase A, chlorate reductase C and tetrathionate reductase in Proteus mirabilis wild type and some chlorate resistant mutants. Arch. Microbiol. 111, 37–43 (1976)
Oltmann, L. F., Schoenmaker, G. S., Stouthamer, A. H.: Solubilization and purification of a cytoplasmic membrane bound enzyme catalyzing tetrathionate and thiosulphate reduction in Proteus mirabilis. Arch. Microbiol. 98, 19–30 (1974)
Oltmann, L. F., Stouthamer, A. H.: Purification of cytoplasmic membranes and outer membranes from Proteus mirabilis. Arch. Mikrobiol. 93, 311–325 (1973)
Peck, H. D., Gest, H.: A new procedure for assay of bacterial hydrogenases. J. Bact. 71, 71–80 (1956)
Pichinoty, F.: Répression par l'oxygène de la biosynthèse de la thiosulfate réductase de Proteus vulgaris. Experientia (Basel) 18, 501–506 (1962)
Pichinoty, F.: Recherche des nitrate-réductases bactériènnes A et B: Méthodes. Bull. Soc. Chim. Biol. 47, 1526–1528 (1965)
Piéchaud, M., Puig, J., Pichinoty, F., Azoulay, E., Le Minor, L.: Mutations affectant la nitrate-réductase A et d'autres enzymes bactériennes d'oxydo-réduction. Etude préliminaire. Ann. Inst. Pasteur 112, 24–37 (1967)
Schnaitman, C. A.: Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J. Bact. 108, 545–552 (1971)
Stouthamer, A. H.: Nitrate reduction in Aerobacter aerogenes. I. Isolation and properties of mutant strains blocked in nitrate assimilation and resistant against chlorate. Arch. Mikrobiol. 56, 68–75 (1967)
Stouthamer, A. H.: Biochemistry and genetics of nitrate reductase in bacteria. Advanc. Microbial Physiol. 14, 315–375 (1976)
Van't Riet, J., Planta, R. J.: Purification and some properties of the membrane-bound respiratory nitrate reductase of Aerobacter aerogenes. FEBS Letters 5, 249–252 (1969)
Van't Riet, J., Planta, R. J.: Purification, structure and properties of the respiratory nitrate reductase of Klebsiella aerogenes. Biochim. biophys. Acta (Amst.) 379, 81–94 (1975)
Van't Riet, J., Stouthamer, A. H., Planta, R. J.: Regulation of nitrate assimilation and nitrate respiration in Aerobacter aerogenes. J. Bact. 96, 1455–1464 (1968)
Weber, K., Osborn, M.: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406–4412 (1969)
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Oltmann, L.F., Reijnders, W.N.M. & Stouthamer, A.H. Characterization of purified nitrate reductase A and chlorate reductase C from Proteus mirabilis . Arch. Microbiol. 111, 25–35 (1976). https://doi.org/10.1007/BF00446546
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00446546