Les nitrate-reductases bacteriennes solubilisation, purification et proprietes de l'enzyme a de Micrococcus halodenitrificans

doi:10.1016/0005-2744(73)90185-X

Biochimica et Biophysica Acta (BBA) - Enzymology

Volume 321, Issue 2, 10 October 1973, Pages 443-455

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Abstract

Solubilization and purification. Crude extract is prepared from cells of Micrococcus halodenitrificans grown anaerobically in the presence of NO₃⁻. Nitrate reductase A is solubilized by an alkali-acetone treatment of particles. The purification consists of four steps: Sephadex gel filtration; elimination of nucleic acids by protamine sulfate precipitation; two successive absorptions and elutions on Ca₃(PO₄)₂ gel. The enzyme is purified 40-fold with a yield of 12%. Polyacrylamide gel electrophoresis) show the preparation to be homogeneous.

Properties. Molecular weight: (electrophoresis 165 000. Flavins: no FMN or FAD. Purified preparations have a brown color. Absorption increases continuously from 600 to 280 nm. The spectrum has a shoulder at 410 nm, but no peak in the visible or near-ultraviolet. The enzyme contains approximately 2 Fe atoms, 1 Mo atom and 4 “labile sulfide groups in acid medium” per mole (M_w = 165 000). It has a high content in aspartic and glutamic acids. Electron donors: the enzyme uses the reduced forms of viologen indicators, FMNH₂ but not NADH and NADPH. It uses NO₃⁻ and ClO₃⁻¹ as substrates. Cyanide and azide inhibit its activity. The inhibition by azide is competitive. The enzyme's affinity for azide is about a thousand times greater than for NO₃⁻ or ClO₃⁻¹. Activity is maximal at pH 6.3 (NO₃⁻¹ and 6.4 (ClO₃⁻¹). NaCl does not activate the enzyme.

Conclusion. Nitrate reductase A of M. halodenitrificans is a non-heme Fe protein containing Mo.

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Les nitrate-reductases bacteriennes solubilisation, purification et proprietes de l'enzyme a de Micrococcus halodenitrificansBacterial nitrate reductases. Solubilization, purification and properties of the enzyme A of Micrococcus halodenitrificans

Abstract

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