Nitrate reductase isozymes in Bradyrhizobium sp. (Lupinus) bacteroids: localisation, biochemical and kinetic characteristics

Bradyrhizobium sp. (Lupinus) strains 750 and IM-43B, possessing nitrate reductase activity (NRA) were used to inoculate Lupinus albus cv. Multolupa plants. Bacteroids from root nodules were sonicated and sedimented to separate membranes from cytosolic fractions. Our results confirmed the existence of constitutive NRA mostly in the membranes, but soluble and membrane-bound NR forms were also induced by nitrate, and both of them used NADH as electron donor. Immunolocalisation of NR using a monoclonal antibody showed the presence of a larger number of epitopes in the soluble region than in membrane of bacteroids, and gold particles increased in those nodules grown with nitrate. Chromatographic purification of NR allowed the detection of two active fractions with an average MW of 91 and 362 kD. A further attempt to separate NR fractions in bacteroids and free-living cells grown with nitrate, led us to detect two more native fractions (180 and 720 kD). SDS-PAGE gel electrophoresis from all of the four native fractions revealed the presence of two common bands of around 20 and 70 kD; these results seem to indicate that all of the native enzymes were associations of one single cong 90 kD monomer molecule. Specific NRA increased from the small to the large proteins, suggesting positive cooperation by monomer associations. There was evidence enough to support an interpretation that constitutive and substrate-induced NRs would be formed by a monomeric and its homotetrameric isozyme. On the other hand, three IEF spots were obtained by 2-D gel electrophoresis and by anionic chromatography with pI 7.0, 7.8 and 8.1, when any of the native fractions were analysed. Kinetic studies of the four MW native isoforms showed that the two smallest molecules behaved according to Michaelis-Menten with similar KM, but the 360 and 720 kD fractions fitted much better to straight lines in a Hill plot, indicating allosterism and positive cooperativity.