Purification and Characterization of Molecular and Immunological Properties of Rice Ferredoxin-Nitrite Reductase

Abstract

Ferredoxin-nitrite reductase [EC 1.7.7.1] has been purified to apparent homogeneity from rice (Oryza satira cv. Kinmaze) leaves by a procedure used for the spinach enzyme [S. Ida and B. Mikami, Biochim. Biophys. Acta, 681, 167 (1986)]. The rice enzyme consists of a single polypeptide of % molecular weight of 60,000 with 536 amino acid residues. The enzyme showed nearly identical absorption, circular dichroism, and magnetic circular dichroism spectra to those of the spinach enzyme, indicating the presence of the same prosthetic groups and protein conformation in both enzymes. The apparent Km values for nitrite and methyl viologen were 360 μm and 63 μm, respectively. The pH optimum was 7.6. These kinetic parameters are indistinguishable from those reported for spinach nitrite reductase. Monospecific antiserum against purified rice enzyme cross-reacted with nitrite reductases from a variety of higher plants and some phylogenetically divergent plants. Immunological comparisons indicated the rice enzyme is much more closely related to the other monocot enzymes in antigenic structure than to the dicot enzyme proteins. The results lend further support to our previous study [S. Ida, Plant Sci., 49, 111 (1987)] that spinach ferredoxin-nitrite reductase is serologically more related to the dicot enzymes than to the monocot nitrite reductases. Conspicuous differences between the rice and spinach enzymes were found in their molecular sizes and antigenicity. Relatedness of amino acid compositions of the enzyme proteins is discussed in relation to antigenic properties of ferredoxin-nitrite reductase.