Monoclonal antibody-based immunoaffinity chromatography for purifying corn and squash NADH: nitrate reductases. Evidence for an interchain disulfide bond in nitrate reductase

Abstract

NADH: nitrate reductase (EC 1.6.6.1) (NR) is present in small amounts in plant tissues and its polypeptide in inherently labile. Consequently, NR is difficult to purify. We have generated 20 monoclonal antibodies (McAb) for corn and squash NR and selected two for use in immunoaffinity chromatography. Squash McAb CM 15(11) and corn McAb ZM 2(69)9, which both bind corn and squash NR, were covalently coupled to Sepharose and used for purification of NR with elution of the purified enzyme by a pH 11 buffer. Although this procedure yielded highly purified NR, its activity was diminished by the pH 11 treatment. When corn leaf crude extract was applied to McAb CM 15(11)-Sepharose, NR bound and could be eluted in homogeneous form by its substrate, NADH. Corn leaf NR prepared by substrate elution retained a high level of NADH: NR activity. Immunoaffinity-purified corn and squash NR were shown to have an interchain disulfide bond as well as a reactive thiol group. These results are discussed in relation to the recently obtained sequences of NR clones and suggestions made for site-directed mutagenesis experiments to aid in identifying the cysteine residues of NR associated with these features of the enzyme.