Distinction of cnxH cofactor gene-specified protomers with monoclonal antibodies to Aspergillus nitrate reductase

Abstract

The nitrate reductase (NADPH) (EC 1.6.6.3) from Aspergillus nidulans is influenced directly by mutations in the structural gene (niaD) for the major subunit of the enzyme and indirectly by mutation in any of several molybdenum cofactor loci (cnx). The cnxE-14 and the cnxH-3 mutants have been noted to contain the enzyme in two distinct forms following induction with nitrate. With the cnxH-3 as a prototype cnxH mutant, 10 other cnxH were found to be devoid of the assembled (dimeric) form of the enzyme. Two monoclonal antibodies specific for the native enzyme of the wild type (biA-1) recognized an epitope on the enzyme from the cnxE-14 and cnxH-3 mutants that was common to both and another that was unique to the cnxH gene specified protomer. Another monoclonal antibody recognized an epitope that occurs only in the assembled dimeric form of the enzyme from the wild type or the cnxE-14 mutant. The experiments further substantiate the cnxH phenotype as one involving unassembled protomers of the nitrate reductase in Aspergillus.