Determination of the subunit size of NADPH-nitrate reductase from Aspergillus nidulans

Abstract

Immunoprecipitation has been used to effect a rapid isolation of NADPH-nitrate reductase (NADPH:nitrate) oxidoreductase, EC 1.6.6.3) from Aspergillus nidulans in order to avoid proteolytic degradation of the enzyme during extensive purification procedures. Using a monospecific antiserum, immunoprecipitates revealed a single band on SDS-polyacrylamide gels which corresponds to a polypeptide with a molecular weight of approx. 91 000. This polypeptide was nitrate-inducible in the wild-type, constitutively produced in an nirA c/d regulatory mutant and was absent from strains carrying large deletions of the niaD gene. Two strains carrying smaller deletions in the niaD gene lacked the 91 kDa polypeptide but each produced two bands corresponding to molecular weights of 58 000 and 55 000. It is proposed that these represent the deletion products of the native gene product and that the 55 kDa polypeptide is derived from the 58 kDa molecule by a proteolytic cleavage in vivo at a particularly susceptible site.