Purification and comparison of nit-1 and wild-type NADPH:nitrate reductases of Neurospora crassa

Abstract

Neurospora crassa NADPH:nitrate oxidoreductase (EC 1.6.6.3) wild-type and mutant nit-1 enzymes have been purified by two different procedures, and the apparent molecular weights of the isolated polypeptides in each preparation have been estimated. Initially, a procedure was developed to purify the nit- I enzyme which resulted in a preparation with very low yields, high specific activities of NADPH:cytochrome c reductase (FAD-dependent), and two protein bands on SDS-polyacrylamide gels having apparent molecular weights of 91 000 and 112 000. A shortened procedure, which minimized proteolysis, resulted in improved yields of the nit- I enzyme, high specific activities of NADPH:cytochrome c reductase (FAD-dependent), and an M r 145 000 polypeptide as the major protein band. Wild-type enzyme prepared similarly also had the M r 145 000 band, in contrast to the previously reported molecular weight values of 115 000 and 130 000 (Pan, S.S. and Nason, A. (1978) Biochim. Biophys. Acta 523, 297–313). After polyacrylamide gel electrophoresis under non-dissociating conditions, all the enzymatic activity present in that preparation was found associated with a single protein band. When this band was reelectrophoresed in SDS-polyacrylamide gels, a single polypeptide was detected with an apparent molecular weight of 145 000. Evidence is presented that the nit-1 enzyme is the apoenzyme of nitrate reductase and that the wild-type enzyme is a homodimer of 145 000 subunits.