PURIFICATION AND CHARACTERIZATION OF THE MEMBRANE-BOUND NITRATE REDUCTASE ISOENZYMES OF BRADYRHIZOBIUM-JAPONICUM

Two respiratory membrane-bound nitrate reductase (NR) isoenzymes, NR(I ) and NR(II), have been purified for the first time from one single mi croorganism, Triton X-100-solubilized NRs mere purified by a three-ste p procedure of differential centrifugation, Q-Sepharose chromatography , and gel filtration on Sephacryl S-300. Both isoenzymes were purified to homogeneity by the criteria of NR activity staining in polyacrylam ide gels run under non-denaturating conditions and coincident staining of the protein band by silver nitrate. NR(I) is composed of three sub units of 116 kDa, 68 kDa, and 56 kDa, whereas NR(II) is composed of fo ur subunits of 116 kDa, 68 kDa, 59 kDa, and 56 kDa. The 116-kDa subuni t of NR(I) and the 59-kDa subunit of NR(II) exhibited immunological cr oss-reactivity with the respiratory NR of Pseudomonas stutzeri strain ZoBell.