IMMUNOLOGICAL APPROACH TO SUBUNIT COMPOSITION OF FERREDOXIN-NITRITE REDUCTASE FROM CHLAMYDOMONAS-REINHARDTII

Antibodies raised against the gel-purified denatured M(r) 63000 polype ptide of the ferredoxin-nitrite reductase from Chlamydomonas reinhardt ii have been used to further investigate the existence of a M(r) 24000 -25000 subunit previously reported to be present in the C. reinhardtii and spinach enzymes. A combination of immunoblotting, immunoprecipita tion and immunoaffinity purification techniques led us to reach the co nclusion that native ferredoxin-nitrite reductase from C. reinhardtii is only composed of a single polypeptide of M(r) 63000. Immunoaffinity purification of C. reinhardtii ferredoxin-nitrite reductase enables t o purify this enzyme 715-fold, giving a specific activity of 193 U/mg protein, Additional experimental evidence is also shown against the pr eviously suggested involvement of a M(r) 25000 subunit in the interact ion of this enzyme with ferredoxin. On the one hand, the ratio of ferr edoxin-dependent to methyl viologen-dependent nitrite reductase activi ties is very significantly affected by salts, but does not correlate w ith the presence of the putative M(r) 25000 component. Secondly, only the M(r) 63000 polypeptide of C. reinhardtii ferredoxin-nitrite reduct ase has been found to cross-react in immunoblot with antibodies raised against the ferredoxin-glutamate synthase from the same organism. Alt hough C. reinhardtii ferredoxin-nitrite reductase is a monomeric enzym e, evidence is presented that proteolytic fragments of the M(r) 63000 Fd-NiR polypeptide, with M(r) 40000 and M(r) 25000, are a common featu re in crude extracts from C. reinhardtii, which could partially explai n the confusion that has arisen with regard to the quaternary structur e of this enzyme. Comparative immunoblots of extracts from different p hotosynthetic organisms indicate that the antibodies raised against C. reinhardtii ferredoxin-nitrite reductase recognize very well the M(r) 63000 polypeptide and similar proteolytic fragments from the green al gae Monoraphidium braunii and Chlorella fusca. Some extent of recognit ion has also been found with a M(r) 63000 polypeptide from spinach and a M(r) 54000 polypeptide from cyanobacteria Synechococcus sp, PCC 794 2 and Anabaena sp. PCC 7120. The conclusion found in this paper of a m onomeric quaternary structure for the ferredoxin-nitrite reductase fro m C. reinhardtii, agrees also with the results recently obtained by ot her authors with regard to the spinach enzyme.