CHLAMYDOMONAS REINHARDTII NITRATE REDUCTASE COMPLEX HAS 105 KDA SUBUNITS IN THE WILD-TYPE STRAIN AND A STRUCTURAL MUTANT

摘要

Reduced bromophenol blue (BPBH) has been characterized as an electron donor for in vitro nitrate reductase NR activity in Chlamydomonas reinhardtii. Assays carried out with crude extracts from C. reinhardtii appeared to be affected by nitrite reductase (NiR) activity. This interference could be eliminated by a short heat treatment which allowed to perform highly sensitive and reliable determinations of NR activity. A method which uses reduced bromophenol blue for NR activity detection after PAGE has been set up. Sensitivity of the assay was much higher than that of the standard assay with benzyl viologen. An identical molecular mass of 210 kDa was determined by gradient native PAGE for the native NR complex from both the structural nitl-305 mutant and the wild type of C. reinhardtii. NR from this mutant was purified to electrophoretic homogeneity. During the purification in the presence of protease inhibitor, an enzymatically active product of proteolytic degradation was accumulated. Native PAGE of the 210 kDa NR showed a single protein band which after SDS PAGE rendered two protein bands of about 105 kDa and 52 kDa. The same two bands were also detected after immunoprecipitation of S-35-labelled NR with a polyclonal antibody raised against the Monoraphidium braunii NR. Our data strongly support that NR from C. reinhardtii is a homodimer of 105 kDa subunits in both the wild type and the structural mutant nitl-305, and that these subunits could be proteolytically cleaved to two halves of 52 kDa. [References: 45]