CDNA cloning, overproduction and characterization of rat adrenodoxin reductase

We isolated a full-length cDNA clone for rat adrenodoxin reductase (AdR). T he precursor of rat AdR was predicted to consist of 34 amino-terminal resid ues of extrapeptide for transport into mitochondria and the following 460 r esidues of the mature peptide region. The deduced amino acid sequence was 7 0.8 and 61.8% homologous to those of bovine and human AdRs in the extrapept ide region, respectively, and 88.5% homologous to both the sequences of bov ine and human AdRs in the mature peptide region. The predicted mature form of rat AdR was directly expressed in Escherichia coil, using cDNA, and was purified with a yield of 32 mg/l of culture. The purified recombinant rat A dR showed an absorption spectrum characteristic of a flavoprotein with peak s at 270, 378 and 450 nm and shoulders at 280, 425 and 474 nm. The extincti on coefficient was estimated to be 10.9 mM(-1) cm(-1) at 450 nm. The absorb ance ratio at 270 nm/450 nm was 7.1. From the theta(208) value in the circu lar dichroism spectrum, the a-helix content in the rat AdR was calculated t o be 30%. In NADPH-cytochrome c reductase activity reconstituted with adren odoxin (Ad), the apparent K-m value of rat AdR for NADPH was 0.32 mu M, a v alue significantly lower than that of bovine AdR (1.4 mu M). The rat AdR sh owed a higher affinity to the heterologous redox partner (bovine Ad, K-m= 9 .3 nM) than to the native partner (rat Ad, K-m = 16.7 nM), whereas the affi nity of bovine AdR was slightly higher to the native partner (bovine Ad, K- m = 37.1 nM) than to the heterologous partner (rat Ad, K-m= 46.8 nM). The K -m values showed a reverse correlation to the difference of pi values betwe en the reeler partners. These results indicate that AdR binds to Ad mainly by ionic interaction. (C) 1999 Elsevier Science B.V. All rights reserved.