Expression of human Cu/Zn superoxide dismutase (SOD) with activity comparable to the human erythrocyte enzyme was achieved in Escherichwa coli by using a vector containing a thermoinducible X PL promoter and a ,8-lactamase-derived ribosomosal binding site. The recombinant human SOD was found in the cytosol of disrupted bacteria and represented >10% of the total bacterial protein. The enzyme was purified to homogeneity by salt precipitation, gel filtration chromatography, and ion exchange chromatography. The active enzyme was obtained in high yield only when 1 mol of copper and 1 mol of zinc were incorporated into each mol of subunit during bacterial growth or by reconstitution of the apoenzyme. Human Cu/Zn SOD produced in bacteria has an apparent subunit molecular mass of 19 kDa on NaDodSO4/polyacrylamide gels. The native enzyme behaves as a dimer of 32 kDa as determined by gel filtration. Sequence analysis of the NH2 terminus revealed that the first 14 amino acids corresponded to authentic human SOD except that the NH2-terminal alanine was not acetylated. Thus, the bacterial processing system readily removes the NH2-terminal methionine residue from recombinant human SOD. and thus provide a defense against oxygen toxicity. There are three known forms of SOD that contain different metalsnamely, iron, manganese, or both copper and zinc. All of these catalyze the same reaction with high efficiency, and all operate by a similar mechanism in which the metal is the catalytic factor in the active site. These enzymes fall into several evolutionary groups. The Fe-containing SODs are found primarily in prokaryotic cells, while Cu/Zn SODs have been demonstrated in all higher eukaryotes. Mn SODs exist throughout the phylogenetic range, from microorganisms to humans (reviewed in ref. 4).Since every biological macromolecule can serve as a target for the damaging action of the abundant oxygen radical, interest has evolved in the therapeutic potential of SOD. A wide range of clinical applications has been suggested. These include prevention of oncogenesis and tumor promotion, reduction of the cytotoxic and cardiotoxic effects of anticancer drugs (5), anti-inflammatory action (6), and protection against reperfusion damage of ischemic tissues (7). In addition, there is much interest in studying the effects of SOD on the aging process (8).The exploration of the therapeutic potential of human SOD has been hindered by its limited availability. The enzyme is a dimeric metalloprotein composed of identical noncovalently linked subunits, each of 16 kDa and containing one atom of copper and one atom of zinc (9). Each subunit is composed of 153 amino acids of known sequence (10, 11). Recently, a cDNA clone containing the entire coding region of human SOD was isolated and sequenced (12, 13). The gene coding for human SOD was introduced by us into an efficient bacterial expression vector. We report here the production of gram quantities of enzymatically active human Cu/Zn SOD in Escherichia coli. MATERIALS AND METHODSBacterial Gro...

Mutagenesis of Azospirillum brasilense with nitrosoguanidine and selection on ethylenediamine yielded prototrophs which fixed nitrogen in the presence of ammonia. Nitrogenase activity in mutant strains exceeded that of the wild type three-to sixfold. The same mutants were also constitutive for histidine transport. Enzyme activities involved in ammonia assimilation were not affected by the mutation. The data suggest that the mutation occurred at a site which regulates nif and histidine tra'nsport functions.

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