Association-dissociation behavior and subunit structure of heat-released nitrate reductase from Escherichia coli.
Nitrate reductase, released from the membrane fraction of Escherichia coli by a neutral heat treatment, was purified to homogeneity by gel filtration chromatography. The purified enzyme behaved as an associating-dissociating system, exhibiting concentration-dependent sedimentation constants which ranged from 24 S at high concentrations in the ultracentrifuge down to 10 S at low concentrations in sucrose gradients. The molecular weight determined at high concentrations by sedimentation equilibrium was 880,000 +/- 30,000. Large and small enzyme species were detected on polyacrylamide disc gels run with diluted samples of enzyme. The ratio of the two species was concentration-dependent and the dissociation was reversible. The purified enzyme appeared to be homogeneous and monodisperse in the ultracentrifuge, on sucrose gradients, during gel filtration on Bio-Gel and on polyacrylamide gels, but it had a heterogeneous subunit composition as determined by sodium dodecyl sulfate gel electrophoresis. Enzyme species with different subunit compositions were partially resolved by gel filtration. The fractions with the highest specific activity contained subunits of 150,000 and 55,000 daltons in a ratio of approximately 1:1. Other fractions contained reduced amounts of the 55,000-dalton subunit and correspondingly increased amounts of 51,000-, 45,000-, and 10,000-dalton subunits, suggesting that the heterogeneity was the result of proteolytic degradation of the 55,000-dalton subunit. The enzyme contained approximately 12 non-heme irons, 12 acid-labile sulfides, 24 cysteine residues, and 1 molybdenum per 200,000 daltons.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D007501:Iron / Q000032:analysis; D046911:Macromolecular Substances; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis; D009565:Nitrate Reductases / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013440:Sulfides / Q000032:analysis
D000596:Amino Acids; D046911:Macromolecular Substances; D013440:Sulfides; D008982:Molybdenum; D007501:Iron; D009565:Nitrate Reductases