An electrophoretic study of the thermal- and reductant-dependent aggregation of the 27 kDa component of ammonia monooxygenase from Nitrosomonas europaea.
Standard protocols for sample preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) typically involve the combined use of heat and a reductant to fully disrupt protein-protein interactions and allow for constant ratios of SDS-binding to individual polypeptides. However, 14C-labeled forms of the membrane-bound, active-site-containing 27 kDa polypeptide of ammonia monooxygenase from Nitrosomonas europaea undergo an aggregation reaction when cells or membranes are heated in the presence of SDS-PAGE sample buffer. The aggregate produced after heating at 100 degrees C is a soluble complex which fails to enter the stacking gel in discontinuous SDS-PAGE gels. The extent of the aggregation reaction is dependent on the temperature of sample preparation, and the reaction exhibits first-order kinetics at 65 degrees C and 100 degrees C (rates constants = 0.07 and 0.35 min-1, respectively). The rate of the aggregation reaction is further dependent on the concentration of reductant used in the sample buffer. However, the concentration of SDS does not significantly affect the rate of aggregation. The aggregated form of the 27 kDA polypeptide can be isolated by gel-permeation chromatography in the presence of SDS. The aggregated protein can also be returned to the monomeric state by incubation at high pH in the presence of SDS. The aggregation reaction also occurs with 14C2H2-labeled polypeptides in other species of autotrophic nitrifiers and a methanotrophic bacterium which expresses the particulate form of methane monooxygenase. We conclude that strongly hydrophobic amino acid sequences present in ammonia monooxygenase are responsible for the aggregation phenomenon.
Hyman
M R
MR
Laboratory for Nitrogen Fixation Research, Oregon State University, Corvallis.
Arp
D J
DJ
D002245:Carbon Dioxide / Q000378:metabolism; D002250:Carbon Radioisotopes; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D009606:Nitrosomonas / Q000201:enzymology*; D010088:Oxidoreductases / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D014241:Trichloroethylene / Q000378:metabolism
D002250:Carbon Radioisotopes; D046911:Macromolecular Substances; D002245:Carbon Dioxide; D014241:Trichloroethylene; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D010088:Oxidoreductases; C045582:ammonia monooxygenase