AN ELECTROPHORETIC STUDY OF THE THERMAL-DEPENDENT AND REDUCTANT-DEPENDENT AGGREGATION OF THE 27 KDA COMPONENT OF AMMONIA MONOOXYGENASE FROMNITROSOMONAS-EUROPAEA

Standard protocols for sample preparation for sodium dodecyl sulfate-p olyacrylamide gel electrophoresis (SDS-PAGE) typically involve the com bined use of heat and a reductant to fully disrupt protein-protein int eractions and allow for constant ratios of SDS-binding to individual p olypeptides. However, C-14-labeled forms of the membrane-bound, active -site-containing 27 kDa polypeptide of ammonia monooxygenase from Nitr osomonas europaea undergo an aggregation reaction when cells or membra nes are heated in the Presence of SDS-PAGE sample buffer. The aggregat e produced after heating at 100-degrees-C is a soluble complex which f ails to enter the stacking gel in discontinuous SDS-PAGE gels.The exte nt of the aggregation reaction is dependent on the temperature of samp le preparation, and the reaction exhibits first-order kinetics at 65-d egrees-C and 100-degrees-C (rates constants = 0.07 and 0.35 min-1, res pectively). The rate of the aggregation reaction is further dependent on the concentration of reductant used in the sample buffer. However, the concentration of S DS does not significantly affect the rate of ag gregation. The aggregated form of the 27 kDA polypeptide can be isolat ed by gel-permeation chromatography in the presence of SDS. The aggreg ated protein can also be returned to the monomeric state by incubation at high pH in the presence of SDS. The aggregation reaction also occu rs with (C2H2)-C-14-labeled polypeptides in other species of autotroph ic nitrifiers and a methanotrophic bacterium which expresses the parti culate form of methane monooxygenase. We conclude that strongly hydrop hobic amino acid sequences present in ammonia monooxygenase are respon sible for the aggregation phenomenon.