Opposite behavior of two isozymes when refolding in the presence of non-ionic detergents

GroEL has a greater affinity for the mitochondrial isozyme (mAAT) of aspart ate aminotransferase than for its cytosolic counterpart (cAAT) (Mattingly J R Jr, Iriarte A, Martinez-Carrion M, 1995, J Biol Chern 270:1138-1148), two proteins that share a high degree of sequence similarity and an almost ide ntical spatial structure. The effect of detergents on the refolding of thes e large, dimeric isozymes parallels this difference in behavior. The presen ce of non-ionic detergents such as Triton X-100 or lubrol at concentrations above their critical micelle concentration (CMC) interferes with reactivat ion of mAAT unfolded in guanidinium chloride but increases the yield of cAA T refolding at low temperatures. The inhibitory effect of detergents on the reactivation of mAAT decreases progressively as the addition of detergents is delayed after starting the refolding reaction. The rate of disappearanc e of the species with affinity for binding detergents coincides with the sl owest of the two rate-limiting steps detected in the refolding pathway of m AAT. Limited proteolysis studies indicate that the overall structure of the detergent-bound mAAT resembles that of the protein in a complex with GroEL . The mAAT folding intermediates trapped in the presence of detergents can resume reactivation either upon dilution of the detergent below its CMC or by adding beta-cyclodextrin. Thus, isolation of otherwise transient product ive folding intermediates for further characterization is possible through the use of detergents.