We describe here an in vitro effect of lithocholic acid (LA), a secondary, hydrophobic bile acid, on the rate of polymerization of mutant, Z and wild-type, M alpha-1-antitrypsin (AAT). Using thioflavine T fluorescence and turbidity assays we demonstrated that the rate of aggregation for the Z AAT in the presence of LA at a molar ratio of 1:5 AAT to LA, in Tris-buffered saline, pH 7.4, is at least twice that of the Z protein alone or the M variant with and without LA. Also, Z AAT incubated for 48 h at room temperature had more than 50% diminished antielastase activity, while M AAT had only a 25% reduction in activity. Analysis of the AAT and AAT-LA samples after cleavage with pancreatic elastase by SDS-PAGE 10% gels showed that interaction between Z or M AAT and LA abolishes their ability to form SDS stable complexes with an enzyme and both of these forms of AAT showed elastase substrate behavior. Furthermore, Z as well as M AAT incubated with LA at 41 degrees C and cleaved with elastase showed only 80 to 60% increased thermal stability compared to 100% stabilization for the cleaved AAT alone in the absence of LA. These observations suggest that a rearrangement of the AAT molecule as a result of interactions with LA increases aggregation of AAT and diminishes its inhibitory activity.
Copyright 1998 Academic Press.