The in vitro effect of lithocholic acid on the polymerization properties of PiZ alpha-1-antitrypsin.
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.
Gerbod
M C
MC
Department of Medicine, Lund University, Malmö, S-20502, Sweden.
Janciauskiene
S
S
Jeppsson
J O
JO
Eriksson
S
S
D052160:Benzothiazoles; D004795:Enzyme Stability / Q000187:drug effects; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008095:Lithocholic Acid / Q000494:pharmacology*; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D013696:Temperature; D013844:Thiazoles / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism*
D052160:Benzothiazoles; D011480:Protease Inhibitors; C043392:SERPINA1 protein, human; D013844:Thiazoles; D000515:alpha 1-Antitrypsin; C009462:thioflavin T; D008095:Lithocholic Acid; D010196:Pancreatic Elastase