In vitro amyloid fibril formation from alpha 1-antitrypsin.
We have previously shown that the interaction between alpha 1-antitrypsin (AAT) and lithocholic acid (LA) results in changes of AAT properties leading to its polymerization and inactivation. To define the structural rearrangements of AAT induced by such interaction, we studied the in vitro binding between AAT and LA at molar ratio 1:5 for varying time intervals at a physiological pH. Complex formation was shown by electrophoretic techniques and autoradiography. Studies of the AAT in complex with LA by using far-UV spectra circular dichroism and fluorescence measurements indicated an increase of beta-structure of AAT and pronounced changes in surroundings of the chromophores. In addition, complexed AAT showed increase in thermal stability, compatible with that after proteolytic cleavage. Characterization of the AAT-LA complexes by Congo red binding, polarization and negative staining electron microscopy provided clear evidence that AAT, under chosen experimental conditions, can self-assemble into amyloid fibrils, compatible with accepted models of fibrillar structures. This propensity of AAT to form stable beta-structures in a hydrophobic surrounding may contribute to improved characterization of various amyloid deposits occurring in vivo and be a guide for understanding details of structure-function relationships in the intact AAT-molecule.
Janciauskiene
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Lund University, Department of Medicine, University Hospital of Malmö, Sweden.
Carlemalm
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Eriksson
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D000682:Amyloid / Q000096:biosynthesis* / Q000737:chemistry; D001345:Autoradiography; D015153:Blotting, Western; D002942:Circular Dichroism; D003224:Congo Red; D004587:Electrophoresis, Agar Gel; D019791:Guanidine; D006146:Guanidines / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D008095:Lithocholic Acid / Q000378:metabolism; D008854:Microscopy, Electron; D009454:Neurofibrils / Q000378:metabolism*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000378:metabolism*
D000682:Amyloid; D006146:Guanidines; D000515:alpha 1-Antitrypsin; D003224:Congo Red; D008095:Lithocholic Acid; D019791:Guanidine