Posttranslational folding of alpha 1-inhibitor 3. Evidence for a compaction process.
alpha 1-inhibitor 3 (alpha 1 I3) is a rodent-specific proteinase inhibitor of about 190 kDa belonging to the alpha 2-macroglobulin family. It consists of five globular domains, three of which are connected by disulfide bridges, and contains an intramolecular thiol ester which can react with attacking proteinases. To explore the folding of newly synthesized alpha 1 I3, we have used rat hepatocytes and pulsechase experiments. In one of the analyses, the radiolabeled protein was isolated from cell lysates by immunoprecipitation and its Asp-Pro bonds cleaved by treatment with formic acid. The size of the major fragment, as assessed by electrophoresis under nonreducing conditions, was found to increase from 100 to 150 kDa upon the chasing. This result, together with knowledge of the positions of the cleavage sites and the disulfide arrangement, indicates that one of the interdomain disulfide bonds is formed after the synthesis of the polypeptide. Analysis of the same material by limited proteolysis and by velocity centrifugation showed that the folded regions became larger and that the protein became more compact; the thiol ester was found to be formed after these conformational changes. These results suggest that the domains of alpha 1 I3 are only partially developed directly after the synthesis of the polypeptide and that they acquire their final structure as the protein condenses and the domains interact with one another.
Wassler
M
M
Department of Medical and Physiological Chemistry, Biomedical Center University of Uppsala, Sweden.
Esnard
F
F
Fries
E
E
D000209:Acute-Phase Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D002474:Cell-Free System; D002478:Cells, Cultured; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D051381:Rats; D013462:Sulfur Radioisotopes; D014461:Ultracentrifugation
D000209:Acute-Phase Proteins; D004220:Disulfides; D010446:Peptide Fragments; D013462:Sulfur Radioisotopes; C017894:alpha 1-inhibitor 3; D008715:Methionine