Secretion, surface localization, turnover, and steady state expression of protein disulfide isomerase in rat hepatocytes.
Protein disulfide isomerase in isolated rat hepatocytes was present at a concentration of 7 micrograms/mg cell protein, representing a approximately 2-fold enrichment compared to isolated hepatic non-parenchymal cells. Though localized mainly in microsomal fractions of hepatocytes, direct immunofluorescence and cell surface radioiodination followed by immunoprecipitation revealed the presence of M(r) 57,000 disulfide isomerase at the cell surface. Electrostatic interaction of the protein with the cell surface was suggested by susceptibility to carbonate washing. Metabolic radiolabeling and immunoprecipitation studies also indicated that some of the newly synthesized M(r) 57,000 disulfide isomerase was secreted. Treatment of cells with colchicine markedly reduced the recovery of disulfide isomerase from the media, indicating microtubular-directed secretion of the protein. Partial staphlococcal V8 proteolytic digestion of the secreted protein revealed a peptide pattern similar to that of the cellular protein. Immunoprecipitation with antibody specific to the -KDEL peptide retention sequence confirmed the presence of this sequence in the secreted protein. Studies of the turnover of disulfide isomerase revealed a half-life of approximately 96 h. Treatment of cells with tunicamycin or heat shock resulted in an increased recovery of newly synthesized disulfide isomerase from cell lysates but diminished recovery from the media. The secretion and cell surface distribution of disulfide isomerase in hepatocytes may be important for the pathogenesis of immune mediated liver injury.
Terada
K
K
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Manchikalapudi
P
P
Noiva
R
R
Jauregui
H O
HO
Stockert
R J
RJ
Schilsky
M L
ML
D000818:Animals; D015153:Blotting, Western; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D007150:Immunohistochemistry; D007535:Isomerases / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008715:Methionine / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology; D008970:Molecular Weight; D009842:Oligopeptides / Q000032:analysis; D010449:Peptide Mapping; D019704:Protein Disulfide-Isomerases; D021382:Protein Sorting Signals; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000201:enzymology; D013462:Sulfur Radioisotopes; D013997:Time Factors
D009842:Oligopeptides; D021382:Protein Sorting Signals; D013462:Sulfur Radioisotopes; C073762:lysyl-aspartyl-glutamyl-leucine; D008715:Methionine; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D003545:Cysteine