Expression and Release of the a and b Subunits for Human Coagulation Factor XIII in Baby Hamster Kidney (BHK) Cells
| Autor: | Hiroshi Kaetsu, Don Foster, Akitada Ichinose, Teruto Hashiguchi |
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| Rok vydání: | 1996 |
| Předmět: |
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Carbonyl Cyanide m-Chlorophenyl Hydrazone Hot Temperature Protein subunit Blotting Western Enzyme-Linked Immunosorbent Assay Cyclopentanes Biology Transfection Biochemistry Cell Line chemistry.chemical_compound Cricetinae Baby hamster kidney cell Animals Humans Secretion Monensin Molecular Biology Secretory pathway Protein Synthesis Inhibitors Brefeldin A Factor XIII Thrombin General Medicine Molecular biology Recombinant Proteins Molecular Weight chemistry Enzyme Induction Chromatography Gel Cell fractionation 2 4-Dinitrophenol |
| Zdroj: | Journal of Biochemistry. 119:961-969 |
| ISSN: | 0021-924X |
| DOI: | 10.1093/oxfordjournals.jbchem.a021336 |
| Popis: | The a subunit of coagulation factor XIII lacks a hydrophobic signal sequence for secretion from cells, while the b subunit has a typical signal sequence. To determine whether the a subunit can be synthesized and released, expression vectors containing the cDNA for either subunit were transfected into baby hamster kidney (BHK) cells. Western blotting analysis and gel filtration chromatography demonstrated that the recombinant a and b subunits (rXIIIa and rXIIIb) had the same molecular weights and subunit structures (a2, b2, and a2b2) as the native molecules. rXIIIa was enzymatically active when activated by thrombin. Most rXIIIb was secreted as measured by ELISA, while most rXIIIa was detected in the cytosol by subcellular fractionation. Co-expression with rXIIIb in the same cells did not promote the release of rXIIIa. Treatment of the cells with brefeldin A, a potent inhibitor of protein transportation, blocked the secretion of rXIIIb, although it had no effect on the release of rXIIIa. Several drugs and heat stress induced the release of rXIIIa, which correlated directly with that of cytoplasmic lactate dehydrogenase. These results suggest that the a subunit is released from cells as a consequence of cell injury, which is independent of the classical secretory pathway. |
| Databáze: | OpenAIRE |
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