Comigration of Two Autophagosome-Associated Dehydrogenases on Two-Dimensional Polyacrylamide Gels
| العنوان: | Comigration of Two Autophagosome-Associated Dehydrogenases on Two-Dimensional Polyacrylamide Gels |
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| المؤلفون: | Monica Fengsrud, Marianne Lunde Sneve, Per Ottar Seglen, Anders Øverbye |
| المصدر: | Autophagy. 1:157-162 |
| بيانات النشر: | Informa UK Limited, 2005. |
| سنة النشر: | 2005 |
| مصطلحات موضوعية: | Male, Proteomics, Autophagosome, Molecular Sequence Data, Dehydrogenase, In Vitro Techniques, Silver stain, 3-alpha-Hydroxysteroid Dehydrogenase (B-Specific), Phagosomes, Organelle, Autophagy, Animals, Electrophoresis, Gel, Two-Dimensional, Amino Acid Sequence, Rats, Wistar, Molecular Biology, Glyceraldehyde 3-phosphate dehydrogenase, biology, Intracellular Membranes, Cell Biology, Molecular biology, Rats, Isoenzymes, Cytosol, Biochemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Hepatocytes, biology.protein, Lysosomes, Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+), Immunostaining |
| الوصف: | Immunoblotting of two-dimensional polyacrylamide gels (pI 3-10) revealed six cytosolic molecular forms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in rat hepatocytes. Two of the four full-length (approximately 37 kDa) forms exhibited some binding to sedimentable cellular elements (but not to mitochondria), whereas one full-length and two short (approximately 35 kDa) forms selectively bound to the membranes of autophagosomes and lysosomes. Tryptic fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmed the identity of the major full-length forms as GAPDH, but attempts to identify the major short form consistently suggested that this spot represented a different enzyme, 3-alpha-hydroxysteroid dehydrogenase (3alphaHSD). Silver staining indicated that this 3alphaHSD form selectively bound to autophagosomal and lysosomal membranes. Immunoblotting of more focused 2D gels (pI 6-9) with an antibody raised against 3alphaHSD demonstrated immunostaining of four 3alphaHSD forms with masses of about 35 kDa. Autophagosomal membrane preparations were highly and selectively enriched with respect to all of these 3alphaHSD forms. One of them comigrated with the major short form of GAPDH, accounting for the paradoxical mass spectrometric identification of 3alphaHSD from this spot. Proteomic analysis by a combination of immunological and mass spectrometric identification methods was thus capable of resolving two comigrating dehydrogenases selectively associated with autophagic organelles. |
| تدمد: | 1554-8635 1554-8627 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ca6a8da4dd2990056ffdde4c6ad79309Test https://doi.org/10.4161/auto.1.3.2037Test |
| رقم الانضمام: | edsair.doi.dedup.....ca6a8da4dd2990056ffdde4c6ad79309 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15548635 15548627 |
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