Production of gastric intrinsic factor, transcobalamin, and haptocorrin in opossum kidney cells
| Autor: | Marilyn M. Gordon, David H. Alpers, Nancy A. Brada, Jipu Wen, Jian-su Shao |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Intrinsic Factor
medicine.medical_specialty Haptocorrin Didelphis Physiology Blotting Western Kidney Cobalamin Epithelium Kidney Tubules Proximal Rats Sprague-Dawley chemistry.chemical_compound Transcobalamin Opossum Internal medicine medicine Animals Secretion RNA Messenger Cells Cultured Transcobalamins Microvilli biology Reverse Transcriptase Polymerase Chain Reaction urogenital system Epithelial Cells Opossums biology.organism_classification Immunohistochemistry Rats Vitamin B 12 Endocrinology medicine.anatomical_structure chemistry Gastric Mucosa Electrophoresis Polyacrylamide Gel |
| Zdroj: | American Journal of Physiology-Renal Physiology. 279:F1006-F1013 |
| ISSN: | 1522-1466 1931-857X |
| DOI: | 10.1152/ajprenal.2000.279.6.f1006 |
| Popis: | Opossum kidney epithelial cells were shown previously to synthesize and secrete two cobalamin (Cbl)-binding proteins, presumed to be haptocorrin (Hc) and transcobalamin II (TCII). The present study examines the hypothesis that renal tubular cells also produce intrinsic factor (IF), and this production provides an explanation for the presence of IF in urine. By using antisera raised against human IF and against TCII, the presence of TCII was confirmed, and that of IF discovered in the media of opossum kidney (OK) cells in culture. The apparent molecular weight of IF and TCII was 68 and 43 kDa, respectively. Immunoreactivity on Western blot of the putative IF protein was blocked by recombinant human IF. When proteins secreted into the media were separated electrophoretically under nondenaturing conditions after binding with [57Co]Cbl, a broad major band migrated at a relative front independently of recombinant IF or TCII, and probably represents Hc, as the Cbl binding is blocked by cobinamide. Small amounts of bound [57Co]Cbl migrated in the position of both IF and TCII, when cobinamide was present. The presence of IF and TCII in OK cells was confirmed by immunohistology. Specific reactivity for IF (blocked by recombinant IF) was found in proximal tubules of opossum kidney, but not in other portions of the nephron, confirming the ability of anti-human IF antiserum to detect opossum IF. A 732-bp fragment of IF, nearly identical in sequence to rat IF, was isolated by RT-PCR from opossum kidney mRNA, and Western blot confirmed the presence of IF protein. The presence of IF was also documented in rat kidney by isolation of an RT-PCR fragment, immunocytochemistry, and Western blot. IF should be added to the list of renal (proximal) tubular antigens that are shared by other epithelia. |
| Databáze: | OpenAIRE |
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