Purification and characterization of folate binding proteins from rat placenta |
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| Institution: | 1. Department of Pediatrics, Hematology and Oncology, Medical University, Gdansk, Poland;2. Department of Pediatrics Adolescent Young Adults, Institut Curie, Paris, France;3. Pediatric Oncology and Hematology, University Children’s Hospital Erlangen, Germany;4. Clinic of Pediatrics, Municipal Hospital Dortmund, Germany;5. Department of Pediatric Surgery, University of Caen Hospital, France;6. Pediatric Oncology Unit, Fondazione IRCCS Istituto Nazionale Tumori, Milano, Italy;7. Pediatric Surgery Unit, Department of Woman’s and Child’s Health, Padova University Hospital, Padova, Italy;8. Division of Hematology-Oncology, Department of Woman’s and Child’s Health, University Hospital of Padova, Italy;1. Transport Department, School of Civil Engineering, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain;2. Gerencia Municipal de Urbanismo, C/ Iris 11 Bajo, 02005 Albacete, Spain;1. Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan;2. Department of Computer Science, School of Computing, Tokyo Institute of Technology, 4259Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan;3. Emergent Bioengineering Materials Research Team, RIKEN Center for Emergent Matter Science, 2-1Hirosawa, Wako, Saitama, 351-0198, Japan;1. Department of Physiology and Toxicology of Reproduction, Institute of Zoology and Biomedical Research, Jagiellonian University in Krakow, Krakow, Poland;2. Department of Developmental Biology and Invertebrate Morphology, Institute of Zoology and Biomedical Research, Jagiellonian University, Krakow, Poland |
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| Abstract: | Rat placenta contains virtually no unsaturated (i.e., apo-form) folate binding protein. However, by lowering the pH of a solubilized membrane preparation of this tissue to 3.5, the endogenous bound folate was dissociated from the protein and adsorbed to charcoal. The apo-form of the folate binding protein thus obtained was purified by affinity chromatography using pteroylglutamic acid covalently coupled to Sepharose 4B. A single protein band with an apparent Mr of 36 000 was observed by SDS-polyacrylamide gel electrophoresis of the eluate from the affinity matrix. Western blot of this preparation using a rabbit antiserum raised with the affinity eluate also identified a single 36 kDa protein band. However, peptide sequencing of the N-terminal region of the proteins in the affinity eluate established that it contained two homologous proteins. Computer alignment of the first 22 N-terminal amino acids of each rat placental protein with human, bovine milk and mouse folate binding proteins showed 50–64% identical homology and 27% homology when the eight proteins were aligned together. The affinity of both rat proteins is highest for pteroylglutamic acid (Ka = 1.6 − 109 l/mol) lower for N5-methyltetrahydrofolate and substantially lower for N5-formyltetrahydrofolate. In the dose-response range studied there was no apparent affinity for methotrexate. The folate binding proteins could be released from a preparation of placental membranes using phospholipase C indicating that these proteins belong to the class of proteins anchored to the plasma membrane by a glycosyl phosphatidylinositol adduct. |
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