Spectroscopic and Biochemical Characterization of Human Membrane-associated Progesterone Receptor Component 1 (PGRMC1_ Human)

• Main Authors: Hsin-Hui Wu, 吳信輝
• Other Authors: Kelvin Huang-Chou Chen
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/bu5894

碩士 === 國立屏東教育大學 === 應用化學暨生命科學系 === 97 === Human progesterone membrane-associated component 1 (PGRMC1_Human) protein belongs to a highly conserved class of putative membrane-associated progesterone binding proteins (MAPR), which Dap1p and inner zone antigen (IZA), the rat homologue of mouse progesterone receptor membrane-associated component 1 protein (mPGRMC1p), recently being reported to bind heme. While primary structure analysis reveals similarities to the cytochrome b5 motif, neither of the two axial histidines responsible for ligation to the heme is present in any of the MAPR proteins. In spite of its name, PGRMC1_Human shares homology with cytochrome b5-related proteins rather than hormone receptors, and heme binding is the sole biochemical activity of PGRMC1_Human. The only known biochemical function of PGRMC1_Human is binding to heme and inducing anti-apoptic signaling or metabolized steroid. So Heme of PGRMC1_Human can be fully loaded is important. The PGRMC1_Human gene was cloned, and the gene product was over-expressed in Escherichia coli. An addition of the heme precursor 5-aminolevulinic acid (ALA) to the medium increased heme content of PGRMC1_Human. The heme loading percentage for PGRMC1_Human can be increased to 87% form trace. In the thesis, CD, UV-Vis, small angle X-ray scattering (SAXS) and general biochemical methods have been used to characterize the nature of heme binding in PGRMC1_Human. CD indicate a well –ordered structure, suggesting the different level of heme loading is probably not due to improperly folded protein. UV-Vis spectrum confirmed a high-spin Fe (Ⅲ) for PGRMC1_Human, indicating one axial amino acid ligand, in contrast to the low-spin Fe (Ⅱ) of cytochrome b5.