Purification and identification of selenoprotein P-like protein from bovine serum as a survival-promoting factor for cultured neurons | Posted on:1997-12-17 | Degree:Ph.D | Type:Dissertation | University:University of Miami | Candidate:Yan, Jun | Full Text:PDF | GTID:1461390014482771 | Subject:Biology | Abstract/Summary: | | The neuronal survival-promoting activity in serum is enriched in a 45-80 kD (kiloDalton) gel filtration fraction (Kaufman and Barrett, 1983). We purified from bovine serum a glycoprotein that promotes survival of cultured rat embryonic neurons from different brain regions including septum in a 7-day survival assay. About 10,000-fold purification was achieved using a combination of ammonium sulfate precipitation, metal-chelating chromatography, Cibacron-blue F3GA dye affinity chromatography, ABx ion exchanger and preparative SDS-PAGE. The concentration required for the half-maximal survival effect {dollar}rm (ECsb{lcub}50{rcub}){dollar} is about 10 ng/ml for the final fraction. This protein has an apparent molecular weight of 56 kD on SDS-PAGE. Amino acid sequencing after cyanogen bromide cleavage yielded two sequences, both of which match with regions of deduced sequence of a bovine selenoprotein P-like protein. The cDNA of this molecule was cloned from the cerebellum (Saijoh et al, 1995), but the protein was not purified previously. The bovine selenoprotein P-like protein is homologous to rat and human selenoprotein P (SPP, Burk & Hill, 1994), and is most likely to be the bovine counterpart for SPP. SPP is found in serum but its function is unknown. We generated antibodies against a synthetic peptide matching 16 amino acids of the predicted bovine SPP-like protein sequence. The antibodies partially neutralized the survival activity of a highly purified fraction. Our results suggest that the bovine SPP-like protein contributes to the neuronal survival-promoting activity of the serum. The SPP-like protein might be involved in antioxidative defense and in maintaining selenium homeostasis. | Keywords/Search Tags: | Serum, Protein, Survival, Bovine, SPP, Activity | | Related items |
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