Purification from bovine serum of a survival-promoting factor for cultured central neurons and its identification as selenoprotein-P.
We purified from bovine serum a glycoprotein that promotes the survival of rat embryonic neurons cultured from septum and other brain regions. A 40,000-fold purification was achieved by using a combination of ammonium sulfate precipitation, Zn2+ affinity chromatography, Cibacron blue 3-GA dye affinity chromatography, ABx ion exchange chromatography, and preparative PAGE. The active protein had an apparent molecular weight of 50-60 kDa. The concentration required for half-maximal survival (EC50) was 12 ng/ml ( approximately 200 pM) for the final fraction. Amino acid sequencing after cyanogen bromide cleavage yielded two sequences that are homologous to regions of deduced sequence of the selenoprotein-P (SPP) family in bovine, rat, and human. Antibodies against a synthetic peptide within the bovine SPP sequence immunoprecipitated and inhibited the survival-promoting activity of a partially purified serum fraction. The purified protein supported neuronal survival more effectively than inorganic selenium. These results suggest that SPP or an SPP-like protein contributes to the neuronal survival-promoting activity of serum.
Yan
J
J
Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101, USA.
Barrett
J N
JN
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001798:Blood Proteins / Q000302:isolation & purification; D001921:Brain / Q000166:cytology / Q000187:drug effects; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006801:Humans; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012643:Selenium / Q000494:pharmacology; D051149:Selenoprotein P; D051140:Selenoproteins; D012688:Septum Pellucidum / Q000166:cytology / Q000187:drug effects; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D001798:Blood Proteins; D011506:Proteins; D051149:Selenoprotein P; D051140:Selenoproteins; D012643:Selenium