Purification and immunological studies of selenoprotein A of the clostridial glycine reductase complex.
One of the essential catalytic components of the clostridial glycine reductase complex is a selenium-containing protein, selenoprotein A. An improved method for the purification of selenoprotein A has been developed that yields milligram quantities of the protein in a few relatively simple steps. Ferredoxin and rubredoxin can be recovered in pure form as by-products of the procedure. The high resolving capabilities of an anion exchange high pressure liquid chromatography step were exploited in these purification protocols. For effective antibody production, the antigenicity of selenoprotein A was increased by coupling the pure protein via its sulfhydryl/selenol group(s) to the amino groups of bovine serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester. The high titer sheep antisera that were elicited were used to study the mechanisms of selenium incorporation into selenoprotein A. Immunoblot analysis of sodium dodecyl sulfate-polyacrylamide gels was employed to monitor the synthesis of selenoprotein A by Clostridium sticklandii as a function of growth conditions. Cells grown under limiting conditions (1 nM) of selenium contained only 1-2% of normal levels of active selenoprotein A and no precursor forms were detected after DEAE-high pressure liquid chromatography fractionation of extracts. Conversely, cells grown with optimal (1 microM) selenium levels contained maximal amounts of seleno-protein A and expressed full glycine reductase activity.
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D003013:Clostridium / Q000378:metabolism*; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D011506:Proteins; D012643:Selenium / Q000378:metabolism; D017279:Selenocysteine; D051140:Selenoproteins
D002352:Carrier Proteins; D009097:Multienzyme Complexes; D011506:Proteins; D051140:Selenoproteins; D017279:Selenocysteine; D000594:Amino Acid Oxidoreductases; C020815:glycine reductase; D012643:Selenium; D003545:Cysteine