The clostridial glycine reductase complex requires a selenium-containing protein, selenoprotein A, as one of its essential components. This enzyme complex catalyzes the reductive deamination of glycine, which is coupled to the esterification of orthophosphate resulting in the formation of ATP. A unique selenocysteine (SEC)-containing peptide from selenoprotein A has been purified and subjected to automated Edman degradation. The sequence of the peptide is VAL-SEC-THR-ALA-ALA-GLY-ALA-MET-ASP-LEU-GLU-ASN-GLN-LYS. In addition to selenocysteine, the protein also contains two other cysteine residues. The mechanism by which Se is incorporated into a specific residue of the polypeptide chain remains unknown. In order to address this question, antibodies to selenoprotein A have been raised. To increase its antigenicity, selenoprotein A (Mr = 12000) was coupled via its sulfhydryl/selenol group(s) to the amino groups of serum albumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester. Using dot-immuno assays and immunoblotting of SDS-polyacrylamide gels, the synthesis of selenoprotein A by Clostridium sticklandii under various growth conditions was examined. Cells grown under limiting conditions (l nM Se) have 50-100 times lower amounts of active selenoprotein A and no precursor forms were detected after DEAE-HPLC fractionation of extracts. Active and inactive forms of selenoprotein A (i.e., lacking Se) have been found in preparations from cells grown with normal Se levels (l MuM). In a collaborative effort with Dr. August Bock from Munich, studies on the structure of a cloned selenoprotein, formate dehdyrogenase, have been initiated. By comparing information obtained by sequencing both the gene and the peptide, the codon and the precursor amino acid will be determined. An alternative amino acid analysis technique, using phenylisothiocyanate, has been introduced to the laboratory. The procedure can be used to quantitate both primary and secondary amines. An added advantage is that selenium amino acids are also easily identified and quantitated.
