Selenium is an essential nutrient that occurs as a component of several prokaryotic and eukaryotic proteins, primarily as a selenocysteine moiety. The incorporation of selenocysteine in prokaryotes requires the products of the selA, selB, selC, and selD genes. The selD gene encodes for selenophosphate synthetase which is required for the synthesis of the reactive selenium donor, selenophosphate. Selenophosphate synthetase has been purified and characterized from an overproducing E. coli strain. Based on cDNA sequences recently obtained from another laboratory, it has been proposed that the enzyme from human and mouse may contain selenocysteine at the active site. Therefore, we chose to study the enzyme from calf liver due to availability and ease of upscaling the purification. Polyclonal antibodies raised against the E. coli enzyme were used to assay for the protein. A cross-reacting protein was partially purified using standard purification techniques, and a partial sequence was obtained after N-terminal sequence analysis. However, no enzymatic activity could be detected. In order to determine if selenophosphate synthetase co-purifies with another protein, an affinity column was made with the antibody against E. coli selenophosphate synthetase. However, no detectable proteins could be eluted from the column after incubation with calf liver extracts. Problems with affinity purification of the calf liver enzyme could result from insufficient homology between the E. coli and mammalian enzymes. Therefore, experiments are currently being carried out with Methanococcus vannielli extracts. Based on Western blot analysis, this organism appears to contain larger quantities of the enzyme. Furthermore, as an archaebacteria, the enzyme from this organism may be more homologous to the E. coli enzyme.
