Monoselenophosphate (SeP) was identified previously and shown to be the biological selenium donor in prokaryotes for specific selenocysteine formation and insertion into proteins and for the conversion of 2- thiouridine to 2-selenouridine residues in tRNAs. Using antibodies raised to Escherichia coli selenophosphate synthetase, the enzyme was detected in various rat tissues and other bacteria by immunoblotting indicating a general biological role for SeP. High levels of SeP synthetase were found in Methanococcus vannielii and the enzyme was purified from this source. The amino acid sequence of the first 25 N- terminal residues of the M. vannielii protein differs from the deduced sequence of the corresponding region of the E. coli enzyme and also from human and murine SeP gene sequences determined in other laboratories. A new 75Se-labeled protein detected in extracts of M. vannielii was obtained as a by-product of the SeP synthetase isolation procedure. This approximately 42 kDa protein contains selenocysteine. The N- terminal amino acid sequence (residues 1-63) was determined and found to be unlike sequences of known proteins. Alternative assays for selenophosphate synthetase activity were developed. The reaction catalyzed by the enzyme (ATP + HSeNa = AMP + SeP + Pi) has been followed by measuring the amount of [14C]AMP formed from [14C]ATP in the presence of selenide after separation of nucleotides by TLC. Using [gamma 32P]ATP the [32P]SeP product is determined after conversion to [32P]Pi by oxidation with iodine and removal of residual labeled ATP on charcoal. 32P in the charcoal filtrate is measured. In a non-radioactivity assay (Song Liu's report) selenide-dependent AMP formation from ATP is estimated by measuring ammonia liberated from the AMP by AMP deaminase. SeP is extremely oxygen labile and difficult to measure directly except by 31P NMR.
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