Alex Shrift

Reduction of dl-selenocystine and isolation of l-seleoncysteine

Cystine, selenocytsine, and several analogs were reduced by dithiothreitol (DTT), beta-mercaptoethanol (ME) and sodium borohydride (NaBH4). DTT was the most effective; DTT to cystine ratios from 10 to 80 were equally effective. With selenocysteine, however, absorption was considerably reduced at all ratios. Selenocysteine was identified as the reduction product by reaction with Gaitondes reagent, comparison of absorption spectra, paper chromatograhy, utilization by cysteinyl-tRNA synthetase fro Paracoccus denitrificans and Vigna radiata, changes in solubility after DTT treatment, and comparison of infrared spectra. During the ATP-PPi exchange assay, DTT and ME convert cysteine and selenocysteine derivatives to cysteine and selenocysteine which serve as substrates for cysteinyl-tRNA synthetase.

Uptake of selenium-75 by PHA-stimulated lymphocytes : Effect on glutathione peroxidase.

To determine which of a variety of inorganic and organic selenium compounds could best stimulate glutathione peroxidase, human lymphocytes were cultured with a number of selenium sources. The phytohemagglutinin-transformed lymphocytes were cultured in the presence of75Se bound to serum proteins (25% v/v) or 10−7M concentrations of [75Se]-selenite, [75Se]-selenate, [75Se]-selenocystine, and [75Se]-selenomethionine. Organic forms of selenium were taken up in preference to inorganic forms. Control cultures, from which exogenous selenium had been omitted, showed a decreased level of glutathione peroxidase activity at the end of a 4 d culture period. Of the Se sources tested, [75Se]-selenocystine and [75Se]-labeled fetal calf serum proteins increased enzyme activity significantly, 79 and 47%, respectively, but selenite increased activity only by 7%. These results indicate that selenium from the two organic sources is most readily available for glutathione peroxidase synthesis.

Use of selenite, selenide, and selenocysteine for the synthesis of formate dehydrogenase by a cysteine-requiring mutant ofEscherichia coli K-12

The forms of Se in the Se-dependent enzyme formate dehydrogenase is known to be selenocysteine, but the way this amino acid enters the polypeptide chain has not been established. Through the use of a cysteine-requiring mutant ofEscherichia coli K-12 that could also grow in the presence of glutathione, we were able to study the effect of selenite, selenide, andl-selenocysteine, each at a concentration of 0.1 μM, on the synthesis of formate dehydrogenase. The three forms of Se served equally well for inducing formate dehydrogenase activity, measured by dichlorophenol-indophenol reduction mediated by phenazine methosulfate. It is known that selenite can be reduced to selenide by the action of glutathione reductase, present inE. coli, and that selenocysteine is converted to elemental Se by the action of selenocysteine lyase, also present in the mutant. Elemental Se is then reduced nonenzymatically to hydrogen selenide. The conversion of both selenite and selenocysteine to selenide and the ability of each form of Se to induce the synthesis of equal levels of formate dehydrogenase suggest that the incorporation of Se into formate dehydrogenase is accomplished by a posttranslational mechanism.

Evidence for the biosynthesis of selenobiotin

Chemically synthesized selenobiotin is, like sulfur biotin, able to bind to avidin. This observation was used to help identify biologically synthesized selenobiotin as an excretion product of Phycomyces blakesleeanus. The identification of [75Se]selenobiotin was based on the highly specific binding of biotin to avidin used as an affinity ligand to Sepharose, on its release from the complex by proteolytic treatment, and its chromatographic behavior relative to [14C]biotin standards. These results represent the first evidence of a biological synthesis of a heterocyclic ring that contains selenium in place of sulfur.

Selenocysteine lyase activity in a cysteine-requiring mutant ofEscherichia coli K-12.

Selenocysteine lyase activity was detected in crude extracts from a cysteine-requiring mutant ofEscherichia coli K-12. The level of activity was the same whether cells had been grown aerobically or anaerobically, with or without selenocysteine. Selenocysteine lyase catalyzes the conversion of selenocysteine to alanine and elemental Se, a reaction that is followed by a nonenzymatic reduction of the Se to hydrogen selenide. Both of these end products were identified in this study. With cysteine as the substrate, alanine and H2S were formed, but only at levels 50% less than the products formed from selenocysteine. Selenocysteine lyase has been identified in a number of mammals and bacteria; its presence in a cysK mutant ofE. coli K-12 suggests a common route whereby hydrogen selenide, derived from selenocysteine, can then be assimilated into selenoproteins.