The specific objectives of the proposed research are to isolate and characterize the selenium containing pooteins and glutathione peroxidase (GPx) from human blood, perform kinetic studies with these purified GPxs, determine the influence of selenium status in rhesus monkeys on the relative levels of various GPxa, study the incorporation of selenium into erythrocyte and plasma proteins in rhesus monkeys, and to determine the GPx and peroxidase activities of hemoglobin from blood of various species of animals. The two GPxa from human erythrocytes, the two GPxs from human plasma and a selenium-containing protein in human plasma are proposed to be purified by gel filtration and ion exchange (both anion and cation) resin chromatography. The purified proteins are proposed to be characterized with respect to molecular weight, the selenium content, amino acid composition, and kinetic properties. The Km, Vmax and any allosteric properties are proposed to be determined with both organic and inorganic peroxides as substrates. The influence of selenium status on the relative activities of the various GPxs in rhesus monkeys erythrocytes is proposed to be determined at the various times after the animals have been placed on a diet containing selenium. The various GPxs will be separated by gel filtration anf the activity of each GPx determined from the sum of each fraction within a given peak. 75Se-selenite will be injected into rhesus monkeys and blood samples taken at various times afterwards to determine the rate of incorporation into hemoglobin and GPx in erthrocytes and into non-GPx proteins and GPx in plasma. The nature of selenium binding to hemoglobin will be investigated by ion exchange chromatography, and the incorporation of selenium into non-Gpx proteins and GPx determined by gel filtration and ion exchange chromatography. In order to obtain some information on why human hemoglobin interferes with the assay of GPx more than that from animals, hemoglobin will be isolated from rat, sheep, rhesus and squirrel monkey, and human blood and the GPx activity determined. It has been assumed that the measurement of GPx in erythrocytes of humans is a measure of selenium status, like it is in animals, but this is apparently not true. The results of the present proposed study should provide basic information which can be used to more accurately assess selenium status in humans, as well as to provide addditional information on the metabolism of this element in people.
| Butler, J A; Whanger, P D; Patton, N M (1988) Effects of feeding selenium deficient diets to rhesus monkeys (Macaca Mulatta). J Am Coll Nutr 7:43-56 |
| Broderick, D J; Deagen, J T; Whanger, P D (1987) Properties of glutathione peroxidase isolated from human plasma. J Inorg Biochem 30:299-308 |
| Deagen, J T; Butler, J A; Beilstein, M A et al. (1987) Effects of dietary selenite, selenocystine and selenomethionine on selenocysteine lyase and glutathione peroxidase activities and on selenium levels in rat tissues. J Nutr 117:91-8 |
| Beilstein, M A; Whanger, P D (1986) Chemical forms of selenium in rat tissues after administration of selenite or selenomethionine. J Nutr 116:1711-9 |
| Beilstein, M A; Whanger, P D (1986) Deposition of dietary organic and inorganic selenium in rat erythrocyte proteins. J Nutr 116:1701-10 |
