The research proposed investigates the nutritional and biochemical function of the essential trace element, selenium. In mammals, almost all of the body selenium is present as selenocysteine in proteins, of which only glutahione (GSH) peroxidase has been well characterized.
The specific aims are: 1. To determine the physical properties, biological or enzymatic activities and physiological functions of mammalian selenocysteine-containing proteins by (a) developing HPLC-based methods for rapid assay of 75Se-labelled selenoproteins in tissue homogenates; (b) developing purification techniques for the major selenoproteins in rat plasma, liver, testes and muscle; (c) defining subcellular locations and type of binding for membrane-bound liver, muscle and testicular selenoproteins; (d) screening selenoproteins for enzymatic activities known to be associated with bacterial selenoproteins, and for antioxidant, antimutagenic and oxidation-reduction activities; (e) using animal models of inflammation and human disease (cancer and genetic muscle dystrophy) to correlate selenoproteins with the disease states; and (f) developing [3H]-iodoacetate labelling of protein selenocysteine as a total tissue selenocysteine assay. 2. To determine the mechanism for incorporation of selenocysteine into GSH peroxidase (a) using in vitro protein synthesis systems and techniques of 75Se-enzyme labelling, chromatography and antibody precipitation; (b) determining the anticodon of selenocysteyl tRNA; and (c) determining the codon that codes for selenocysteine incorporation into GSH peroxidase. 3. To define relationships between penicillamine and gold, which are drugs used to treat arthritis and which are strong inhibitors of GSH peroxidase, and selenoproteins and rheumatoid arthritis by (a) in vitro gel filtration studies of the binding of the two drugs to purified selenoproteins; (b) in vitro and (c) in vivo inhibiton studies of GSH peroxidase from tissues affected by adjuvant arthritis in rats; (d) identification of selenoproteins that bind in vivo to radiolabelled gold in adjuvant-treated rats; (e) determination of the effect of dietary selenium on arthritis in adjuvant-treated rats; and (f) investigation with radiolabelled arachidonic acid of the effect of penicillamine and gold inhibition of GSH peroxidase on the production of hydroxyeicosatetraenoic acid and leukotriene B4, both chemotoxic agents for phagocytic cells involved in arthritic inflammation. This research is related to human health through (i) the potential findings of a relationship of selenium in selenoproteins to cancer and genetic muscular dystrophy; (ii) potential definition of selenoproteins involved in reactions that lead to arthritis; (iii) potential development of in vitro methods for evaluation of antiarthritic drugs; and (iv) determination of a relationship between selenium nutrition and arthritis.
