Distribution of an 18 kDa-selenoprotein in several tissues of the rat

Abstract

By combining methods for trace element analysis, tracer techniques and various biochemical and electrophoretical procedures, information on the characteristics of an 18 kDa-selenoprotein was obtained. By labeling of rats in vivo with [ 75Se]-selenite and gel electrophoretic separation of the proteins in tissues and subcellular fractions, a larger number of selenium-containing proteins could be distinguished. In most of the tissues investigated a labeled 18 kDa-band was present. After co-electrophoresis of the 18 kDa-bands from kidney, liver and brain we found that they all migrated in the same way. Using ultracentrifugational fractionation the 18 kDa-band was localized in the mitochondrial and microsomal membranes. Two-dimensional electrophoresis showed that it consists of a single selenium-containing protein with an isoelectric point of about 4.9–5.0. By means of proteolytic cleavage of the 18 kDa-protein and separation of its peptides by tricine-SDS-PAGE six selenium-containing peptides with molecular masses of 17, 16, 14, 12, 10, and 8 kDa were detected. After electrophoretic separation of the mitochondrial and/or microsomal proteins and acid hydrolysis of the electroeluted protein its amino acid composition was analyzed by RP-HPLC. In this way it was shown that selenium is present in the 18 kDa-protein in form of selenocysteine which is a characteristic of a genetically encoded selenoprotein.