Distribution of an 18 kDa-selenoprotein in several tissues of the rat.
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.
Kyriakopoulos
Antonios
A
Hahn-Meitner-Institut Berlin, Department Molecular Trace Elements Research in the Life Science, Germany. Kyriakopoulos@hmi.de
Bertelsmann
Holger
H
Graebert
Alexandra
A
Hoppe
Barbara
B
Kühbacher
Markus
M
Behne
Dietrich
D
D000818:Animals; D001345:Autoradiography; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D008297:Male; D008861:Microsomes / Q000737:chemistry; D008928:Mitochondria / Q000737:chemistry; D008970:Molecular Weight; D009928:Organ Specificity; D011506:Proteins / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D012643:Selenium / Q000172:deficiency; D012644:Selenium Radioisotopes; D051140:Selenoproteins; D014018:Tissue Distribution; D014461:Ultracentrifugation
D011506:Proteins; D012644:Selenium Radioisotopes; D051140:Selenoproteins; D012643:Selenium