In the present studies radiotracer techniques have been combined with biochemical separation procedures to investigate the selenium-containing proteins in the culture cells of the lung, trachea and their subcellular fractions. Subcellular separation of the lung and trachea tissues has been achieved by differential ultracentrifugation. The selenium-containing proteins in these compartments have been investigated by labeling of lung and trachea cultured cells in vitro with Se-75, gel electrophoretic separation of the proteins and autoradiographic detection of the tracer. The protein separation by gel electrophoresis using mono-dimensional (1D)- and two-dimensional (2D)-SDS-PAGE has been successfully applied for the selenium research. It has resulted in the detection of a large number of selenium-containing proteins. Two-dimensional gel electrophoresis (2-DE) was also helpful in the identification of the proteins of interest according to their molecular mass and isoelectric point. In this way more than 30 selenium-containing proteins could be distinguished in the lung and trachea samples. Some of them such as Gpx1, Trx1, SelP, SelT and Sel15 could be identified by means of immunoassays, their molecular weight and pI values and localized in the cellular compartments.

In the present article the radiotracer techniques have been combined with biochemical separation procedures to investigate the effects of changes in the selenium status on the expression of the selenium-containing proteins in the lung and their subcellular fractions. Subcellular separation of the lung has been achieved by differential ultracentrifugation. The selenium-containing proteins in these compartments have been investigated by labeling of rats in vivo with (75)Se, gel electrophoretic separation of the proteins, and autoradiographic detection of the tracer. In the lung of the selenium-deficient animals, the selenium administered was used predominantly to restore the levels of the selenoproteins, while in the lung of the selenium-sufficient animals most of the selenium retained was incorporated into the glutathione peroxidase. Also, higher activity of this enzyme has been found in the lung of the selenium-sufficient animals. The differences in the specific incorporation of the element in the selenium deficiency into different compounds suggested that there are different metabolic pathways for selenium, strongly dependent on its status.

The concentrations of arsenic, chromium, cobalt, iron, manganese, rubidium, selenium and zinc were determined by instrumental neutron activation analysis (INAA) in the homogenate and the subcellular fractions of lungs from rats fed either a selenium-adequate or a selenium-deficient diet. Feeding of the selenium-deficient diet led to a considerable decrease in the selenium levels in all samples investigated but had no significant effect on the concentrations of the other elements. All elements were distributed inhomogeneously among the subcellular fractions. Selenium, iron and zinc had their highest concentrations in the microsomal fraction, chromium and cobalt in the nuclear fraction and arsenic and rubidium in the cytosol. Information about the trace element-containing proteins in the lung cytosol was obtained by size exclusion chromatography and online multi-element analysis of the separated protein fractions by mass spectrometry in conjunction with an inductively coupled plasma (ICP-MS). The results suggested that arsenic, cadmium, cobalt, copper, iron, manganese, molybdenum, nickel, selenium and zinc are present in the rat lung cytosol in several protein-bound forms.

Abstract: By a combination of trace techniques and various biochemical methods, information about the characteristics of a 15‐kDa selenoprotein was obtained. After labeling of rats in vivo with [75Se]selenite, subcellular fractionation of the homogenates of the prostate, lung, brain, thyroid gland, and large intestine, and gel electrophoretic separation of the proteins and subcellular fractionation, 15‐kDa 75Se was found in the cytosols of the tissues prostate > brain > lung > thyroid gland > large intestine after autoradiography. After coelectrophoresis of the separated 15‐kDa labeled band obtained from each cytosolic fraction, the 15‐kDa 75Se band migrated in the same way as the combined bands isolated from the five tissue cytosols. After proteolytic cleavage in the gel of the 15‐kDa labeled band obtained from the cytosol of each tissue and re‐electrophoresis, the same labeled peptide pattern was found in each gel slice after autoradiography. By means of reversed‐phase HPLC, we characterized a selenocysteine‐containing protein that has enzymatic activity like that of glutathione peroxidase.