Speciation of selenium in the mammalian organism.

As almost all of the selenium present in the mammalian organism is protein-bound, speciation is mostly concerned with the determination of the different selenium-containing proteins. Information on their distribution and their concentrations in the different tissues of the rat was obtained by means of tracer procedures which, after application of 75Se-selenite with a very high specific activity to selenium-depleted animals and electrophoretic separation of the labelled proteins, allow the determination of these compounds in the pmol to fmos range. A method was developed for the determination of selenocysteine and selenomethionine in the selenium-containing proteins. The identification of specific selenoproteins was achieved by analysis of their selenoamino acid residues and by studies on their characteristics and possible biological functions. This is being followed by the development of methods for the quantitative analysis of the selenoproteins in questions in the tissues of animals and man. In this paper the strategies and procedures used in the identification, characterization and determination of the selenium species present in the mammalian organism will be discussed.

Two new selenoproteins found in the prostatic glandular epithelium and in the spermatid nuclei.

After labeling of rats in vivo with 75Se and protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis more than 25 Se-containing bands could be distinguished. Of those proteins which were detected only in certain compartments and might therefore have tissue-specific functions, two were chosen for detailed investigation. A 15 kDa-protein was found in the prostatic epithelium where it accounted for about two thirds of the protein-bound 75Se. It was mainly present in the cytosol but was not released into the prostatic secretion. After gel chromatography it was found in the fraction which contained proteins with molecular masses of about 300 kDa. Using two-dimensional electrophoresis a pI-value of about 4.5 was determined. In the testis a specific Se-containing 34 kDa-protein was observed which appeared after the onset of puberty. It was localized in the spermatid nuclei where it contained about 80% of the Se tracer present and was found to be bound to the DNA. After extraction it partly disintegrated into a 20 kDa-protein. Both compounds contain Se in the form of selenocysteine. The fact that their formation had priority over that of glutathione peroxidase during insufficient Se intake is an indication of their biological significance. Special interest in the prostatic epithelial selenoprotein derives from a possible inverse relationship between the Se status and the incidence of prostate cancer observed in epidemiological studies, whereas with the 34 kDa-selenoprotein its appearance during the condensation phase of the spermatid nuclei might suggest its participation in some processes of sperm maturation.

Identification of selenocysteine and selenomethionine in protein hydrolysates by high-performance liquid chromatography of their o-phthaldialdehyde derivatives.

A method for the identification of selenocysteine and selenomethionine in protein hydrolysates was developed. The proteins were subjected to acid hydrolysis after they had been carboxymethylated to prevent decomposition of selenocysteine during this process. After precolumn derivatization of the amino acids with o-phthaldialdehyde, the hydrolysate was chromatographed on C18 columns. The selenoamino acids were detected either by the fluorescence of their o-phthaldialdehyde derivatives (detection limit 30 pmol for selenomethionine and 170 pmol for selenocysteine) or by selenium determination in the eluate using atomic absorption spectrometry (detection limit 0.3 pmol) or, with 75Se-labelled compounds, the measurement of the tracer activity. With the latter procedure the detection limit, which depends on the specific activity of the Se tracer, could be decreased to the femtomole range. The method was successfully applied to the identification of selenocysteine in several newly found mammalian selenium-containing proteins.

Protein-bound selenium in the seeds of coco de mono (Lecythis ollaria).

High selenium concentrations (7-12 g/kg dry mass) were found in the seeds of the selenium-accumulator plant coco de mono (Lecythis ollaria). In order to obtain information on the protein-bound part of selenium in extracts of these seeds, dialysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used combined with neutron activation analysis. Extractions were carried out at pH 4.5 and 7.5. In both cases about 90% of the element was dissolved. Of the extracted selenium only 9% was shown to be firmly bound to proteins at pH 4.5 and 29% at pH 7.5. For the protein-bound selenium, concentrations of 0.7 g and 2.4 g per kg of seeds and 40 and 25 g per kg of extractable protein were determined at pH 4.5 and 7.5, respectively. By analyzing the protein fractions separated by SDS-PAGE the element was found to be present in extremely selenium-rich proteins with molecular masses below 20 kDa.

A new selenoprotein found in the glandular epithelial cells of the rat prostate.

An inverse relationship between the Se status and the incidence of prostate cancer suggests a significant role of Se in this organ. After labeling of rats with 75Se and sodium dodecyl sulfate-polyacrylamide gel electrophoresis a strongly labeled prostatic 15-kDa protein band was found which was equally distributed among the different lobes. It was localized in the epithelial cells of isolated acini but did not appear in the prostatic secretion. By two-dimensional electrophoresis the band was resolved into three spots with pI-values around 4.5. The most strongly labeled spot stemmed from a cytosolic selenoprotein with an apparent native molecular mass of about 300 kDa which contained Se in the form of selenocysteine. The fact that with insufficient Se intake the element is preferentially incorporated into this compound as compared with glutathione peroxidase implies an important function of this newly found prostatic epithelial selenoprotein (PES).

Heme stimulation of globin synthesis in a cell free system.

In a cell-free system of pigeon erythrocyte nuclei high concentrations of oxygen inhibit globin synthesis because of the inhibition of heme synthesis, which is required for globin synthesis. The effect of heme in overcoming the inhibition of globin synthesis by oxygen is manifested only during the first few minutes of incubation. This period represents the time during which the system is being warmed from 4 degrees to 37 degrees C. The effect of heme seems to be upon some initial assembly or structural process.