M. A. Beilstein

Selenoprotein W of rat muscle binds glutathione and an unknown small molecular weight moiety

When purified from rat muscle, selenoprotein W is fractionated into four forms distinguished by slightly different chromatographic behavior. Precise masses of the four forms were determined by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. The mass distribution of the forms (9549, 9592, 9853, and 9898 d) suggests that they occur through derivatization of the lowest mass form with two moieties of approximate masses 44 and 305 d. The apparent 305 d moiety was demonstrated to be glutathione (307 d) by reductive release from the 9853 d protein form with 1000-fold excess of dithiothreitol at 50 degrees C. Milder conditions failed to remove the glutathione. The reduction produced nearly stoichiometric amounts of free glutathione as determined by HPLC of a fluorometric derivative. HPLC retention of the protein changed to match that of the 9549 d form, and a change of mass to 9550 d was observed by MALDI mass spectrometry. The identity of the 44 d moiety is unknown. The presence of glutathione in isolated selenoprotein W may suggest its involvement in the metabolism of this tripeptide.

Tissue distribution and influence of selenium status on levels of selenoprotein W.

Rabbits were immunized with two synthetic peptides based on hydrophilic regions of selenoprotein W from rat muscle. The resulting polyclonal antibodies were used in Western blots to determine the compartmentation and tissue distribution of selenoprotein W, and to determine the influence of selenium on the levels of this selenoprotein in rat muscle. Selenoprotein W exists mainly in cytosol, but very small amounts were associated with membranes. Western blots revealed selenoprotein W in muscle, spleen, testis, and brain of rats. Rats were fed diets of either no addition of selenium (0 ppm Se) or additions of 0.1 and 4.0 mg selenium/g (0.1 ppm Se and 4.0 ppm Se) diet for 6 wk. Selenoprotein W was undetectable in skeletal muscle of rats fed the basal diet, detectable in those fed 0.1 ppm selenium in the diet, and much higher in muscle from rats fed 4 ppm selenium diet. In a species comparison, Western blots indicated the presence of selenoprotein W in muscle of rabbits, sheep, and cattle.—Yeh, J‐Y., Beilstein, M. A., Andrews, J. S., Whanger, P. D. Tissue distribution and influence of selenium status on levels of selenoprotein W. FASEB J. 9, 392–396 (1995)

Chemical forms of selenium in selenium containing proteins from human plasma

The chemical forms of selenium (Se) were determined in human plasma fractions. Human plasma was subjected to gel filtration using Sephadex G-150, and the first Se peak from this column was subsequently chromatographed on DEAE-Sephacel. The form of Se in the Se peak which eluted from this column was shown to be selenocysteine (SeCys). In a second approach human plasma was again subjected to gel filtration and the first Se peak was chromatographed on Affigel blue. SeCys was shown to be the form of Se in both the retained and unretained Se on this column. The second gel filtration Se peak was also chromatographed on Reactive Blue 2-Sepharose CL-6B and the form of Se which was not retained was also shown to be SeCys. However, the form which was retained was shown to be selenomethionine. Evidence is presented that there are three Se containing proteins in human plasma, which are selenoprotein P, glutathione peroxidase, and albumin.

Selenium metabolism and glutathione peroxidase activity in cultured human lymphoblasts

The metabolism of selenite, selenocysteine (SeCys), and selenomethionine (SeMet) was studied in three human lymphoblast cell lines with defects in the transsulfuration pathway and in control cells without this defect. There were very little differences in the induction of glutathione peroxidase (GPX) activity by selenite and SeCys among these cells. However, markedly higher levels of SeMet were required to induce GPX activity in transsulfuration defective cells than in control cells. Surprisingly, the addition of pyridoxal phosphate (PLP) to the media resulted in elevated GPX activity in all cells regardless of the chemical form of Se used. There is no explanation for this effect of PLP, but it is not through direct reaction with GPX or on the alteration of sulfhydryl groups.

Chemical forms of selenium present in rat and ram spermatozoa.

In vivo and in vitro studies were conducted to investigate the chemical forms by ion-exchange chromatography of selenium (Se) present in rat and ovine spermatozoa. After injection with 75Se-selenite, the form of 75Se in rat sperm was selenocysteine, but selenocysteine and selenomethionine (SeMet) were present in ovine sperm. Presumably, synthesis of SeMet by rumen microbes are responsible for its presence in ovine sperm. In vitro incubation of ram sperm with selenocysteine or SeMet produced no changes, but incubation with selenite produced a compound that eluted one fraction before SeMet from the ion-exchange column. After treatment of this fraction with mercaptoethanol, it eluted in a later fraction upon rechromatography, suggesting it to be selenodicysteine. This compound is apparently formed because of high levels of cysteine in semen. Cysteine, reduced glutathione, and oxidized glutathione were also found in semen. The significance of the results is discussed.

Low Molecular Weight Cadmium and Selenium Containing Proteins unlike Metallothionein in Animals

Since the assumed metallothionein (MT) fractions from testis did not respond to cadmium and zinc exposure like MT in other tissues, they were purified using gel filtration and ion exchange resins to determine if they were indeed MT or other proteins. The major amino acids were found to be aspartate, glutamate and glycine with a low cysteine (less than 3%) content. The amino acid content is remarkably similar to a low molecular weight (MW) cadmium-binding protein isolated from oysters with properties unlike MT. Selenium has been shown to be present in a low MW protein called the G protein in various tissues including the testis. The major amino acids in a partially purified preparation were found to be aspartate, glutamate, glycine and lysine with a low methionine (about 2.5%) but a very low cysteine (less than 0.3%) content. The selenium is present in this protein as selenocysteine. The metabolic significance of this selenoprotein is not known, but it should be indicated that selenium will alter the binding of cadmium in testicular proteins.

Development of a ponasterone A-inducible gene expression system for application in cultured skeletal muscle cells

The goal of this study was to develop an inducible gene expression system to assess functions of specific proteins in differentiated cultured skeletal muscle. We utilized and modified the ecdysone inducible system because others have used this system to express exogenous genes in vitro and in transgenic animals. A limitation of the commercially-available ecdysone system is its constitutive expression in all tissues. Hence, its application in vivo would result in expression of a cloned gene in undifferentiated and differentiated tissues. To target its expression to muscle, we removed the constitutively-active CMV promoter of pVgRXR and replaced it with a skeletal muscle alpha-actin promoter so that the regulatory features of the system would be expressed in differentiated muscle cells. We transfected our newly designed expression system into L8 muscle myoblasts and established stable cell lines via antibiotic selection. We determined that reporter gene activity was induced by ponasterone A in myotubes, a differentiated muscle phenotype, but not in myoblasts (undifferentiated cells). This proved the validity of the concept of an inducible muscle-specific expression system. We then determined that beta-galactosidase expression was dependent upon the dose of ponasterone A and duration of exposure to inducer. This creates potential to regulate both the level of expression and duration of expression of a cloned gene in differentiated muscle.

Selenium-Containing Proteins in Plasma of Higher Primates

Only one major selenium-containing peak was found when animal plasma was subjected to gel filtration (Sephadex G-150) (Beilstein and Whanger 1983). The selenium peak eluted slightly ahead of the glutathione peroxidase (GPX) activity peak, suggesting that the majority of the plasma selenium may not be associated with GPX. In subsequent work (Beilstein and Whanger 1988), less than 10% of the selenium in plasma was calculated to be associated with GPX. Surprisingly, when plasma from humans was subjected to gel filtration, two major selenium peaks were obtained but neither one of them cochromatographed with GPX activity (Deagen et al. 1988). Because of this difference in the gel filtration patterns of plasma between animals and humans, further work was undertaken to investigate the reasons for this disagreement.