Purification, characterization, and glutathione binding to selenoprotein W from monkey muscle.
Selenoprotein W was purified from monkey skeletal muscle to investigate its binding of glutathione. The purification was accomplished by concentration of the cytosol with an Amicon cell, gel filtration using Sephadex G-50, cation-exchange chromatography with CM-Sephadex, and reverse-phase high-pressure liquid chromatography using a C-18 Vydac column. Selenoprotein W was monitored during purification by slot blots. These steps resulted in an electrophoretically pure selenoprotein W preparation that was estimated by gel filtration to have molecular weight of about 10 kDa. N-terminal amino acid sequencing was used to confirm that the pure proteins were selenoprotein W. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) revealed that the proteins existed in three masses of 9635 +/- 7, 9371 +/- 11, and 9330 +/- 5 Da. The theoretical mass of the protein predicted from the cDNA sequence is 9330 Da. The 9635-Da form of the protein was shown to contain bound glutathione (306 Da), which could be released by reduction with dithiothreitol at 50 degreesC. The form with a mass of 9371 Da is assumed to result from binding of an unidentified 41-Da moiety to the 9330-Da form of the protein. MALDI peptide mapping with endoproteinase Glu-C suggested that glutathione is bound to the 36th amino acid (cysteine) of selenoprotein W.
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QP
Department of Agricultural Chemistry, Oregon State University, Corvallis, Oregon, 97331, USA.
Beilstein
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MA
Barofsky
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E
Ream
W
W
Whanger
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PD
D000818:Animals; D001665:Binding Sites; D005978:Glutathione / Q000378:metabolism*; D008253:Macaca mulatta; D008970:Molecular Weight; D009124:Muscle Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry; D010449:Peptide Mapping; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D051151:Selenoprotein W; D051140:Selenoproteins
D009124:Muscle Proteins; D011506:Proteins; D051151:Selenoprotein W; D051140:Selenoproteins; D005978:Glutathione