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-press...

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Bibliographic Details
Published in:Archives of biochemistry and biophysics 1999-01, Vol.361 (1), p.25-33
Main Authors: Gu, Qiu-Ping, Beilstein, Michael A., Barofsky, Elisabeth, Ream, Walt, Whanger, Philip D.
Format: Article
Language:English
Subjects:
Animals
Binding Sites
endoproteinase C
gel filtration
Glutathione - metabolism
HPLC
ion-exchange resins
Macaca mulatta
mass spectrometry
Molecular Weight
Muscle Proteins - chemistry
Muscle Proteins - isolation & purification
Muscle Proteins - metabolism
Muscle, Skeletal - chemistry
Peptide Mapping
Protein Binding
Proteins - chemistry
Proteins - isolation & purification
Proteins - metabolism
Selenoprotein W
Selenoproteins
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Summary: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°C. 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.
ISSN:0003-9861
1096-0384
DOI:10.1006/abbi.1998.0949