Isolation and identification of selenium-labeled proteins in the mouse kidney.
Selenium is a potent chemopreventive agent; however, the mechanisms for its chemopreventive activities remain elusive. Selenium binds to several proteins, some of which require selenium for functional activity. In this study, two 58kDa selenium-labeled proteins were identified in mouse kidney using a 75Se labeling method. The proteins were partially purified using Sephadex G.150 gel filtration, DEAE-Sephadex A-50 ion-exchange chromatography and one- / two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-/2D-SDS-PAGE). The two proteins migrated at 58kDa on 2D-SDS-PAGE and differed only slightly in their pI values; i.e., 6.2 and 6.6, respectively. The polyclonal antibodies raised in rabbits against the 58kDa proteins electro-eluted from the 1D-SDS-PAGE of the DEAE purified fraction, recognized both protein spots on 2D-SDS-PAGE gel. The in situ enzymatic digestion of the two proteins separated in 2D-SDS-PAGE gels, followed by microsequencing of the peptides, resulted in the identification of these two proteins as related to human lipoamide dehydrogenase and thiol: protein disulfide oxidoreductase (TPDO). In common, both these proteins have a bis (cysteinyl) sequence motif cys-X-X-cys (for lipoamide dehydrogenase it is cys-X-X-X-X-cys) which is also an integral part of several other proteins such as thioredoxin, protein disulfide isomerase, endoplasmic reticulum protein (ERp72), selenoprotein W, 56kDa acetaminophen binding protein and formate dehydrogenase. This sequence motif acts as an active redox center for majority of the proteins mentioned above, that may be controlling the oxidation/reduction of proteins in vivo. How and why selenium is binding to proteins with this common sequence motif needs further investigation.
Jamba
L
L
Department of Biophysics, Panjab University, Chandigarh, India.
Nehru
B
B
Medina
D
D
Bansal
M P
MP
Sinha
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003545:Cysteine; D003600:Cytosol / Q000378:metabolism; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007535:Isomerases / Q000737:chemistry; D007668:Kidney / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D019704:Protein Disulfide-Isomerases; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012643:Selenium / Q000378:metabolism*; D012644:Selenium Radioisotopes; D051151:Selenoprotein W; D051140:Selenoproteins; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D010446:Peptide Fragments; D011506:Proteins; C495240:SELENOW protein, human; D012644:Selenium Radioisotopes; D051151:Selenoprotein W; D051140:Selenoproteins; D008058:Dihydrolipoamide Dehydrogenase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D012643:Selenium; D003545:Cysteine