ISOLATION AND IDENTIFICATION OF SELENIUM-LABELED PROTEINS IN THE MOUSE KIDNEY

Selenium is a potent chemopreventive agent; however, the mechanisms fo r its chemopreventive activities remain elusive. Selenium binds to sev eral proteins, some of which require selenium for functional activity, In this study, two 58kDa selenium-labeled proteins were identified in mouse kidney using a Se-75 labeling method. The proteins were partial ly purified using Sephadex G-150 gel filtration, DEAE-Sephadex A-50 io n-exchange chromatography and one- / two-dimensional sodium dodecyl su lfate polyacrylamide gel electrophoresis (1D-/2D-SDS-PAGE). The two pr oteins 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 antib odies raised in rabbits against the 58kDa proteins electro-eluted from the 1D-SDS-PAGE of the DEAE purified fraction, recognized both protei n spots on 2S-SDS-PAGE gel. The in situ enzymatic digestion of the two proteins separated in 2D-SDS-PAGE gels, followed by microsequencing o f the peptides, resulted in the identification of these two proteins a s related to human lipoamide dehydrogenase and thiol: protein disulfid e oxidoreductase (TPDO). In common, both these proteins have a bis (cy steinyl) 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 prot eins such as thioredoxin, protein disulfide isomerase, endoplasmic ret iculum protein (ERp72), selenoprotein W, 56kDa acetaminophen binding p rotein and formate dehydrogenase. This sequence motif acts as an activ e redox center for majority of the proteins mentioned above, that may be controlling the oxidation/reduction of proteins in vivo. How and wh y selenium is binding to proteins with this common sequence motif need s further investigation.