Journal
JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY
Volume 14, Issue -, Pages S290-S296Publisher
AMER SOC NEPHROLOGY
DOI: 10.1097/01.ASN.0000078024.50060.C6
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Biotin-cysteine was used to study protein S-thiolation in isolated rat kidneys subjected to ischemia and reperfusion. After 40 min of ischemia, total protein S-thiolation increased significantly (P < 0.05), by 311%, and remained significantly elevated (P < 0.05), 221% above control, after 5 min of postischemic reperfusion. Treatment of protein samples with 2-mercaptoethanol abolished the S-thiolation signals detected, consistent with the dependence of the signal on the presence of a disulfide bond. With the use of gel filtration chromatography followed by affinity purification with streptavidin-agarose, S-thiolated proteins were purified from CHAPS-soluble kidney homogenate. The proteins were then separated by SDS-PAGE and stained with Coomassie blue. With a combination of matrix-assisted laser desorption ionization time of flight mass spectrometry and LC/MS/MS analysis of protein bands digested with trypsin, a number of S-thiolation substrates were identified. These included the LDL receptor-related protein 2, ATP synthase a chain, heat shock protein 90 beta, hydroxyacid oxidase 3, serum albumin precursor, triose phosphate isomerase, and lamin. These represent proteins that may be functionally regulated by S-thiolation and thus could undergo a change in activity or function after renal ischemia and reperfusion.
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