Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates

Two tyrosine residues (Tyr⁴ and Tyr⁷⁶) of succinyl-CoA:3-oxoacid CoA transferase (SCOT) are sensitive to nitric oxide (NO) stress, as assessed by mass spectrometry and site-direct mutagenesis. However, monitoring the SCOT nitration in tissue or cells is challenging. Herein, we describe the development of an assay to detect nitrated SCOT directly using site-specific antibodies; the monoclonal antibodies were generated and screened against nitrated peptides of SCOT. After stringent filtration, two antibodies, anti-SCOT4N and anti-SCOT76N, which specifically recognise Tyr⁴ or Tyr⁷⁶ of SCOT, respectively, were successfully selected. In a cell model over-expressing iNOS in the mitochondria, nitrated SCOT was significantly increased compared with control cells. In addition, in a mouse model of diabetes, nitrated Tyr⁴ and Tyr⁷⁶ in the heart and kidney were higher compared to the control animals. Our results using monoclonal antibodies against nitrated SCOT peptides are in good agreement with the proteomic data.