Evidence for expression of a single distinct form of mammalian cysteine dioxygenase

Summary.

Cysteine dioxygenase (CDO) plays a critical role in the regulation of cellular cysteine concentration. Because multiple forms of CDO (∼23 kDa, ∼25 kDa, and ∼68 kDa) have been claimed based upon separation and detection using SDS-PAGE/western blotting (with antibodies demonstrated to immunoprecipitate CDO), we further investigated the possibility of more than one CDO isoform. Using either rabbit antibody raised against purified rat liver CDO or against purified recombinant his₆-tagged CDO (r-his₆-CDO) and using 15% (wt/vol) polyacrylamide for the SDS-PAGE, we consistently detected the ∼25 kDa band, but never detected a ∼68 kDa band, in rat liver, kidney, lung and brain. Nondenatured gel electrophoresis of r-his₆-CDO yielded a molecular mass estimate of 25.7 kDa and no evidence of dimerization. Mass spectrometry of r-his₆-CDO yielded two peaks with molecular masses of 24.1 kDa and 24.3 kDa. Anion-exchange FPLC of r-his₆-CDO also gave two peaks, with the first containing CDO that was 7.5-times as active as the more anionic form that eluted second. When the two peaks recovered from FPLC were run on SDS/PAGE, the first (more active) CDO fraction yielded two bands (perhaps as an artifact of SDS/PAGE), whereas the second (less active) CDO fraction yielded only the ∼23 kDa band. We conclude that the physiologically active form of CDO is the ∼25 kDa (i.e., 23.5 kDa based on mass spectrometry) monomer and that this active form is probably derived by post-translational modification of the 23 kDa gene product.