Evidence for expression of a single distinct form of mammalian cysteine dioxygenase.
Cysteine dioxygenase (CDO) plays a critical role in the regulation of cellular cysteine concentration. Because multiple forms of CDO ( approximately 23 kDa, approximately 25 kDa, and approximately 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 his(6)-tagged CDO (r-his(6)-CDO) and using 15% (wt/vol) polyacrylamide for the SDS-PAGE, we consistently detected the approximately 25 kDa band, but never detected a approximately 68 kDa band, in rat liver, kidney, lung and brain. Nondenatured gel electrophoresis of r-his(6)-CDO yielded a molecular mass estimate of 25.7 kDa and no evidence of dimerization. Mass spectrometry of r-his(6)-CDO yielded two peaks with molecular masses of 24.1 kDa and 24.3 kDa. Anion-exchange FPLC of r-his(6)-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 approximately 23 kDa band. We conclude that the physiologically active form of CDO is the approximately 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.
Stipanuk
M H
MH
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853-6301, USA. mhs6@cornell.edu
Londono
M
M
Hirschberger
L L
LL
Hickey
C
C
Thiel
D J
DJ
Wang
L
L
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D050558:Cysteine Dioxygenase; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D010928:Placental Lactogen; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D011993:Recombinant Fusion Proteins; D010928:Placental Lactogen; D049308:Dioxygenases; D050558:Cysteine Dioxygenase