Evaluation and modification of an assay procedure for cysteine dioxygenase activity: high-performance liquid chromatography method for measurement of cysteine sulfinate and demonstration of physiological relevance of cysteine dioxygenase activity in cysteine catabolism.
Conflicting reports in the literature of appropriate assay procedures for measurement of cysteine dioxygenase activity led us to evaluate the procedure for assay of cysteine dioxygenase activity in rat liver preparations. Cysteine dioxygenase activity was largely in the soluble fraction of liver and was stimulated by addition of NAD+ and Fe2+. The pH optimum of the enzyme was 6.1. Addition of an inhibitor of pyridoxal 5-phosphate-dependent enzymes was necessary to prevent rapid removal of the reaction product cysteine sulfinate. Cysteine sulfinate and cysteic acid were separated by anion-exchange HPLC on a polymer-based column with trimethylamino active groups, and the reaction products were quantitated by measurement of 35S radioactivity or by formation and measurement of fluorescent derivatives. This assay of cysteine dioxygenase under optimal conditions provides a physiologically relevant measure of cysteine dioxygenase activity in liver and hepatocytes based on the observation that this activity was highly correlated with the capacity for cysteine catabolism and taurine production by isolated hepatocytes.
Bagley
P J
PJ
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853, USA.
Hirschberger
L L
LL
Stipanuk
M H
MH
D000818:Animals; D002364:Caseins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003544:Cysteic Acid / Q000737:chemistry; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D050558:Cysteine Dioxygenase; D049308:Dioxygenases; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005296:Ferrous Compounds / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D008297:Male; D009243:NAD / Q000494:pharmacology; D018377:Neurotransmitter Agents; D010105:Oxygenases / Q000378:metabolism* / Q000502:physiology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D013654:Taurine / Q000096:biosynthesis; D013997:Time Factors
D002364:Caseins; D005296:Ferrous Compounds; D018377:Neurotransmitter Agents; D000644:Quaternary Ammonium Compounds; D009243:NAD; D013654:Taurine; C038178:ammonium ferrous sulfate; D003544:Cysteic Acid; D010105:Oxygenases; D049308:Dioxygenases; D050558:Cysteine Dioxygenase; D003545:Cysteine; C013461:cysteine sulfinic acid