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 th e procedure for assay of cysteine dioxygenase activity in rat liver pr eparations. Cysteine dioxygenase activity was largely in the soluble f raction of liver and was stimulated by addition of NAD(+) and Fe2+. Th e pH optimum of the enzyme was 6.1. Addition of an inhibitor of pyrido xal 5-phosphate-dependent enzymes was necessary to prevent rapid remov al 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 we re quantitated by measurement of S-35 radioactivity or by formation an d measurement of fluorescent derivatives. This assay of cysteine dioxy genase under optimal conditions provides a physiologically relevant me asure of cysteine dioxygenase activity in liver and hepatocytes based on the observation that this activity was highly correlated with the c apacity for cysteine catabolism and taurine production by isolated hep atocytes. (C) 1995 Academic Press, Inc.