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
Pj. Bagley et al., 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, Analytical biochemistry, 227(1), 1995, pp. 40-48
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.