Rapid spectrophotometric differentiation between glutathione-dependent and glutathione-independent gentisate and homogentisate pathways.
A total of four pathways are known for the catabolism by microorganisms of gentisate (2,5-dihydroxybenzoate) and homogentisate (2,5-dihydroxyphenylacetate). Both of these dihydric phenols can be degraded by either a glutathione-dependent or a glutathione-independent reaction sequence. We found that it is not always possible to unequivocally assign glutathione dependence or independence to a particular catabolic sequence by using the well-established spectrophotometric assays at 330 nm (gentisate pathway) or 320 nm (homogentisate pathway). This paper reports a modification of the classical spectrophotometric assays that allowed an unequivocal differentiation between glutathion-dependent and glutathione-independent pathways, even when crude cell extracts contained significant quantities of cell-derived, reduced glutathione. This was accomplished by performing assays in the presence of an approximately 10(-3) M solution of the sulfhydryl-binding agent N-ethylmaleimide.
D001419:Bacteria / Q000378:metabolism*; D001673:Biodegradation, Environmental; D005033:Ethylmaleimide; D005841:Gentisates / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006713:Homogentisic Acid / Q000378:metabolism*; D013045:Species Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D005841:Gentisates; D005978:Glutathione; D006713:Homogentisic Acid; D005033:Ethylmaleimide