THE IMMUNOREGULATORY MEDIATOR MACROPHAGE-MIGRATION INHIBITORY FACTOR (MIF) CATALYZES A TAUTOMERIZATION REACTION

Background: Recent studies of melanin biosynthesis have uncovered an u nusual enzymatic activity which converts the non-naturally occurring D -isomer of 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) into 5 ,6-dihydroxyindole-2-carboxylic acid (DHICA). The aim of the present i nvestigation was to isolate and characterize the enzyme catalyzing thi s tautomerization reaction. Materials and Methods: After we performed a tissue survey of D-dopachrome tautomerase activity, 10 bovine lenses were homogenized and used as a source of enzyme. A soluble fraction w as obtained by high-speed centrifugation and subjected to successive F PLC chromatography on Phenyl-sepharose, Mono S cation-exchange, and Su perdex gel-filtration. The isolated enzyme was electrophoresed, blotte d onto PVDF membrane, and the N terminus analyzed by gas phase micro-s equencing. Results: The protein catalyzing the conversion of D-dopachr ome to DHICA was purified to homogeneity in 14% yield and showed a mol ecular weight of 12 kD when analyzed by SDS-PAGE. The first 27 amino a cid residues of this protein were sequenced and found to be identical with those of bovine macrophage migration inhibitory factor (MIF). The catalytic activity of native MIF was confirmed by studies of purified recombinant human MIF, which showed the same tautomerase activity. Wh ile L-dopachrome was not a substrate for this reaction, the methyl est ers of the L- and D-isomers were found to be better substrates for MIF than D-dopachrome. Conclusions: MTF has been described recently to be an anterior pituitary hormone and to be released from immune cells st imulated by low concentrations of glucocorticoids. Once secreted, MIF acts to control, or counter-regulate, the immunosuppressive effects of glucocorticoids on the immune system. Although the tested substrate, D-dopachrome, does not occur naturally, the observation that MIF has t automerase activity suggests that MIF may mediate its biological effec ts by an enzymatic reaction. These data also offer a potential approac h for the design of small molecule pharmacological inhibitors of MIF t hat may modulate its potent immunoregulatory effects in vivo.