Background: Recent studies of melanin biosynthesis have uncovered an unusual 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 investigation was to isolate and characterize the enzyme catalyzing this 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 was obtained by high-speed centrifugation and subjected to successive FPLC chromatography on Phenyl-sepharose, Mono S cation-exchange, and Superdex gel-filtration. The isolated enzyme was electrophoresed, blotted onto PVDF membrane, and the N terminus analyzed by gas phase micro-sequencing. Results: The protein catalyzing the conversion of Ddopachrome to DHICA was purified to homogeneity in 14% yield and showed a molecular weight of 12 kD when analyzed by SDS-PAGE. The first 27 amino acid 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 MIX was confirmed by studies of purified recombinant human MIW, which showed the same tautomerase activity. While L-dopachrome was not a substrate for this reaction, the methyl esters of the Land D-isomers were found to be better substrates for MIF than D-dopachrome. Conclusions: MIW has been described recently to be an anterior pituitary hormone and to be released from immune cells stimulated 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 tautomerase activity suggests that MIF may mediate its biological effects by an enzymatic reaction. These data also offer a potential approach for the design of small molecule pharmacological inhibitors of MIF that may modulate its potent immunoregulatory effects in vivo.

A macrophage migration inhibitory factor (MIF), originally described as a product of activated lymphocytes, has been defined as a 12 kDa protein, expressed in a wide variety of tissues. Here MIF is identified as a phenylpyruvate tautomerase (EC 5.3.2.1) having /»-hydroxyphenylpyruvate and phenylpyruvate as its natural substrates. The definition of MIF as an enzyme may yield insight into the mechanism of action of this proinflammatory and immunomodulating cytokine.

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The influence of cysteine on the oxidation of tyrosine, dopa, and monocysteinyldopas by mushroom tyrosinase was reexamined. During oxidation of tyrosine in the presence of cysteine the concentration of dopa increased slowly, whereas the concentration of cysteinyldopas increased more rapidly. When the concentration of cysteine decreased the cysteinyldopas were rapidly consumed and dopa concentrations increased sharply. Experiments on the oxidation of dopa by tyrosinase in the presence of cysteine showed that this thiol does not inhibit the oxidation. Dopa concentrations decreased more rapidly in the presence of cysteine because cysteine addition to dopaquinone prevented reformation of dopa from dopaquinone. Both 2-S-cysteinyldopa and 5-S-cysteinyldopa are substrates for tyrosinase. The oxidation of cysteinyldopas was inhibited at high cysteine concentrations. The greater part of 2,5-S,S-dicysteinyldopa formed during the oxidation of monocysteinyldopas in the presence of cysteine is derived from 5-S-cysteinyldopa, which is a better substrate for tyrosinase than 2-S-cysteinyldopa. The fact that cysteine binds more rapidly to 5-S-cysteinyldopaquinone than to 2-S-cysteinyldopaquinone further stresses the importance of 5-S-cysteinyldopa in the formation of 2,5-S,S-dicysteinyldopa. Oxidation of dopa in the presence of cysteine and glutathione or methionine showed that glutathione is added to dopaquinone but less rapidly than cysteine. Methionine showed insignificant addition to dopaquinone. When dopa or 5-OH-dopa is added to an incubate of cysteinyldopa and tyrosinase the oxidation of cysteinyldopa is accelerated owing to oxidation of cysteinyldopa by dopaquinone or 5-OH-dopaquinone.