SPECIFIC REDUCTION OF INSULIN DISULFIDES BY MACROPHAGE-MIGRATION INHIBITORY FACTOR (MIF) WITH GLUTATHIONE AND DIHYDROLIPOAMIDE - POTENTIAL ROLE IN CELLULAR REDOX PROCESSES

The molecular mechanism of action of MIF, a cytokine that plays a crit ical role in the host immune and inflammatory response, has not yet be en identified. We recently demonstrated that MIF is an enzyme that exh ibits oxidoreductase activity by a cysteine thiol-mediated mechanism. Here,ve further investigated this function by examining the reduction of insulin disulfides by wild-type human MIF (wtMIF) using various sub strates, namely glutathione (GSH), dihydrolipoamide, cysteine, beta-me rcaptoethanol and dithiothreitol. The activity of wtMIF was compared t o that of the relevant cysteine mutants of MIF and to two carboxy-trun cated mutants. Only GSH and dihydrolipoamide were found to serve as re ductants, whereas the other substrates were not utilized by MIF. Reduc tion of insulin disulfides by MIF was closely dependent on the presenc e of the Cys(57)-Ala-Leu-Cys(60) (CALC) motif-forming cysteines C57 an d C60, whereas C81 was not involved (activities: 51 +/- 13%, 14 +/- 5% , and 70 +/- 12% of wtMIF, respectively, and 20 +/- 3% for the double mutant C57S/C60S). Confirming the notion that the activity of MIF was dependent on the CALC motif in the central region of the MIF sequence, the C-terminal deletion mutants MIF(1-105) and MIF(1-110) were found to be fully active. The favored use of GSH and dihydrolipoamide indica ted that MIF may be involved in the regulation of cellular redox proce sses and was supported further by the finding that,MIF expression by t he cell lines COS-1 and RAW 264.7 was significantly induced upon treat ment with the oxidant hydrogen peroxide. (C) 1998 Federation of Europe an Biochemical Societies.