Intersubunit cross-linking by cis-dichlorodiammineplatinum(II) stabilizes an .alpha.2-macroglobulin "nascent" state: evidence that thiol ester bond cleavage correlates with receptor recognition site exposure

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Abstract
Treatment of human .alpha.2-macroglobulin (.alpha.2M) with proteinase results in cleavage of the .alpha.2M subunits and subsequently in a conformational change in the inhibitor. This change irreversibly traps the proteinase and is accompanied by the generation of four thiol groups as well as exposure of receptor recognition sites. cis-Dichlorodiammineplatinum(II) (cis-DDP) causes extensive intersubunit cross-linking of .alpha.2M. Incubation of .alpha.2M or cis-DDP-treated .alpha.2M with trypsin results in complete subunit cleavage; however, trypsin treatment of cis-DDP-.alpha.2M does not result in a conformational change as determined by nondenaturing polyacrylamide gel electrophoresis (PAGE), receptor recognition site exposure, or appearance of thiol groups from the inhibitor. These results are marked constrast to previous studies which demonstrate that incubation of cis-DDP-treated .alpha.2M with CH3NH2 resulted in thiol ester bond cleavage and receptor recognition site exposure. cis-DDP-treated .alpha.2M bound only 0.13 mol of 125I-trypsin/mol of cis-DPP-.alpha./2M. Incubation of trypsin-treated cis-DDP-.alpha.2M with diethyldithiocarbamate (DDC), a potent chelator of platinum compounds, results in the removal of the intersubunit cross-links and completion of the .alpha.2M conformational change as determined by nondenaturing PAGE. Complete receptor recognition site exposure and the appearance of 3.3 thiol groups/mol of .alpha.2M also occur following this treatment. These results demonstrate that cross-linking of .alpha.2M by cis-DDP prevents a conformational change in the inhibitor which is necessary for thiol ester bond activation and cleavage. Removal of intersubunit cross-links by incubation with DDC allows the completion of this conformational change. These studies also indicate that there is a strong correlation between thiol ester bond cleavage and exposure of receptor recognition sites on .alpha.2M. It is further suggested that incubation of cis-DDP-treated .alpha.2M with trypsin results in a "primed" form of .alpha.2M which is very similar to "nascent" .alpha.2M originally described by Sottrup-Jensen et al. [Sottrup-Jensen, L., Petersen, T. E., and Magnusson, S. (1981) FEBS Lett. 128, 127-132]. Incubation of trypsin-treated cis-DDP-.alpha.2M with DDC results in the generation of a form of .alpha.2M with complete subunit cleavage and complete thiol ester bond cleavage but essentially no proteinase binding.