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.
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 in marked contrast to previous studies which demonstrated 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-DDP-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.(ABSTRACT TRUNCATED AT 250 WORDS)
D002945:Cisplatin / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D006801:Humans; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008970:Molecular Weight; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000378:metabolism*; D013438:Sulfhydryl Compounds; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism*
D003432:Cross-Linking Reagents; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011971:Receptors, Immunologic; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins; D014357:Trypsin; D002945:Cisplatin