FIGURE 7.

The PGP cross-link is essential for interactions between the protein components of IαI. Differential ChonABC treatment of IαI released bikunin (HC1·HC2) or completely removed the CS (IαI dissociated). The samples were cross-linked with BS3 in the presence of either Mg²⁺ or EDTA. After cross-linking mild alkaline treatment dissociated the protein components from the CS and the samples were analyzed by SDS-PAGE and Western blotting using antibodies against HC1 (panel A) and HC2 (panel B). SDS-PAGE under non-reducing conditions and trypsin inhibitor counterstaining was used to visualize bikunin (panel C). The Western blots reveal that BS3 cross-linking is only possible when the CS chain links the protein components. Moreover, a full-length CS chain is essential for the effect of divalent cations. Remarkably, upon complete dissociation of IαI, HC2 forms polymers (panel B, lanes 6 and 9). Apart from these polymers, it is evident that the interactions between IαI protein components depend on the presence of the CS. The data represent 3 independent experiments.