Spatially related amelogenin interactions in developing rat enamel as revealed by molecular cross-linking studies

A cleavable cross-linker (dithiobis[succinimidyl propionate], DTSP) was use d to investigate the subunit structure of the developing enamel matrix. Int act matrix was cross-linked under conditions chosen to simulate those found in vivo. The cross-linked complexes were isolated by preparative sodium do decyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and their subu nit composition determined by analytical SDS-PAGE following reductive cleav age of the cross-links. Western blotting using antiamelogenin antibodies wa s used to confirm the identity of the proteins involved. The results showed that nascent amelogenins tended to be cross-linked to other nascent amelog enins while amelogenin-processing products tended to be cross-linked to oth er processed molecules at the same stage of processing. The results suggest that nascent amelogenins are in close association after secretion and duri ng extracellular processing, and that processed products are not free to as sociate with nascent molecules, presumably due to diffusion constraints in the tissue. This conclusion implies that individual amelogenin molecules wi thin supramolecular aggregates (nanospheres) are processed in situ and rema in in the same nanosphere while all the individual component amelogenins un dergo processing. The biological function of amelogenin processing remains unclear but the fact that amelogenin-amelogenin associations are maintained during processing indicates that matrix stability is an important factor w hile the enamel layer is being deposited. (C) 2000 Elsevier Science Ltd. Al l rights reserved.