Self-assembly and cross-linking of Volvox extracellular matrix glycoproteins are specifically inhibited by Ellman's reagent

A major impediment to the biochemical characterization of extracellular mat rices from algae (as well as higher plants) is the extensive covalent cross -linking that exists in the matrix, rendering most components insoluble and resistant to conventional extraction procedures. In the multicellular gree n alga Volvox, biogenesis of the extracellular matrix (ECM) is initiated im mediately after the process of embryonic inversion. At this stage of develo pment, the sulfhydryl reagent 5,5'-dithio-bis(2-nitrobenzoic acid), known a s Ellman's reagent, interferes in a highly specific manner with ECM biogene sis. Treated post-inversion embryos are no longer able to assemble an intac t ECM and consequently dissociate into a suspension of single cells. Dissoc iated cells remain viable and continue to secrete ECM proteins into the gro wth medium, as documented by the identification of several members of the p herophorin family. Cross-linked ECM polymers such as sulfated surface glyco protein 185 remain in a soluble state. Thus, treatment with Ellman's reagen t opens a simple approach for the isolation and characterization of otherwi se inaccessible monomeric precursors.