Cross-linking Phytochrome to its Receptor in situ using Imidoesters

Phytochrome can be cross-linked to a particulate fraction usingimidoesters, namely dimethy adipimidate (DMA) and dimethyl suberimidate(DMS). DMS was more effective than DMA. Cross-linking of phytochrometo its in situ receptor effected by DMS occurred in red light-irradiatedcoleoptiles. If DMS cross-linking was carried out prior to redlight irradiation there was very little formation of particulatephytochrome. Phytochrome in the particulate fraction obtainedby in situ DMS cross-linking was totally resistant to the solubilizingeffect of washing with solutions of high salt concentrationand high pH and was indistinguishable spectro-scopically fromthe phytochrome in untreated coleoptiles. DMS cross-linkingof phytochrome to its assumed receptor in situ preferentiallyprotected it from destruction following red light irradiationand also prevented it from dissociating from its receptor followingR/FR¹ irradiation when incubated subsequently in the dark. Thesecharacteristics of phytochrome in DMS-treated coleoptiles matchedthose observed using glutaraldehyde as the cross-linking reagent.It is therefore concluded that earlier results obtained usingglutaraldehyde are not peculiar to that reagent and can be duplicatedreadily using more defined bifunctional cross-linkers.