SYNTHESIS AND APPLICATION OF BIDENTATE PHOTOAFFINITY CROSS-LINKING REAGENTS - NUCLEOTIDE PHOTOAFFINITY PROBES WITH 2 PHOTOACTIVE GROUPS

Two ''targeted bidentate'' photoaffinity cross-linking reagents, the m onoanhydride of 8-N3ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-P-32]8-N3ATPgammaBP) and the monoanhydride of 8-N3GDP with N-( 4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-P-32] 8-N3GTPgammaBP), were developed for studying the inter- and intramolecular interactions of nucleotide-binding proteins. Experiments using these bidentate rea gents with two photoactive groups led to specific cross-linking: [gamm a-P-32]8-N3GTPgammaBP and [gamma-P-32]8-N3ATPgammaBP showed intersubun it cross-linking of glutamate dehydrogenase and [gamma-P-32]8-N3GTPgam maBP appeared to cross-link the alpha- and beta-subunits of tubulin. T he non-azido ''monodentate'' versions of these reagents, the monoanhyd ride of ADP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-P-3 2]ATPgammaBP) and the monoanhydride of GDP with N-(4-(benzoyl)phenylme thyl)-phosphoramide ([gamma-P-32]GTPgammaBP), were also synthesized an d characterized. The ability of these monodentate reagents with one ph otoactive group to serve as photoaffinity probes was established by ph otolabeling specifically the exchangeable GTP-binding domain of tubuli n with [gamma-P-32[GTPgammaBP and the ATP-binding domain of purified a denylate kinase and several nucleotide-binding proteins in human brain homogenate with [gamma-P-32]ATPgammaBP.