K. Rajagopalan et al., SYNTHESIS AND APPLICATION OF BIDENTATE PHOTOAFFINITY CROSS-LINKING REAGENTS - NUCLEOTIDE PHOTOAFFINITY PROBES WITH 2 PHOTOACTIVE GROUPS, The Journal of biological chemistry, 268(19), 1993, pp. 14230-14238
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.