TY - JOUR
T1 - Photoaffinity Heterobifunctional Cross-Linking Reagents Based on N-(Azidobenzoyl) tyrosines
AU - Imai, Nobuyuki
AU - Kometani, Tadashi
AU - Crocker, Peter J.
AU - Bowdan, Jean B.
AU - Demir, Ayhan
AU - Dwyer, Lori D.
AU - Mann, Dennis M.
AU - Vanaman, Thomas C.
AU - Watt, David S.
PY - 1990/3/1
Y1 - 1990/3/1
N2 - New heterobifunctional cross-linking reagents that possessed a photoactive terminus, an electrophilic terminus, and a linking arm between the two termini that had a radiolabeled, enzymatically cleavable bond were synthesized. In a model study, succinimidyl N-[N′-(4-azidobenzoyl)tyrosyl]-β-alanate (16A) was coupled to n-butylamine (a Lys surrogate), iodinated, and cleaved with chymotrypsin in the presence of tyrosylamide to afford the desired adduct N-(N′-(4-azidobenzoyl)-3-iodotyrosyl)tyrosinamide, thereby demonstrating the feasibility of the enzymatic cleavage. In a biochemical study, succinimidyl N-[N′-(3-azido-5-nitrobenzoyl)tyrosyl]-β-alanate (16C) was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was successfully photo-cross-linked, in a calciumdependent manner, to the human erythrocyte plasma membrane Ca2+,Mg2+-ATPase and to a synthetic fragment (M13) containing the CaM-binding region of myosin light-chain kinase. In the latter case, densitometry readings indicated 20% cross-linking efficiency.
AB - New heterobifunctional cross-linking reagents that possessed a photoactive terminus, an electrophilic terminus, and a linking arm between the two termini that had a radiolabeled, enzymatically cleavable bond were synthesized. In a model study, succinimidyl N-[N′-(4-azidobenzoyl)tyrosyl]-β-alanate (16A) was coupled to n-butylamine (a Lys surrogate), iodinated, and cleaved with chymotrypsin in the presence of tyrosylamide to afford the desired adduct N-(N′-(4-azidobenzoyl)-3-iodotyrosyl)tyrosinamide, thereby demonstrating the feasibility of the enzymatic cleavage. In a biochemical study, succinimidyl N-[N′-(3-azido-5-nitrobenzoyl)tyrosyl]-β-alanate (16C) was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was successfully photo-cross-linked, in a calciumdependent manner, to the human erythrocyte plasma membrane Ca2+,Mg2+-ATPase and to a synthetic fragment (M13) containing the CaM-binding region of myosin light-chain kinase. In the latter case, densitometry readings indicated 20% cross-linking efficiency.
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U2 - 10.1021/bc00002a008
DO - 10.1021/bc00002a008
M3 - Article
C2 - 2151309
AN - SCOPUS:0025398283
VL - 1
SP - 138
EP - 143
IS - 2
ER -