Photoaffinity heterobifunctional cross-linking reagents based on N-(azidobenzoyl)tyrosines.
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]-beta-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]-beta-alanate (16C) was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was successfully photo-cross-linked, in a calcium-dependent 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.
Imai
N
N
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506.
Kometani
T
T
Crocker
P J
PJ
Bowdan
J B
JB
Demir
A
A
Dwyer
L D
LD
Mann
D M
DM
Vanaman
T C
TC
Watt
D S
DS
D000345:Affinity Labels / Q000138:chemical synthesis* / Q000737:chemistry; D001386:Azides / Q000737:chemistry*; D017301:Ca(2+) Mg(2+)-ATPase / Q000097:blood; D000252:Calcium-Transporting ATPases / Q000097:blood; D002147:Calmodulin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000138:chemical synthesis* / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry*; D004910:Erythrocyte Membrane / Q000201:enzymology; D006801:Humans; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009579:Nitrobenzoates / Q000737:chemistry*; D011485:Protein Binding
D000345:Affinity Labels; D001386:Azides; D002147:Calmodulin; D003432:Cross-Linking Reagents; D004151:Dipeptides; D007202:Indicators and Reagents; D009579:Nitrobenzoates; C069109:succinimidyl N-(N'-(3-azido-5-nitrobenzoyl)tyrosyl)-beta-alanate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases