Abstract
The potential of histidines to modulate the binding of agonists and antagonists to human platelet thromboxane A 2 (TXA 2) receptors was investigated. TXA 2 receptors were purified from crude platelet membranes via affinity and wheat germ lectin chromatography. Radioligand binding studies were conducted using the TXA 2, mimetic [ 125I]BOP ( I-BOP = [1S-(lα,2β(5 Z),3α(1E,3 R ∗),4α)]-7-[3-(3- hydroxy -4-(4′- iodophenoxy)-1- butenyl)7- oxabicyclo-[2.2.1] heptan-2- yl]-5- heptenoic acid) and the TXA, receptor antagonist [ 125I]SAP (I-SAP = 7-[(1 R,2 S,3 S,5 R)-6,6-dimethyl-3-(4-iodobenzenesulfonylamino)-bicyclo-[3.1.1] hept-2-yl]-(5Z)-heptenoic acid). The histidine modifying reagent diethylpyrocarbonate (DEPC) produced a concentration (30–100 μM) dependent inhibition of binding of both [ 125I]BOP and [ 125I]SAP. DEPC treatment significantly ( P < 0.05, N = 6) decreased the affinity of the receptor for [ 125I]SAP ( K d = 2.4 ± 0.4 and 5.4 ± 0.4 nM, control and DEPC, respectively) without significantly decreasing the B max. The effects of DEPC were reversed by hydroxylamine. The inhibition of [ 125I]BOP and [ 125I]SAP binding produced by DEPC was reduced significantly by prior incubation of the purified receptors with the TXA 2 receptor agonist U-46619 or the TXA 2 receptor antagonist SO 29548. The results strongly support the notion that one or more histidines reside in a domain that can modulate ligand binding to the TXA 2 receptor.
Published Version
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