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Selective binding interactions of deramciclane to the genetic variants of human α1-acid glycoprotein
Ilona Fitos Júlia VisyMiklós Simonyi György MádyFerenc Zsila 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
α1-Acid glycoprotein (AGP) plays a decisive role in the serum protein binding of several drugs.Genetic variants of AGP have different ligand binding properties. The binding of deramciclane (DER), a chiral anxiolytic agent, has been studied on A and F1/S genetic variants of AGP.Methods
The effects of DER and reference drugs on the binding of specific fluorescent and circular dichroism (CD) probes of AGP were determined. Dicumarol (DIC) binding was measured by CD and equilibrium dialysis.Results
DER effectively displaced probes bound to variant A, while it was less effective at displacing probes bound to variant F1/S. DER increased the binding and inverted the induced CD spectrum of DIC in the solution of variant F1/S. This phenomenon could not be brought about by the enantiomer of DER.Conclusion
DER has high-affinity binding (Ka ≥ 2×106 M-1) to variant A, while its binding to the variant F1/S is about thirty times weaker. During simultaneous binding of DER and DIC to variant F1/S a ternary complex having about four times higher affinity is formed, in which the opposite chiral conformation of DIC is favored.General significance
The binding interactions found prove that AGP can simultaneously accommodate different ligand molecules. Even weakly bound ligands can provoke unexpected allosteric protein binding interactions. 相似文献2.
An improved resazurin-based cytotoxicity assay for hepatic cells 总被引:2,自引:0,他引:2
McMillian MK Li L Parker JB Patel L Zhong Z Gunnett JW Powers WJ Johnson MD 《Cell biology and toxicology》2002,18(3):157-173
A simple resazurin-based cytotoxicity assay is presented for screening of cytotoxicity in hepatocytes and liver cell lines.
Human hepatoma (HepG2) cells in 96-well culture plates were exposed to known toxic (cisplatin, 5-fluorouracil, ethionine,
flufenamic acid, and diflunisal) and control (transplatin, 5-chlorouracil, methionine, and acetylsalicylic acid) compounds
for 1–3 days, and resazurin (5 μmol/L) was added. A conventional short-term (1 h) assay was first performed, where cytotoxicity
is indicated by decreased reduction of resazurin to its fluorescent product resorufin. Our improved assay consists of additionally
measuring fluorescence 2–4 days later, when cytotoxicity is indicated by a striking increase in the concentration of resorufin, resulting from two distinct processes. First, viable liver-derived cells slowly convert
resorufin to nonfluorescent metabolites. Fluorescence of control cell wells decreased to background during a 2- to 4-day exposure
to resazurin. This metabolism of resorufin was largely blocked by dicumarol and to lesser extents by disulfiram and SKF525a.
Second, dead or dying cells slowly convert resazurin to resorufin but do not further metabolize resorufin; thus this fluorescent
metabolite accumulates to high levels in wells with dead cells by 2 to 4 days. A similar increase in fluorescence associated
with cytotoxicity was observed in primary cultures of rat hepatocytes using the long-term resazurin-based assay. In addition
to an improved signal relative to the short-term assay, the inversion of the fluorescent signal from high = alive short-term
to high = dead long-term allows determination of two independent cytotoxicity endpoints after addition of one innocuous vital
dye.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
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Mironov AA Colanzi A Polishchuk RS Beznoussenko GV Mironov AA Fusella A Di Tullio G Silletta MG Corda D De Matteis MA Luini A 《European journal of cell biology》2004,83(6):263-279
Dicumarol (3,3'-methylenebis[4-hydroxycoumarin]) is an inhibitor of brefeldin-A-dependent ADP-ribosylation that antagonises brefeldin-A-dependent Golgi tubulation and redistribution to the endoplasmic reticulum. We have investigated whether dicumarol can directly affect the morphology of the Golgi apparatus. Here we show that dicumarol induces the breakdown of the tubular reticular networks that interconnect adjacent Golgi stacks and that contain either soluble or membrane-associated cargo proteins. This results in the formation of 65-120-nm vesicles that are sometimes invaginated. In contrast, smaller vesicles (45-65 nm in diameter, a size consistent with that of coat-protein-I-dependent vesicles) that excluded cargo proteins from their lumen are not affected by dicumarol. All other endomembranes are largely unaffected by dicumarol, including Golgi stacks, the ER, multivesicular bodies and the trans-Golgi network. In permeabilized cells, dicumarol activity depends on the function of CtBP3/BARS protein and pre-ADP-ribosylation of cytosol inhibits the breakdown of Golgi tubules by dicumarol. In functional experiments, dicumarol markedly slows down intra-Golgi traffic of VSV-G transport from the endoplasmic reticulum to the medial Golgi, and inhibits the diffusional mobility of both galactosyl transferase and VSV-G tagged with green fluorescent protein. However, it does not affect: transport from the trans-Golgi network to the cell surface; Golgi-to-endoplasmic reticulum traffic of ERGIC58; coat-protein-I-dependent Golgi vesiculation by AlF4 or ADP-ribosylation factor; or ADP-ribosylation factor and beta-coat protein binding to Golgi membranes. Thus the ADP-ribosylation inhibitor dicumarol induces the selective breakdown of the tubular components of the Golgi complex and inhibition of intra-Golgi transport. This suggests that lateral diffusion between adjacent stacks has a role in protein transport through the Golgi complex. 相似文献
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