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首页> 外文期刊>Biochimica et biophysica acta. Biomembranes >Steady-state binding of [~3H]ATP to rat liver plasma membranes and competition by various purinergic agonists and antagonists
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Steady-state binding of [~3H]ATP to rat liver plasma membranes and competition by various purinergic agonists and antagonists

机译:[〜3H] ATP与大鼠肝细胞膜的稳态结合以及各种嘌呤能激动剂和拮抗剂的竞争

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摘要

Steady-state analysis of nucleotide-binding sites on rat liver plasma membranes was carried out using ~3H-labelled ATP as radioligand under complete inhibition of ecto-ATPase activity be excess EDTA. Binding of [~3H]ATP to the membranes is saturable, reversible and apparently involves one population of specific binding sites with K_d of about 90 nM and binding capacity (B_(max)) of 15 pmol/mg protein. A broad spectrum of purinergic agonists and antagonists was examined as potential inhibitors of the measured binding. The displacement studies showed the following rank order of inhibitory potency for [~3H]ATP-binding sites (pIC_(50) values in parentheses): ATPγS (7.49) > 2-MeSATP (7.18) > ATP (6.91) ADPβS (6.64) ≥ ADP (6.56) RB2 (6.14) suramin (5.40) Ap_4A (4.57) > α,β-MeATP (4.19) ≥ β,γ-MeATP(3.97). AMP, adenosine, Ap_5A, PPADS, β-glycerophosphate as well as non-adenine nucleoside triphosphates GTP, UTP and CTP did not exert any effect on the measured binding at concentration ranges of 10~(-6)-10~(-4) M. In order to ascertain whether ATP and its analogues are capable of interacting with the same binding domain, 2-MeSATP and ADP were treated as alternative ligands that could compete with unlabelled ATP for its binding sites. A 2-fold increase of K_d value for ATP-receptor interaction was observed in the presence of 2-MeSATP (60 nM) or ADP (250 nM) without any modulation of B_(max) value, confirming that inhibitory effects of these compounds are competitive in nature. These studies demonstrate that ATP and its analogues are able to interact with a single binding domain on liver plasma membranes, which may be identified as ligand-binding component of P2 purinoceptors of the P2Y_1 subtype.
机译:使用〜3H标记的ATP作为放射性配体,在过量EDTA完全抑制胞外ATP酶活性的情况下,对大鼠肝脏质膜上核苷酸结合位点进行稳态分析。 [〜3H] ATP与膜的结合是可饱和的,可逆的,并且显然涉及一组特定的结合位点,其K_d约为90 nM,结合能力(B_(max))为15 pmol / mg蛋白。研究了广泛的嘌呤能激动剂和拮抗剂作为所测结合的潜在抑制剂。置换研究表明,对[〜3H] ATP结合位点的抑制力的排名顺序如下(括号中的pIC_(50)值):ATPγS(7.49)> 2-MeSATP(7.18)> ATP(6.91)ADPβS(6.64) ≥ADP(6.56) RB2(6.14)苏拉明(5.40) Ap_4A(4.57)>α,β-MeATP(4.19)≥β,γ-MeATP(3.97)。 AMP,腺苷,Ap_5A,PPADS,β-甘油磷酸以及非腺嘌呤核苷三磷酸GTP,UTP和CTP在10〜(-6)-10〜(-4)浓度范围内对测得的结合没有任何影响M.为了确定ATP及其类似物是否能够与相同的结合域相互作用,将2-MeSATP和ADP视为可以与未标记的ATP竞争其结合位点的替代配体。在存在2-MeSATP(60 nM)或ADP(250 nM)的情况下,ATP受体相互作用的K_d值增加了2倍,而对B_(max)值没有任何调节,证实了这些化合物的抑制作用本质上具有竞争力。这些研究表明,ATP及其类似物能够与肝脏质膜上的单个结合域相互作用,该结合域可能被鉴定为P2Y_1亚型的P2嘌呤受体的配体结合成分。

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