REAL-TIME ANALYSIS OF THE ASSEMBLY OF LIGAND, RECEPTOR, AND G PROTEIN BY QUANTITATIVE FLUORESCENCE FLOW-CYTOMETRY
REAL-TIME ANALYSIS OF THE ASSEMBLY OF LIGAND, RECEPTOR, AND G PROTEIN BY QUANTITATIVE FLUORESCENCE FLOW-CYTOMETRY
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DOI：
10.1021/bi00234a033
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发表时间：
1991-05-21
期刊：
影响因子：
2.9
通讯作者：
SKLAR, LA
中科院分区：
文献类型：
--
作者：
FAY, SP;POSNER, RG;SKLAR, LA
我们描述了使用荧光流式细胞术定量分析荧光肽配体(L)，受体(R)和G蛋白(G)之间相互作用的一般方法。该方案依赖于使用市售荧光微珠作为标准来校准溶液中荧光肽的浓度和悬浮细胞上的受体数量。我们描述了一个荧光甲酰基肽家族，并分析了地黄苷渗透人中性粒细胞中配体甲酰基肽-受体相互作用的稳态和动态方面。详细的受体结合研究是用五肽n -甲酰基- met - leu - ph - ph - lys -荧光素进行的。平衡研究表明，GTP[S]导致了大约两个数量级的结合亲和力损失，分别从大约0.04 nM （LRG）到大约3 nM （LR）。动力学研究表明，这种亲和力的变化主要是由于解离速率常数从大约1 X 10(-3) s-1 （LRG）增加到大约1 X 10(-1) s-1 （LR）。相比之下，鸟嘌呤核苷酸存在和不存在时的关联速率常数（约3 X 10(7) s-1 M-1）在统计学上无法区分，接近扩散极限。在鸟嘌呤核苷酸（LR）存在的情况下，单步可逆结合模型充分拟合了动力学数据。在缺乏鸟嘌呤核苷酸的情况下，并非所有受体都能快速进入G形成LRG三元复合物。从数学上讲，那些快速接近G的R要么是预耦合到R，要么是G与R的关联比L与R的关联快。
We describe a general approach for the quantitative analysis of the interaction among fluorescent peptide ligands (L), receptors (R), and G proteins (G) using fluorescence flow cytometry. The scheme depends upon the use of commercially available fluorescent microbeads as standards to calibrate the concentration of fluorescent peptides in solution and the receptor number on cells in suspension. We have characterized a family of fluoresceinated formyl peptides and analyzed both steady-state and dynamic aspects of ligand formyl peptide-receptor interactions in digitonin-permeabilized human neutrophils. Detailed receptor-binding studies were performed with the pentapeptide N-formyl-Met-Leu-Phe-Phe-Lys-fluorescein. Equilibrium studies showed that GTP[S] caused a loss of binding affinity of approximately two orders of magnitude, from approximately 0.04 nM (LRG) to approximately 3 nM (LR), respectively. Kinetic studies revealed that this change in affinity was principally due to an increase in the dissociation rate constants from approximately 1 X 10(-3) s-1 (LRG) to approximately 1 X 10(-1) s-1 (LR). In contrast, the association rate constants in the presence and absence of guanine nucleotide (approximately 3 X 10(7) s-1 M-1) were statistically indistinguishable and close to the diffusion limit. In the presence of guanine nucleotide (LR), the kinetic data were adequately fit by a single-step reversible-binding model. In the absence of guanine nucleotides, not all receptors have rapid access to G to form the LRG ternary complex. Mathematically, those R that have rapid access to G are either precoupled to R or the association of G with R is fast compared to the association of L with R. The physiological consequences of coupling heterogeneity are discussed.