Kinetic evidence for an activation step following binding of human interferon alpha 2 to the membrane receptors of Daudi cells.
Kinetic evidence for an activation step following binding of human interferon alpha 2 to the membrane receptors of Daudi cells.
复制标题
人干扰素 α2 与 Daudi 细胞膜受体结合后激活步骤的动力学证据。
DOI：
10.1111/j.1432-1033.1983.tb07575.x
复制
发表时间：
1983
期刊：
影响因子：
--
通讯作者：
M. Bandu
中科院分区：
文献类型：
--
作者：
K. Mogensen;M. Bandu
用~(125)I高掺入法标记一种单一的人干扰素α，用于B淋巴母细胞系Daudi的结合研究，该细胞系的生长被低剂量的干扰素抑制，这种作用在大约100U/ml(25 Pm)时达到饱和。放射性标记的干扰素被证明是完全活性的，并且通过碘标记显示与细胞部位的结合亲和力没有变化。实验条件被标准化，使得结合和细胞生长实验可以在相同的细胞初始培养上进行。将125I标记的干扰素α2(从携带人α2基因的大肠杆菌中制备的干扰素α)加入到指数生长的培养物中(平均比生长率0.77±0.07d-1)，平均浓度为235000+/-20000个细胞·ml-1。在生长中的培养物上可以观察到两种类型的结合：第一种是短暂的结合峰，然后是有效位点的丢失或释放；第二种是在加入干扰素后大约3小时达到平衡。表观离解常数有很大的差异。在“稳态”结合的亲和力似乎要高得多。结合干扰素的置换率分析表明，在研究的整个剂量范围内(1-100U/ml；0.25-25 pm干扰素)，这两种反应是连续发生的。在这个剂量范围内，我们发现Daudi细胞在所有剂量下最终都会停止生长，细胞生长减速率与干扰素的剂量成双倒数关系图(即类似于Michaelis-Menten动力学)。结合和抑制生长的平衡常数之间有很好的一致性(分别为2.65和2.39)。因此，稳定状态下的干扰素结合量决定了生长受到抑制的速率。我们认为，第一个反应是干扰素与表面受体的结合，第二个反应是干扰素转移到细胞膜上的活化复合体。讨论了合适的模型及其对干扰素作用的普遍适用性。
A single species of human interferon alpha (IFN alpha) was labelled with 125I to high incorporation for binding studies on the B-lymphoblastoid cell line, Daudi, whose growth is inhibited by low doses of IFN, the effect being saturated at about 100 U/ml (25 pM). The radiolabelled IFN was shown to be fully active and the binding affinity to cellular sites was shown to be unchanged by iodination. Experimental conditions were standardized such that binding and cell growth experiments could be performed on the same initial culture of cells. 125I-labelled IFN alpha 2 (IFN alpha prepared from Escherichia coli carrying human alpha 2 gene) was added to exponentially growing cultures (mean specific growth rate 0.77 +/- 0.07 days-1) at a mean concentration of 235000 +/- 20000 cells ml-1. Two types of binding could be discerned on growing cultures: the first with a transient peak followed by a loss or discharge of available sites, the second reaching equilibrium some 3 h after the addition of IFN. Large differences in the apparent dissociation constants were evident. The affinity of binding at the 'steady-state', appeared to be much higher. An analysis of the displacement rates for bound IFN suggested that the two reactions were occurring consecutively over the whole of the dose range studied (1-100 U/ml; 0.25-25 pM IFN). In this dose range we found that Daudi cells would eventually stop growing at all doses and that the rates of deceleration of cellular growth were linearly proportional to the dose of IFN in a double-reciprocal plot (i.e. in analogy to Michaelis-Menten kinetics). A good congruence was found between the equilibrium constants for binding and for growth inhibition (2.65 pM and 2.39 pM, respectively). The amount of IFN bound at steady state thus determines the rate at which growth is inhibited. We propose that the first reaction represents binding of IFN to surface receptors, and the second transfer of IFN to an activation complex on the cell membrane. Appropriate models and their general applicability to IFN action are discussed.
DOI：
--
发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Joshi,AR;Sarkar,FH;Gupta,SL
通讯作者：
Gupta,SL
DOI：
--
发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Branca,AA;Baglioni,C
通讯作者：
Baglioni,C
干扰素与细胞受体的相互作用。
DOI：
--
发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Branca,AA;Faltynek,CR;D'Alessandro,SB;Baglioni,C
通讯作者：
Baglioni,C