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首页> 美国卫生研究院文献>Biochemical Journal >Interaction of phosphorylase b with eosin. Influence of substrate and effectors on eosin-enzyme complexes.
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Interaction of phosphorylase b with eosin. Influence of substrate and effectors on eosin-enzyme complexes.

机译：磷酸化酶b与曙红的相互作用。底物和效应物对曙红-酶复合物的影响。

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The interactions of rabbit muscle glycogen phosphorylase b with Eosin (2',4',5',7'-tetrabromofluorescein) was studied. Eosin was found to be an effective inhibitor of the enzyme. The inhibition constants for the dye were estimated to be approx. 36 and 60 microM with respect to AMP and glucose 1-phosphate respectively. The binding of Eosin to phosphorylase b is accompanied by a red-shift of about 12 nm in the dye absorption-spectrum maximum, indicating low-polarity binding sites on the enzyme molecule for the dye. The absorbance in the difference absorption maximum at 537 nm was utilized to follow the conjugation of phosphorylase b with Eosin. Scatchard plots of the titration data revealed the existence of at least two classes of binding sites on the protein molecule for Eosin, and the dissociation constants measured in Tris/HCl buffer, pH 7.0 (IO.091), were 7.7 and 41.7 microM respectively. The influence of the substrates and effectors on Eosin-enzymes complexes was used to study the ligand-phosphorylase b interactions. IMP displaced the dye completely from the enzyme, indicating that there are two IMP-binding sites per phosphorylase b monomer. AMP binding to the enzyme with respect to Eosin concentration is of two types: a non-competitive one for the high-affinity site for AMP and a competitive one for the low-affinity site for the activator. The effects of glucose 6-phosphate, ATP, Pi and glycerol 2-phosphate in the system are in according dance with a partially competitive model. Glucoes 1-phosphate and UDP-glucose appear to affect only the high-affinity site for Eosin, whereas glucose and glycogen have no effect on Eosin-phosphorylase b complexes. Our results suggest that Eosin can be used as an efficient optical probe for studying the phosphorylase b system.
机译：研究了兔肌肉糖原磷酸化酶b与曙红（2'，4'，5'，7'-四溴荧光素）的相互作用。发现曙红是该酶的有效抑制剂。该染料的抑制常数估计为约。对于AMP和1-磷酸葡萄糖分别为36和60 microM。曙红与磷酸化酶b的结合伴随着染料吸收光谱最大值中约12 nm的红移，表明该染料的酶分子上的低极性结合位点。利用在537nm处的最大吸收差异吸收来跟踪磷酸化酶b与曙红的缀合。滴定数据的Scatchard图揭示了在曙红蛋白分子上存在至少两类结合位点，在Tris / HCl缓冲液pH 7.0（IO.091）中测得的解离常数分别为7.7和41.7 microM。底物和效应物对曙红-酶复合物的影响用于研究配体-磷酸化酶b的相互作用。 IMP完全使染料脱离了酶，表明每个磷酸化酶b单体有两个IMP结合位点。关于曙红浓度，AMP与酶的结合有两种类型：一种是针对AMP的高亲和力位点的非竞争性，另一种是针对激活剂的低亲和力位点的竞争性。系统中6-磷酸葡萄糖，ATP，Pi和2-磷酸甘油的影响与部分竞争性模型一致。 1-磷酸葡糖和UDP-葡萄糖似乎只影响曙红的高亲和力位点，而葡萄糖和糖原对曙红-磷酸化酶b复合物没有影响。我们的结果表明，曙红可以用作研究磷酸化酶b系统的有效光学探针。

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