Preparation and properties of two active forms of ribonuclease dimer.
Preparation and properties of two active forms of ribonuclease dimer.
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核糖核酸酶二聚体两种活性形式的制备和性质。
DOI：
10.1016/s0021-9258(18)97123-1
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发表时间：
1965
期刊：
影响因子：
--
通讯作者：
A. M. Crestfield
中科院分区：
文献类型：
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作者：
R. Fruchter;A. M. Crestfield
当牛胰腺核糖核酸酶A从507，冰醋酸和产物在磷酸盐缓冲液中溶解时，通过Sephadex G-75(1)的凝胶过滤，可以分离出具有酶活性的二聚体、四聚体和更高的聚集体。用超速离心法测定了从Sephadex柱中分离得到的组分的相对分子质量。在0.2M磷酸盐缓冲液中，单体是酶最稳定的形式，因为当聚集体的混合物加热到65英寸以上，然后冷却时，得到的溶液只含有单体。聚集体在磷酸根离子存在下比在氯化钠或醋酸盐缓冲溶液中更稳定。比活度数据表明，从Sephadex柱中分离的二聚体每个二聚体分子含有两个活性中心。与单体(2)中的活性部位类似，假定每个活性部位由一个组氨酸-12残基和一个组氨酸-119残基组成。原则上，这样的二聚体可以通过两个单体的并置形成，每个单体的活性部位保持不变，或者通过两个核糖核酸酶分子相互缠绕，从而使一个分子的关键组氨酸-12残基与另一个分子的基本组氨酸-119残基相邻。本文提出了一个与Vithayathil和Richards(3)提出的类似于Vithayathil和Richards(3)所提出的具有这种复合活性中心的二聚体的形成机制，以解释核糖核酸酶S-肽和无活性的1-羧甲基组氨酸-119型核糖核酸酶如何在碱中部分解离形成活性产物。他提出的聚集机制的一个优点是，它可以通过几种方式进行实验测试。例如，在非活性的1-羧甲基、组氨酸-119和3-羧甲基组氨酸-12衍生物的混合物的50yc的冰醋酸冷冻干燥后，应该可以形成活性的杂化聚集体。已经证实了这种活性杂化产物的形成，这种杂化产物在热分解时失去活性(4)。它也应该可以通过与碘乙酸酯反应使二聚体失活，并预测这样形成的不活跃的烷基化产物的性质。本文件和所附文件中介绍的工作就是为了测试这一可能性。作为第一步，有必要对二聚体部分的均一性进行进一步的研究，因为在初步实验中。S，在离子交换层析中发现有两个组分分离。本文介绍了导致这一现象的研究结果。
When bovine pancreatic ribonuclease A is lyophilized from 507, acetic acid and the product is dissolved in phosphate buffer, enzymically active dimers, tetramers, and higher aggregates may be isolated by gel filtration on Sephadex G-75 (1). The molecular weights of the fractions isolated from the Sephadex column were established by ultracentrifugation. In 0.2 M phosphate buffer the monomer is the most stable form of the enzyme, for when the mixture of aggregates is heated above 65” and then cooled, the resulting solution contains only monomer. The aggregates are more stable in the presence of phosphate ions than in solutions of NaCl or acetate buffer. The specific activity data indicate that the dimer isolated from Sephadex columns contains two active sites per dimeric molecule. Each active site is assumed to consist of a histidine-12 residue and a histidine-119 residue, by analogy with the active site in monomer (2). Such a dimer can be formed, in principle, either by the juxtaposition of two monomers, each with its active site intact, or by the intertwining of 2 molecules of ribonuclease so as to bring the crucial histidine-12 residue from 1 molecule adjacent to the essential histidine-119 residue of the other. A mechanism was suggested for the formation of a dimer with such composite active sites (1) which is similar to the one proposed by Vithayathil and Richards (3) to explain how an active product is formed upon mixing ribonuclease S-peptide with inactive 1-carboxymethylhistidine-119 ribonuclease which had been partially unfolded by t. reatment with alkali. An advantage of t, he proposed mechanism of aggregation is that it can be tested experimentally in several ways. For example, it should be possible to form active hybrid aggregates upon lyophilization from 50yc acetic acid of a mixture of the inactive 1-carboxymet, hylhistidine-119 and 3-carboxymethylhistidine-12 derivatives. The formation of such active hybrids, which lose activity on thermal dissociation, has been demonstrated (4).It should also be possible to inactivate the dimer by reaction with iodoacetate and to predict the nature of the inactive, alkylated products so formed. The work presented in, this and the accompanying paper was undertaken in order to test this possibility. As a first step, it was necessary to carry out further studies on the homogeneity of the dimer fraction because, in preliminary experiment. s, it was found that a separation into two components occurred upon ion exchange chromatography. In this paper are presented the results of studies leading to the