Identification of intracellular degradation intermediates of aldolase B by antiserum to the denatured enzyme.
Identification of intracellular degradation intermediates of aldolase B by antiserum to the denatured enzyme.
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通过针对变性酶的抗血清鉴定醛缩酶 B 的细胞内降解中间体。
DOI：
10.1073/pnas.82.18.6114
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发表时间：
1985
影响因子：
11.1
通讯作者：
Gershon,D
中科院分区：
文献类型：
--
作者：
Reznick,AZ;Rosenfelder,L;Shpund,S;Gershon,D
已经开发了一种使我们能够鉴定果糖-二磷酸醛缩酶B（D-果糖-1，6-二磷酸D-甘油醛-3-磷酸-裂解酶，EC 4.1.2.13）的细胞内降解中间体的方法。该方法基于使用针对彻底变性的纯化的醛缩酶的抗体。这种抗体已被证明只识别变性分子，它不与“天然”酶相互作用。将肝和肾匀浆的上清液（24，000 × g，30分钟）与变性酶的抗血清一起孵育。将由此形成的抗原-抗体沉淀物进行NaDodSO 4/PAGE，然后电转移到硝酸纤维素纸上，并用变性酶的抗血清和125 I标记的蛋白A进行免疫修饰。在肝脏中可鉴定出7个分子量在38，000（完整亚基的分子量）至18，000之间的肽段，其与变性果糖二磷酸醛缩酶发生抗原性交叉反应。最长的三个肽也存在于肾脏中。这些肽是均质化产物的可能性被排除如下：在肝脏均质化之前，将125 I标记的标记的纯化的天然醛缩酶加入到缓冲液中。然后对匀浆进行NaDodSO 4/PAGE，然后进行放射自显影，标记的酶显示保持完整。该方法建议在其他细胞蛋白质的降解产物的搜索中普遍使用。
A method has been developed that enables us to identify intracellular degradation intermediates of fructose-bisphosphate aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). This method is based on the use of antibody against thoroughly denatured purified aldolase. This antibody has been shown to recognize only denatured molecules, and it did not interact with "native" enzyme. supernatants (24,000 X g for 30 min) of liver and kidney homogenates were incubated with antiserum to denatured enzyme. The antigen-antibody precipitates thus formed were subjected to NaDodSO4/PAGE, followed by electrotransfer to nitrocellulose paper and immunodecoration with antiserum to denatured enzyme and 125I-labeled protein A. Seven peptides with molecular weights ranging from 38,000 (that of the intact subunit) to 18,000, which cross-reacted antigenically with denatured fructose-bisphosphate aldolase, could be identified in liver. The longest three peptides were also present in kidney. The possibility that these peptides were artifacts of homogenization was ruled out as follows: 125I-labeled tagged purified native aldolase was added to the buffer prior to liver homogenization. The homogenates were than subjected to NaDodSO4/PAGE followed by autoradiography, and the labeled enzyme was shown to remain intact. This method is suggested for general use in the search for degradation products of other cellular proteins.