Ciechanover A, Elias S, Heller H, Hershko A
J Biol Chem. 1982 Mar 10;257(5):2537-42.
We have previously described an enzyme that activates ubiquitin, the heat-stable polypeptide of the ATP-dependent proteolytic system from reticulocytes (Ciechanover, A., Heller, H., Katz-Etzion, R., and Hershko, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 761-765). It carries out ubiquitin-dependent PPi-ATP and AMP-ATP exchange reactions and binds to the activated polypeptide by a thiolester linkage. We describe here a procedure for the purification of this enzyme by its binding to ubiquitin-Sepharose. Binding of the enzyme to the affinity column requires ATP and Mg2+, and bound enzyme cannot be displaced by high salt but can be eluted by raising the pH, by increased concentrations of a thiol compound, or by the joint supplementation of AMP and pyrophosphate. Another form of the enzyme that cannot carry out AMP-ATP exchange (but catalyzes ubiquitin-dependent PPi-ATP exchange) does not bind to the affinity column. These data indicate that a covalent, possibly thiolester intermediate, is formed between the activating enzyme and Sepharose-bound ubiquitin. It is suggested designating this procedure of enzyme isolation "covalent affinity" chromatography. The purified enzyme has an apparent Mr = 210,000 and appears to be composed of two subunits of Mr = 105, 000. ATP-dependent binding of ubiquitin to the purified enzyme and to its subunit is demonstrated.
我们之前描述过一种能激活泛素的酶，它是来自网织红细胞的ATP依赖型蛋白水解系统中的热稳定多肽（Ciechanover, A., Heller, H., Katz-Etzion, R., and Hershko, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 761 - 765）。它能进行依赖泛素的焦磷酸-ATP和AMP-ATP交换反应，并通过硫酯键与活化的多肽结合。我们在此描述一种通过其与泛素-琼脂糖结合来纯化这种酶的方法。酶与亲和柱的结合需要ATP和Mg2+，结合的酶不能被高盐洗脱，但可以通过提高pH值、增加硫醇化合物的浓度或联合添加AMP和焦磷酸来洗脱。另一种不能进行AMP-ATP交换（但催化依赖泛素的焦磷酸-ATP交换）的酶形式不与亲和柱结合。这些数据表明，在活化酶与琼脂糖结合的泛素之间形成了一种共价的、可能是硫酯的中间体。建议将这种酶分离方法命名为“共价亲和”色谱法。纯化后的酶表观分子量为210,000，似乎由两个分子量为105,000的亚基组成。已证明泛素对纯化后的酶及其亚基的ATP依赖型结合。
