The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis.
The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis.
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DOI：
10.1016/s0021-9258(18)34024-9
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发表时间：
1982-09
期刊：
影响因子：
--
通讯作者：
A. Haas;I. A. Rose
中科院分区：
文献类型：
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作者：
A. Haas;I. A. Rose
材料和方法通过扩展以前的方法，从人红细胞中纯化出均一的subiquitin（14，15）。相对于通过氨基酸分析（5）确定的标准物测量泛素储备溶液的浓度，并基于8565的分子量计算。用氯胺-T法用I2 ′ I标记一部分纯化的泛素（3）。天然泛素和碘化泛素对于活化酶的功能等效性先前已通过在使用I-泛素、[γ-P] ATP或[γ-H] ATP的化学计量研究中发现的良好一致性得到证实（13）。无载体NaI 2 ′ I获自阿默舍姆;[ynZP] ATP、[G-11 HIAMP]、[2，8- 11 HIATP]和Nar ′ PP获自新英格兰核公司。通过苯肼诱导成年雄性家兔获得富含网织红细胞的全血（16），并用于制备如前所述的组分I1（1）。通过共价亲和色谱法从级分I1中纯化泛素活化酶至表观均一性，调节至0.1mM二硫苏糖醇和1 mg/ml牛血清白蛋白以增强稳定性，然后如所述充分透析（13）。将酶分成小的等分试样并在-80 ℃下储存。尽管当在二硫苏糖醇和牛血清白蛋白存在下于-80 ℃储存时，该酶保持完全活性至少8个月，但在反复冷冻和解冻时观察到活性略有损失。因此，所有实验均使用仅解冻一次的等分试样进行。在[：'H] ATP和焦磷酸酶存在下，通过泛素[，' H]腺苷酸形成的程度来定量活化酶（13）。在37 ℃下在含有50 mMTris-Cl的50 μ l终体积中形成，同位素交换试验-所有同位素交换试验均为perpH7.6、10 mMMgCl 2、0.1mM二硫苏糖醇和0.27 μ mol活化酶。ATP、AMP、PP和泛素的浓度如附图图例所述。选择孵育时间以仅测量进展曲线的线性部分内的初始交换速度，因此，不需要对接近平衡进行校正。对于ATP：PP，交换，通过将ATP吸附到木炭上来确定12 PP掺入ATP中。通过添加含有4 mM载体PP的0.5ml 5%（w/v）三氯乙酸，随后添加0.3ml 10%（w/v）木炭在2%三氯乙酸中的浆液来淬灭反应。离心后，用3 × 1 ml 2%三氯乙酸洗涤活性炭颗粒，并通过切伦科夫辐射直接测定结合放射性（13）。数据针对对照测定进行校正，其中酶已被省略，并且通常表示< 0.2%的添加的放射性（4-6 X 107总cpm）。测定重复进行两次，并始终在其平均值的5%范围内一致。在不存在泛素的情况下没有观察到交换（未显示）。对于ATP：AMP交换，用55 μ 1含有0.3M MgCl 2的0.2M磷酸钾缓冲液（pH 2.0）淬灭反应，
MATERIALS AND METHODSUbiquitin was purified to homogeneity from human erythrocytes by an extension of previous methods (14, 15). Concentrations of ubiquitin stock solutions were measured relative to a standard determined by amino acid analysis (5) and calculated based on a molecular weight of 8565. A portion of the purified ubiquitin was labeled with I2'I by the chloramine-T method (3). The functional equivalence of native and iodinated ubiquitin for the activating enzyme has been demonstrated previously by the good agreement found in stoichiometry studies using'" I-ubiquitin,[y-"'P] ATP, or [:'H] ATP (13). Carrierfree NaI2'I was obtained from Amersham;[ynZP] ATP,[G-" HIAMP,[2, 8-" HIATP, and Nar'" PP, were obtained from New England Nuclear. Reticulocyte-rich whole blood was obtained by phenylhydrazine induction of adult male rabbits (16) and used to prepare fraction I1 as previously described (1). Ubiquitin activating enzyme was purified to apparent homogeneity from fraction I1 by covalent affinity chromatography, adjusted to 0.1 mM dithiothreitol and 1 mg/ml of bovine serum albumin to enhance stability, then extensively dialyzed as described (13). The enzyme was divided into small aliquots and stored at-80" C. Although the enzyme retains full activity for at least 8 months when stored at-80" C in the presence of dithiothreitol and bovine serum albumin, a slight loss of activity is observed with repeated freezing and thawing. Therefore, all experiments were performed with aliquots thawed only once. Activating enzyme was quantitated by the extent of ubiquitin [,'H] adenylate formation in the presence of [:'H] ATP and pyrophosphatase (13). formed at 37" C in a final volume of 50 pl containing 50 mM Tris-C1, Isotope Exchange Assays-All isotope exchange assays were perpH 7.6, 10 mM MgCI2, 0.1 mM dithiothreitol, and 0.27 pmol of activating enzyme. Concentrations of ATP, AMP, PP,, and ubiquitin were as described in the accompanying figure legends. Incubation times were chosen to measure only initial exchange velocities within the linear portion of the progress curve and, therefore, required no correction for approach to equilibrium.For ATP: PP, exchange, the incorporation of" 2PP, into ATP was determined by adsorbing the latter onto charcoal. Reactions were quenched by addition of 0.5 ml5%(w/v) trichloroacetic acid containing 4 mM carrier PP, followed by 0.3 ml of a lo%>(w/v) slurry of charcoal in 2% trichloroacetic acid. After centrifugaticn the charcoal pellet was washed with 3 X 1 ml 2% trichloroacetic acid and bound radioactivity determined directly by Cerenkov radiation (13). Data were corrected for a control assay from which enzyme had been omitted and that generally represented< 0.2% of added radioactivity (4-6 x 10'total cpm). Assays were performed in duplicate and consistently agreed within 5% of their mean value. No exchange was observed in the absence of ubiquitin (not shown). For ATP: AMP exchange, reactions were quenched with 55 pl of 0.2 M potassium phosphate buffer, pH 2.0, containing 0.3 M MgCI2, and