DETECTION, RESOLUTION, AND NOMENCLATURE OF MULTIPLE UBIQUITIN CARBOXYL-TERMINAL ESTERASES FROM BOVINE CALF THYMUS
DETECTION, RESOLUTION, AND NOMENCLATURE OF MULTIPLE UBIQUITIN CARBOXYL-TERMINAL ESTERASES FROM BOVINE CALF THYMUS
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DOI：
10.1021/bi00427a024
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发表时间：
1989-01-10
期刊：
影响因子：
2.9
通讯作者：
WILKINSON, KD
中科院分区：
文献类型：
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作者：
MAYER, AN;WILKINSON, KD
在体内，泛素可以自由存在，也可以通过其羧基末端与α结合。和。-多种细胞蛋白质的氨基。泛素羧基末端水解活性可能是泛素辅因子从泛素、sbd、蛋白偶联物再生的必要步骤。此外，这种类型的活性需要从唯一已知的基因产物：多蛋白前体和各种泛素融合蛋白中产生活性的单体泛素。因此，这种活性对利用泛素作为辅助因子的系统至关重要。一个通用的底物，泛素乙酯，先前开发[威尔金森，k.d., Cox, m.j., Mayer, a.n .，和Frey， T.（1986）生物化学25,6644.sbd。[6649]并在此用于监测小牛胸腺中这些活性的分离。通过快速高效液相色谱法，鉴定和分离了四种不同的泛素特异性酯酶。除了从兔网织红细胞中纯化的泛素羧基末端水解酶的牛同源物外，先前未描述的活性已被分解和表征。另外两种活性类似于以前在粗提取物中检测到但未纯化的解结活性。这些活性似乎形成了一个机械相关的水解酶家族。所有四种活性都被碘乙酰胺抑制，表明必需巯基的存在，并在不同程度上被锰抑制。从观察到的低Km值（0.6 sbd）判断，它们都具有特异性的泛素结合位点。30 .mu.M)。泛素的羧基末端醛是这些酶活性的有效抑制剂，其Ki值比相应的Km值低约1000倍。泛素乙酯的使用为这些酶活性的纯化和这一重要酶群成员之间已知和假定的差异的比较提供了一种快速、定量的分析方法。为了便于比较不同实验室的研究，提出了这些酶活性及其底物的系统命名。这种命名法清楚地表明了催化反应的性质和用于检测各种活性的底物，并应避免先前出现的多种命名法的特质。这些结果将极大地促进从单一哺乳动物来源中纯化和表征这一重要酶家族的蛋白质和基因。
In vivo, ubiquitin exists both free and conjugated through its carboyl terminus to the .alpha.- and .epsilon.-amino groups of a wide variety of cellular proteins. Ubiquitin carboxyl-terminal hydrolytic activity is likely a necessary step in the regeneration of the ubiquitin cofactor from ubiquitin.sbd.protein conjugates. In addition, this type of activity is required to generate the active, monomeric ubiquitin from the only known gene products: the polyprotein precursor and various ubiquitin fusion proteins. Thus, this activity is of vital importance to systems that utilize ubiquitin as a cofactor. A generic substrate, ubiquitin ethyl ester, was previously developed [Wilkinson, K. D., Cox, M. J., Mayer, A. N., and Frey, T. (1986) Biochemistry 25, 6644.sbd.6649] and utilized here to monitor the fractionation of these activities from calf thymus. By use of a rapid HPLC assay, four distinct, ubiquitin-specific esterases were identified and separated. A previously undescribed activity has been resolved and characterized, in addition to the bovine homologue of ubiquitin carboxyl-terminal hydrolase purified from rabbit reticulocytes. Two other activities resemble deconjugating activities previously detected in crude extracts but not previously purified. These activities appear to form a family of mechanistically related hydrolases. All four activities are inhibited by iodoacetamide, indicating the presence of an essential thiol group, and are inhibited to various extents by manganese. All have specific ubiquitin binding sites as judged by the low observed Km values (0.6.sbd.30 .mu.M). The carboxyl-terminal aldehyde of ubiquitin is a potent inhibitor of these enzyme activities, with Ki values approximately 1000-fold lower than the respective Km values. The use of ubiquitin ethyl ester provides a rapid, quantitative assay for purification of these enzyme activities and for the comparison of the known and postulated differences between the members of this important group of enzymes. A systematic nomenclature for these enzyme activities and their substrates is proposed in order to facilitate comparison of studies by various laboratories. This nomenclature clearly indicates the nature of the reactions catalyzed and the substrates used to detect the varous activities and should avoid the idiosyncrasies of the multiple nomenclatures which have arisen previously. These results should greatly facilitate the purification and characterization of the proteins and the genes of this important family of enzymes from a single mammalian source.