Fibla J, Atrian S, Gonzàlez-Duarte R
Departament de Genètica, Universitat de Barcelona, Spain.
Eur J Biochem. 1993 Jan 15;211(1-2):357-65. doi: 10.1111/j.1432-1033.1993.tb19905.x.
With the use of monoclonal antibodies against alcohol dehydrogenase (ADH) we detected ADH proteolysis in different Drosophila melanogaster tissues during development [Visa, N., Fiblas, J., Santa-Crus, M. C. & Gonzàlez-Duarte R. (1992) J. Histochem. Cytochem. 40, 39-49]. We now report the analysis of this proteolytic activity in crude homogenates and in purified ADH preparations of several Drosophila species. Our results indicate that in non-denaturing IEF gels the proteolytic activity comigrates with native ADH electromorphs of all the species analyzed. In addition, we show that it copurifies with ADH and is responsible for the instability of apparently homogeneous ADH preparations in the presence of SDS. When purified ADH preparations were analyzed, the endogenous proteolytic activity yielded the same banding pattern as that obtained with crude homogenates. Even after rechromatography on Sephacryl S-200, the usual last step in our standard purification protocol, the proteolytic activity remained associated with the ADH fractions. Among the various agents which could explain the ADH-linked proteolytic effect, a pre-existing nicked state of the enzyme or chemical proteolysis have been ruled out. The kinetics observed on pure ADH preparations, the effect of specific protease inhibitors and substrate specificity have led us to ascribe this activity to the subtilase serine-protease family. Given that proteolysis is evident even in rechromatographed Sephacryl S-200 fractions, if incubated in SDS for enough time, we propose two alternative hypotheses to explain this phenomenon. First, the proteolytic activity may come from a protease which is inseparable from the ADH active forms and second, the ADH itself may behave as a subtilase when it adopts a particular conformation. Moreover, the previously reported differential banding pattern during development suggests a role for this activity in vivo, in which fatty acids could produce the inducer effect attributed to SDS in vitro.
利用抗乙醇脱氢酶（ADH）的单克隆抗体，我们在发育过程中检测了不同黑腹果蝇组织中的ADH蛋白水解作用[维萨，N.，菲布拉斯，J.，圣克鲁斯，M. C. & 冈萨雷斯 - 杜阿尔特，R.（1992）《组织化学与细胞化学杂志》40，39 - 49]。我们现在报告对几种果蝇物种的粗匀浆和纯化ADH制剂中这种蛋白水解活性的分析。我们的结果表明，在非变性IEF凝胶中，蛋白水解活性与所分析的所有物种的天然ADH电变体共迁移。此外，我们表明它与ADH共纯化，并且是SDS存在下明显均匀的ADH制剂不稳定的原因。当分析纯化的ADH制剂时，内源性蛋白水解活性产生的条带模式与粗匀浆获得的相同。即使在我们标准纯化方案中通常的最后一步，在Sephacryl S - 200上再色谱分离后，蛋白水解活性仍与ADH组分相关。在可以解释与ADH相关的蛋白水解作用的各种因素中，酶预先存在的切口状态或化学蛋白水解已被排除。在纯ADH制剂上观察到的动力学、特异性蛋白酶抑制剂的作用和底物特异性使我们将这种活性归因于枯草杆菌蛋白酶丝氨酸蛋白酶家族。鉴于即使在再色谱分离的Sephacryl S - 200组分中蛋白水解作用也很明显，如果在SDS中孵育足够长的时间，我们提出两个替代假说来解释这种现象。首先，蛋白水解活性可能来自与ADH活性形式不可分离的蛋白酶，其次，ADH本身在采用特定构象时可能表现为枯草杆菌蛋白酶。此外，先前报道的发育过程中不同的条带模式表明这种活性在体内起作用，其中脂肪酸可能产生体外归因于SDS的诱导作用。
