Identification of a segment containing a reactive cysteine residue in human liver cytoplasmic aldehyde dehydrogenase (isoenzyme E1).
Identification of a segment containing a reactive cysteine residue in human liver cytoplasmic aldehyde dehydrogenase (isoenzyme E1).
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鉴定人肝细胞质醛脱氢酶（同工酶 E1）中含有反应性半胱氨酸残基的片段。
DOI：
10.1021/bi00269a032
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发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Jörnvall,H
中科院分区：
文献类型：
--
作者：
Hempel,J;Pietruszko,R;Fietzek,P;Jörnvall,H
材料和方法蛋白质。人肝乙醛脱氢酶同工酶Ej是按照描述的方法分离出来的(Hempel等人，1982)。该酶用于碘[L-14C]乙酰胺(13.1mCi/mmo1，纽格兰德核)的选择性烷基化反应，在所用的测定系统中的比活力为0.35U/mg(Feldman&Weiner，1972)。通过在30 mM磷酸二钠(pH 7.0)-1 mM EDTA-0.7 mM NAD和碘[14C]乙酰胺中孵育E，进行标记。6.5h后，仍有25%的残留酶活性，这与先前的结果一致(Hempel&Pietruszko，1981)。加入巯基乙醇终止孵育，透析后发现含有1.3当量的羧胺甲基。冷冻干燥后，在6M盐酸胍-0.1M Tris-2 mM EDTA，pH 8.0中，剩余的蛋白SH基用巯基乙醇还原，并用未标记的碘乙酰胺甲基化，如所述(Hempel&Pietruszko，1981)。通过加入0.2体积的巯基乙醇来停止反应，并通过对1，2，4，4，6，6，6，6，6，6，6，6，6，6，6，6，6，6，6，6，6，4，4，4，4，4，4，4，4，4，6-三者的广泛透析，冷冻干燥回收蛋白质。
Materials and MethodsProtein. Human liver aldehyde dehydrogenase isoenzyme Ej was isolated as described (Hempel et al., 1982). The enzyme used for selective alkylation by iodo [l-14C] acetamide (13.1 mCi/mmol, NewEngland Nuclear) had a specific ac-tivity of 0.35 unit/mg in the assay system used (Feldman & Weiner, 1972). Labeling was performed by incubation of E,(2 mg/mL) in 30 mM sodium phosphate (pH 7.0)—1 mM EDTA-0.7 mM NAD with iodo [14C] acetamide. After 6.5 h, 25% residual enzyme activity was present, in agreement with previous results (Hempel & Pietruszko, 1981). The incubation was terminated by addition of mercaptoethanol, and after dialysis 1.3 equiv of carboxamidomethyl groups was found to be incorporated.After lyophilization, the remaining protein SH groups were reduced with mercaptoethanol and carboxamidomethylated with unlabeled iodoacetamide, in 6 M guanidine hydrochloride-0.1 M Tris-2 mM EDTA, pH 8.0, as described (Hempel & Pietruszko, 1981). The reaction was stopped by addition of 0.2 volume of mercaptoethanol, and the protein was recovered by lyophilization after extensive dialysis against 1