Isolation and characterization of aminopeptidase mutants of Saccharomyces cerevisiae
Isolation and characterization of aminopeptidase mutants of Saccharomyces cerevisiae
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DOI：
10.1128/jb.156.1.36-48.1983
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发表时间：
1983-10
影响因子：
3.2
通讯作者：
R. Trumbly;G. Bradley
中科院分区：
文献类型：
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作者：
R. Trumbly;G. Bradley
分离出酿酒酵母突变体，其水解亮氨酸β-萘酰胺（氨基肽酶的显色底物）的能力降低。淀粉凝胶电泳显示突变影响四种不同氨肽酶中的一种。影响给定酶的突变属于单个互补组。这四个基因分别标记为lap1、lap2、lap3和lap4，以及相应的酶LAPI、LAPII、LAPIIII和LAPIV。 lap1和lap4都被映射到XI染色体的左臂，lap3被映射到XIV染色体的左臂。构建了仅具有四种亮氨酸氨基肽酶（LAP）中的一种的菌株。这些菌株的粗提物用于研究各个酶的特性。 EDTA 透析大大降低了除 LAPIIII 之外的所有 LAP 的活性。在测试的阳离子中，Co2+ 在恢复活性方面最有效。 LAPIV 是唯一被 Zn2+ 重新激活的 LAP。从细胞匀浆中，LAPI 纯化了 331 倍，LAPII 纯化了 126 倍。两种纯化的酶对二肽和三肽均具有较强的活性。 LAP 的活性水平强烈依赖于分批培养的生长阶段，在早期稳定阶段活性水平最高。缺乏所有四种 LAP 的菌株的生长速度略低于野生型菌株。在缺乏所有四种 LAP 的菌株中，亮氨酸营养缺陷型细胞在含有亮氨酸的二肽和三肽上生长的能力不会受到损害。
Mutants of Saccharomyces cerevisiae were isolated which have decreased ability to hydrolyze leucine beta-naphthylamide, a chromogenic substrate for amino-peptidases. The mutations were shown by starch gel electrophoresis to affect one of four different aminopeptidases. Mutations affecting a given enzyme belong to a single complementation group. The four genes were symbolized lap1, lap2, lap3, and lap4, and the corresponding enzymes LAPI, LAPII, LAPIII, and LAPIV. Both lap1 and lap4 were mapped to the left arm of chromosome XI, and lap3 was mapped to the left arm of chromosome XIV. Strains which possessed only one of the four leucine aminopeptidases (LAPs) were constructed. Crude extracts from these strains were used to study the properties of the individual enzymes. Dialysis against EDTA greatly reduced the activity of all the LAPs except for LAPIII. Of the cations tested, Co2+ was the most effective in restoring activity. LAPIV was the only LAP reactivated by Zn2+. LAPI was purified 331-fold and LAPII was purified 126-fold from cell homogenates. Both of the purified enzymes had strong activity on dipeptides and tripeptides. The activity levels of the LAPs are strongly dependent on growth stage in batch culture, with the highest levels in early-stationary phase. Strains lacking all four LAPs have slightly lower growth rates than wild-type strains. The ability of leucine auxotrophs to grow on dipeptides and tripeptides containing leucine is not impaired in strains lacking all four LAPs.