Rapid assay and purification of a unique signal peptidase that processes the prolipoprotein from Escherichia coli B.
Rapid assay and purification of a unique signal peptidase that processes the prolipoprotein from Escherichia coli B.
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快速测定和纯化处理大肠杆菌 B 的前脂蛋白的独特信号肽酶。
DOI：
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发表时间：
1984
影响因子：
4.8
通讯作者：
P. Ray
中科院分区：
文献类型：
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作者：
I. K. Dev;P. Ray
一种简单而准确的测定前脂蛋白信号肽酶活性的方法已经被描述，该方法是基于信号肽在80%丙酮中的溶解度。在沉淀物中定量回收未加工的前体和成熟形式的脂蛋白。利用纯化的信号肽酶从丙酮上清液中得到的信号肽在其NH 2末端含有标记的甲硫氨酸，并且具有Mr = 2200（S.E. = 69）。一种特殊的信号肽酶，处理的布朗氏前脂蛋白的修改形式，其正确的成熟形式已被纯化。该酶对球霉素敏感，通过在pH4.0下用2%Triton X-100提取并加热，然后在室温下进行常规柱层析，已从大肠杆菌的膜中纯化了35，000倍。这种前脂蛋白信号肽酶的最适pH为6.0，不受EDTA抑制，需要1 mM二硫苏糖醇才能稳定。该特异性信号肽酶的单体分子量为17，800（S.E. = 900）。
A simple and accurate assay for prolipoprotein signal peptidase activity has been described that is based on the solubility of the signal peptide in 80% acetone. The unprocessed precursor and the mature form of the lipoprotein are quantitatively recovered in the precipitate. The signal peptide, from the acetone supernatant utilizing the purified signal peptidase, contains labeled methionine at its NH2 terminus and has Mr = 2200 (S.E. = 69). A specific signal peptidase that processes the modified form of Braun's prolipoprotein to its correct mature form has been purified. This enzyme is globomycin sensitive and has been purified 35,000-fold from the membranes of Escherichia coli by extraction at pH 4.0 with 2% Triton X-100 and heating, followed by conventional column chromatography at room temperature. This prolipoprotein signal peptidase has a pH optimum at 6.0, is not inhibited by EDTA, and requires 1 mM dithiothreitol for stability. The monomer molecular weight of this specific signal peptidase is 17,800 (S.E. = 900) as determined by sodium dodecyl sulfate-gel electrophoresis.