Amanuma H, Strominger J L
J Biol Chem. 1980 Dec 10;255(23):11173-80.
Penicillin-binding proteins (PBPs) 5 and 6 in the cytoplasmic membranes of Escherichia coli K12, which had previously been co-purified as a penicillin-sensitive D-alanine carboxypeptidase IA (Tamura, T., Imae, Y., and Strominger, J. L. (1976) J. Biol. Chem. 251, 414-423), were each purified to protein homogeneity. Purification involved selective solubilization of PBPs 1a, 5, and 6 from membranes by Triton X-100 at low ionic strength, covalent penicillin affinity chromatography, and CM-cellulose column chromatography. Purified PBP 5 and PBP 6 each catalyzed a D-alanine carboxypeptidase I activity using various natural and synthetic substrates including linear uncross-linked peptidoglycan. PBP 5 showed 3- to 4-fold higher specific activities toward these substrates than PBP 6. Both PBPs also catalyzed a model transpeptidase activity using glycine as a transpeptidation acceptor, and showed similar pH profiles and MgCl2 sensitivities for their D-alanine carboxypeptidase I activities. Both PBPs bound a stoichiometric amount of [14C]penicillin G at saturation. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after partial proteolysis by proteases and cyanogen bromide demonstrated that these PBPs are distinct polypeptides.
大肠杆菌K12细胞膜中的青霉素结合蛋白（PBPs）5和6，此前曾作为一种对青霉素敏感的D - 丙氨酸羧肽酶IA共同纯化（田村，T.，今江，Y.，和斯特罗明格，J. L.（1976年）《生物化学杂志》251卷，414 - 423页），现已分别纯化至蛋白质均一性。纯化过程包括在低离子强度下用Triton X - 100从膜中选择性溶解PBPs 1a、5和6、共价青霉素亲和层析以及CM - 纤维素柱层析。纯化后的PBP 5和PBP 6各自使用包括线性未交联肽聚糖在内的各种天然和合成底物催化D - 丙氨酸羧肽酶I活性。PBP 5对这些底物的比活性比PBP 6高3至4倍。两种PBPs还使用甘氨酸作为转肽受体催化一种模型转肽酶活性，并且在其D - 丙氨酸羧肽酶I活性方面表现出相似的pH谱和MgCl2敏感性。两种PBPs在饱和时结合化学计量的[14C]青霉素G。在蛋白酶和溴化氰部分蛋白水解后，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳进行肽图谱分析表明，这些PBPs是不同的多肽。
