Johnston L S, Neuhaus F C
Biochemistry. 1977 Apr 5;16(7):1251-7. doi: 10.1021/bi00626a001.
Membrane preparations from Gaffkya homari catalyzed the in vitro biosynthesis of soluble uncross-linked spin-labeled peptidoglycan, a uniformly labeled polynitroxide, from the spin-labeled nucleotide UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-2,2,5,5-tetramethyl-1-pyrrolin-1-oxyl-3-carbonyl)-DAla-DAla (I) and UDP-GlcNAc. Soluble spin-labeled peptidoglycan was separated from membrane fragments and its spin-labeled precursor by centrifugation and gel filtration. The molecular weight distribution of the polymer was examined by agarose gel filtration. Spin-labeled [14C]peptidoglycan was polydisperse with a peak of radioactivity corresponding to a molecular weight of 5.0 X 10(5). The electron spin resonance spectrum of spin-labeled peptidoglycan was extensively broadened by spin-spin exchange interactions. These interactions were modified by changes in temperature, reduction by ascorbate, hydrolysis by lysozyme, and complexation with the antibiotic, vancomycin. Spin-spin exchange was reduced or eliminated in spin-labeled peptidoglycan by the random reduction of free radicals by ascorbate. A rotational correlation time of 0.37 ns was calculated for the probe in partially reduced spin-labeled peptidoglycan. This compares to a correlation time of 0.13 ns for the substrate (I). Raising the temperature increases spin-spin exchange line broadening. No transition points were observed for spin-labeled peptidoglycan as measured by this method. Degradati on of spin-labeled peptidoglycan by lysozyme eliminated the observed spin-spin exchange and yielded products with a mobility similar to I. Complexation of spin-labeled peptidoglycan with vancomycin resulted in both pronounced free-radical immobilization and a decrease in spin-spin exchange. The exchange effects are consistent with distance measurements in molecular models for peptidoglycan.
来自龙虾加夫基菌的膜制剂催化了可溶性未交联自旋标记肽聚糖（一种均匀标记的多硝基氧）的体外生物合成，该肽聚糖由自旋标记的核苷酸UDP - MurNAc - Ala - D - Glu - Lys（Nε - 2,2,5,5 - 四甲基 - 1 - 吡咯啉 - 1 - 氧基 - 3 - 羰基） - D - Ala - D - Ala（I）和UDP - GlcNAc合成。通过离心和凝胶过滤将可溶性自旋标记肽聚糖与膜片段及其自旋标记前体分离。通过琼脂糖凝胶过滤检查聚合物的分子量分布。自旋标记的[14C]肽聚糖是多分散的，放射性峰值对应于分子量5.0×10(5)。自旋标记肽聚糖的电子自旋共振谱因自旋 - 自旋交换相互作用而大幅加宽。这些相互作用通过温度变化、抗坏血酸还原、溶菌酶水解以及与抗生素万古霉素络合而改变。抗坏血酸对自由基的随机还原减少或消除了自旋标记肽聚糖中的自旋 - 自旋交换。计算出部分还原的自旋标记肽聚糖中探针的旋转相关时间为0.37 ns。相比之下，底物（I）的相关时间为0.13 ns。升高温度会增加自旋 - 自旋交换线加宽。用这种方法测量自旋标记肽聚糖时未观察到转变点。溶菌酶对自旋标记肽聚糖的降解消除了观察到的自旋 - 自旋交换，并产生了与I迁移率相似的产物。自旋标记肽聚糖与万古霉素络合导致自由基明显固定化以及自旋 - 自旋交换减少。这些交换效应与肽聚糖分子模型中的距离测量结果一致。
