Perlin M H, Lerner S A
J Bacteriol. 1981 Aug;147(2):320-5. doi: 10.1128/jb.147.2.320-325.1981.
A plasmid-encoded enzyme reported by us to phosphorylate amikacin in a laboratory strain of Escherichia coli has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing E. coli transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthetase, and beta-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H(3) (32)PO(4) were shown to utilize internally generated adenosine 5'-triphosphate in the phosphorylation of amikacin. The absence of (32)P-phosphorylated amikacin after incubation of [gamma-(32)P]adenosine 5'-triphosphate with spheroplasts confirmed that exogenous adenosine 5'-triphosphate was not used in the reaction. These results suggest an internal location for APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.
我们曾报道过一种由质粒编码的酶，它可在大肠杆菌的实验室菌株中使阿米卡星磷酸化，该酶已在细菌细胞中定位。通过乙二胺四乙酸 - 溶菌酶处理含 APH 的大肠杆菌转导子（已知该转导子易于形成原生质球）形成的原生质球中，保留了超过 88%的这种阿米卡星磷酸转移酶（APH）活性。相比之下，原生质球保留了两种内部标记物——94%的脱氧核糖核酸聚合酶 I 和 98%的谷氨酰 - 转移核糖核酸合成酶，而作为周质标记物的酸性磷酸酶活性在原生质球中的含量不到 10%。用不能穿过质膜的化学探针处理转导子的全细胞会消除酸性磷酸酶活性，而内部标记物脱氧核糖核酸聚合酶 I、谷氨酰 - 转移核糖核酸合成酶和β - 半乳糖苷酶在处理 5 分钟后几乎不受影响；超过 60%的 APH 活性仍然存在。作为对照，对超声提取物进行类似的化学处理，其中酶不受细菌区室化的保护，会使包括 APH 在内的所有测试酶的活性有更广泛的降低。通过薄层色谱法显示，预先用腺苷三磷酸酶孵育的原生质球可使阿米卡星磷酸化。在含有 H(3) (32)PO(4)的条件下生长的细胞形成的原生质球，被证明在阿米卡星磷酸化过程中利用内部产生的腺苷 5'-三磷酸。用[γ-(32)P]腺苷 5'-三磷酸与原生质球孵育后未出现(32)P - 磷酸化的阿米卡星，这证实了反应中未使用外源腺苷 5'-三磷酸。这些结果表明 APH 位于细胞内部。这一结论对这类酶在革兰氏阴性菌氨基糖苷类耐药性中的作用具有重要意义。
