Coombe R G, George A M
Biochemistry. 1982 Mar 2;21(5):871-5. doi: 10.1021/bi00534a009.
An aminoglycoside 3-acetyltransferase [AAC(3)], possibly a new isoenzymic species of the 3-N-acetyltransferase group, was purified to apparent homogeneity from a crude extract of Pseudomonas aeruginosa, a gentamicin-resistant clinical isolate. The method of purification was consecutive column chromatography--(i) gel filtration, (ii) affinity chromatography, and (iii) ion-exchange chromatography--to give two protein peaks, one of which was coincident with activity and which indicated a purification of 600 (specific activity = 9.743 units mg-1 at pH 7.2, 34 degrees C). Polyacrylamide disc gel electrophoresis indicated a single protein band coincident with enzymic activity. The molecular weight of the enzyme was about 39 000. AAC(3)-V (provisonal designation) was further characterized by stability, substrate, pH, and kinetic studies. The Km was 0.724 microM (sisomicin), and the Vmax was 0.102 mumol min-1 mg-1 (sisomicin) at pH 7.2 and 34 degrees C. Substrate inhibition was exhibited by kanamycin A and tobramycin. Studies showed that enzyme activity was significantly stabilized when preparations contained substrate.
从庆大霉素耐药的临床分离株铜绿假单胞菌的粗提物中纯化出一种氨基糖苷3 - 乙酰转移酶【AAC(3)】，它可能是3 - N - 乙酰转移酶组中的一种新的同工酶。纯化方法是连续柱层析——(i)凝胶过滤，(ii)亲和层析，以及(iii)离子交换层析——得到两个蛋白峰，其中一个与活性一致，纯化倍数为600（在pH 7.2、34℃时比活性 = 9.743单位mg⁻¹）。聚丙烯酰胺圆盘凝胶电泳显示一条与酶活性一致的单一蛋白带。该酶的分子量约为39000。通过稳定性、底物、pH和动力学研究对AAC(3) - V（暂定名称）进行了进一步表征。在pH 7.2和34℃时，Km为0.724μM（西索米星），Vmax为0.102μmol min⁻¹ mg⁻¹（西索米星）。卡那霉素A和妥布霉素表现出底物抑制作用。研究表明，当制剂含有底物时，酶活性显著稳定。
