Dey S, Dou D, Rosen B P
Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan 48201.
J Biol Chem. 1994 Oct 14;269(41):25442-6.
Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli results from active efflux of these anions out of the cell. Extrusion is an active process mediated by an ATP-dependent pump composed of two types of subunits, the integral membrane ArsB protein and the catalytic ArsA subunit. An in vitro assay for transport in everted membrane vesicles of E. coli was developed. Uptake of 73AsO2- by everted vesicles was time- and temperature-dependent and required both pump subunits. Transport required ATP; no other nucleotide, including GTP, CTP, UTP, or the nonhydrolyzable analog adenosine 5'-O-(thiotriphosphate), could substitute for ATP. Protonophores, ionophores, or inhibitors of other types of ion-motive ATPases did not inhibit arsenite uptake. The sulfhydryl reagent N-ethylmaleimide was a potent inhibitor of ATP-dependent arsenite accumulation in vesicles. The apparent Km values for ATP and arsenite were approximately 2 and 0.1 mM, respectively. Antimonite, the most potent activator of the ArsA ATPase, inhibited arsenite uptake with an apparent Ki of 10 microM.
大肠杆菌对砷和锑的毒性含氧阴离子的抗性源于这些阴离子从细胞中的主动外排。外排是一个由ATP依赖性泵介导的主动过程，该泵由两种类型的亚基组成，即整合膜蛋白ArsB和催化亚基ArsA。开发了一种用于大肠杆菌外翻膜囊泡转运的体外测定法。外翻囊泡对73AsO2-的摄取具有时间和温度依赖性，并且需要两个泵亚基。转运需要ATP；没有其他核苷酸，包括GTP、CTP、UTP或不可水解的类似物腺苷5'-O-(硫代三磷酸)，可以替代ATP。质子载体、离子载体或其他类型离子驱动ATP酶的抑制剂均不抑制亚砷酸盐的摄取。巯基试剂N-乙基马来酰亚胺是囊泡中ATP依赖性亚砷酸盐积累的有效抑制剂。ATP和亚砷酸盐的表观Km值分别约为2 mM和0.1 mM。ArsA ATP酶最有效的激活剂亚锑酸盐抑制亚砷酸盐的摄取，表观Ki为10 μM。
