PURIFICATION AND CHARACTERIZATION OF HISP, THE ATP-BINDING SUBUNIT OFA TRAFFIC ATPASE (ABC TRANSPORTER), THE HISTIDINE PERMEASE OF SALMONELLA-TYPHIMURIUM - SOLUBILITY, DIMERIZATION, AND ATPASE ACTIVITY

The nucleotide-binding subunit, HisP, of the histidine permease, a tra ffic ATPase (ABC transporter), has been purified as a soluble protein and characterized. Addition of a 6-histidine extension (HisP((His6))) allows a rapid and effective metal affinity purification, giving a 30- fold purification with a yield of 50%. HisP((his6)) is indistinguishab le from underivatized HisP when incorporated into the permease membran e-bound complex, HisQMP(2). Purified HisP((his6)) has a strong tendenc y to precipitate; 5 mM ATP and 20% glycerol maintain it in solution at a high protein concentration. HisP((his6)) is active as a dimer, bind s ATP with a K-d value of 205 mu M, and hydrolyzes it at a rate compar able to that of HisQMP(2); in contrast to the latter, it does not disp lay cooperativity for ATP. HisP((his6)) has been characterized with re spect to substrate and inhibitor specificity and Various physico-chemi cal characteristics. Its pH optimum is 7 and it requires a cation for activity, with Co(2+)and Mn2+ being more effective than Mg2+ at lower concentrations but inhibitory in the higher concentration range. In co ntrast to the intact complex, HisP((his6)) is not inhibited by vanadat e but is inhibited by N-ethylmaleimide. Neither the soluble receptor, HisJ, nor the transport substrate, histidine, has any effect on the ac tivity.