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
K. Nikaido et al., 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 Journal of biological chemistry, 272(44), 1997, pp. 27745-27752
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.