Purification and characterization of the membrane-bound complex of an ABC transporter, the histidine permease.
The bacterial histidine permease, an ABC transporter, from Salmonella typhimurium is composed of a membrane-bound complex, HisQMP2, comprising two hydrophobic subunits (HisQ and HisM), two copies of an ATP-hydrolyzing subunit, HisP, and a soluble receptor, HisJ. We describe the purification and characterization of HisQMP2 using a 6-histidines extension at the carboxy terminus of HisP [HisQMP2(his6)]. The purification is rapid and effective, giving a seven-fold purification with a yield of 85 and 98% purity. Two procedures are described differing in the detergent used (decanoylsucrose and octylglucoside, respectively) and in the presence of phospholipid. HisQMP2(his6) has ATPase and transport activities upon reconstitution into proteoliposomes (PLS). HisQMP2(his6) has a low level ATPase activity (intrinsic activity), which is stimulated to a different extent by the receptor--liganded and unliganded. Its pH optimum is 7.8-8.0, it requires a cation for activity and it displays cooperativity for ATP. The effect of various ATP analogs was analyzed. Determination of the molecular size of HisQMP2(his6) indicates that it is a monomer. The permeability properties of two kinds of reconstituted PLS preparations are described.
Ames
G F
GF
Department of Molecular and Cell Biology, University of California at Berkeley, 94720-3202, USA.
Nikaido
K
K
Wang
I X
IX
Liu
P Q
PQ
Liu
C E
CE
Hu
C
C
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D026923:Amino Acid Transport Systems, Basic / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D001693:Biological Transport, Active; D003902:Detergents; D008566:Membranes / Q000737:chemistry; D008970:Molecular Weight; D010539:Permeability; D010743:Phospholipids / Q000494:pharmacology; D021122:Protein Subunits; D011510:Proteolipids; D012486:Salmonella typhimurium / Q000378:metabolism; D012995:Solubility
D018528:ATP-Binding Cassette Transporters; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D003902:Detergents; D010743:Phospholipids; D021122:Protein Subunits; D011510:Proteolipids; C020485:proteoliposomes; C043828:histidine permease, Bacteria; D000251:Adenosine Triphosphatases