Overexpression in Escherichia coli and purification of an ATP-binding peptide from the yeast plasma membrane H(+)-ATPase.
The two major hydrophilic domains from the Saccharomyces cerevisiae plasma membrane H(+)-ATPase fused to glutathione S-transferase have been expressed in Escherichia coli. The GST-L peptide contained the hydrophilic region from Ala340 to Ser660. The GST-SL peptide contained in addition the hydrophilic region Glu162 to Val276. After solubilization of the inclusion bodies with urea, renaturation, and affinity chromatography, 3 mg of highly purified peptides were recovered per liter of E. coli culture. The purified peptides interacted with 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP), the fluorescence of which was enhanced identically upon binding of either GST-L or GST-SL. ATP competitively displaced the TNP-ATP binding. The observed dissociation constants for TNP-ATP (6.5 microM) and ATP (3 mM) are close to those found for the complete native H(+)-ATPase protein. The fluorescence of TNP-ATP was sensitive to Mg2+ indicating the existence of a Mg(2+)-binding site on the peptide. Apparent affinity for this Mg2+ site was found to vary from 50 microM at pH 7.5 to 400 microM at pH 5.5.
Capieaux
E
E
Unité de Biochimie Physiologique, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.
Rapin
C
C
Thinès
D
D
Dupont
Y
Y
Goffeau
A
A
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D009838:Oligodeoxyribonucleotides; D010455:Peptides; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D006180:Proton-Translocating ATPases