OVEREXPRESSION IN ESCHERICHIA-COLI AND PURIFICATION OF AN ATP-BINDINGPEPTIDE FROM THE YEAST PLASMA-MEMBRANE H-ATPASE()

The two major hydrophilic domains from the Saccharomyces cerevisiae pl asma membrane H+-ATPase fused to glutathione S-transferase have been e xpressed in Escherichia coli. The GST-L peptide contained the hydrophi lic region from Ala340 to Ser660. The GST-SL peptide contained in addi tion the hydrophilic region Glu162 to Val276. After solubilization of the inclusion bodies with urea, renaturation, and affinity chromatogra phy, 3 mg of highly purified peptides were recovered per liter of E. c oli culture. The purified peptides interacted with -O-(2,4,6-trinitrop henyl)adenosine-5'-triphosphate (TNP-ATP), the fluorescence of which w as enhanced identically upon binding of either GST-L or GST-SL. ATP co mpetitively displaced the TNP-ATP binding. The observed dissociation c onstants for TNP-ATP (6.5 muM) and ATP (3 mM) are close to those found for the complete native H+-ATPase protein. The fluorescence of TNP-AT P was sensitive to Mg2+ indicating the existence of a Mg2+-binding sit e on the peptide. Apparent affinity for this Mg2+ site was found to va ry from 50 muM at pH 7.5 to 400 muM at pH 5.5.