Catalytic activity of an isolated domain of Na,K-ATPase expressed in Escherichia coli.
Fusion proteins of glutathione-S-transferase and fragments from the large cytoplasmic domain of the sheep Na,K-ATPase alpha1-subunit were expressed in Escherichia coli. The Na,K-ATPase sequences begin at Ala345 and terminate at either Arg600 (DP600f), Thr610 (DP610f), Gly731 (DP731f), or Glu779 (DP779f). After affinity purification on glutathione-Sepharose, the fusion proteins were labeled with [alpha-32P]-2-N3-ATP, and incorporation of the radiolabel into the fusion proteins was measured by scintillation counting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kd values of 220-290 microM for 2-N3-ATP binding to the fusion proteins were obtained from the photolabeling experiments. Approximately 1 mol of 2-N3-ATP was calculated to be incorporated per mole of fusion protein after correction for photochemical incorporation efficiency. Labeling of all of the fusion proteins by 25 microM 2-N3-ATP was reduced in the presence of MgATP, Na2ATP, MgCl2, 2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and p-nitrophenylphosphate, and Ki values of 2-11 mM for Na2ATP, 0.2-5 mM for MgCl2, 0.1-5 mM for MgATP, and 20-300 microM for p-nitrophenylphosphate were calculated for these ligands. All of the fusion proteins catalyze the hydrolysis of p-nitrophenylphosphate. The reaction requires MgCl2 and is inhibited by inorganic phosphate, which is similar to the hydrolysis of p-nitrophenylphosphate by native Na,K-ATPase. Based on these observations, it appears that the soluble fragments from the large cytoplasmic domain of Na,K-ATPase expressed in bacterial cells are folded in an E2-like conformation and are likely to retain much of the native structure.
Tran
C M
CM
Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90033 USA.
Farley
R A
RA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D007668:Kidney / Q000201:enzymology; D008024:Ligands; D009596:Nitrophenols / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics
D000345:Affinity Labels; D001386:Azides; D004791:Enzyme Inhibitors; D008024:Ligands; D009596:Nitrophenols; D009711:Nucleotides; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; C008644:nitrophenylphosphate; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase