The conformations of a functional spin-labeled derivative of gastric H/K-ATPase investigated by EPR spectroscopy.
A spin-labeled derivative of porcine gastric H/K-ATPase with high ATP hydrolyzing activity (77 mumol of Pi/(mg.h)) has been prepared. Over 65% of initial ATPase activity (115 mumol of Pi/(mg.h)) was preserved after complete reaction of the enzyme with the lysine reactive nitroxide spin-labeled TEMPO isothiocyanate (TITC). In contrast, rapid and complete loss of ATPase activity occurred after reaction of the enzyme with the lysine directed fluorescent probe FITC. Conventional EPR spectra of TITC labeled H/K-ATPase reflected mainly the slow rotational diffusion of the enzyme in the membrane. An upper limit enzyme intramembranous radius of 108 A was calculated on the basis of rotational correlation times estimated from saturation transfer (ST) EPR spectral lineshapes. Conventional EPR spectra exhibited two major components corresponding to at least two populations of strongly constrained spin-labels. Difference spectroscopy revealed that the proportion of these two components changed markedly with temperature. Moreover, the proportion of the components was sensitive to the presence of the activating ionic ligands Mg2+ and ATP, which induce enzyme conformational transitions, and to the reversible inhibitor SCH 28080, which binds to the K+ sensitive form of the enzyme. These findings show that EPR spectroscopy is able to report functionally coupled conformational changes of gastric H/K-ATPase and imply that the spin-labels are attached to lysines within functionally important regions of the enzyme.
Middleton
D A
DA
Department of Biochemistry, University of Oxford, U.K.
Reid
D G
DG
Watts
A
A
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000302:isolation & purification / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D011487:Protein Conformation; D013113:Spin Labels; D013552:Swine; D013816:Thermodynamics
D013113:Spin Labels; D017301:Ca(2+) Mg(2+)-ATPase; D017506:H(+)-K(+)-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate