Rubidium occlusion within tryptic peptides of the H,K-ATPase.
86Rb+ binding to the H,K-ATPase was measured in the Mg(2+)-vanadate-inhibited enzyme at 4 degrees C. The concentration dependence of 86Rb+ binding in detergent-free preparations exhibited two components, one saturable with a K0.5 (Rb+) of 0.76 +/- 0.3 mM and a binding capacity of 2626 +/- 690 pmol of Rb+/mg of protein and the second nonsaturable, but linearly dependent, upon the 86Rb+ concentration. The concentration dependence of 86Rb+ binding was unaffected by digitonin treatment with a K0.5 (Rb+) of 0.63 +/- 0.09 mM and a binding capacity of 2824 +/- 152 pmol of Rb+/mg of protein, but the amplitude of the nonsaturable component was eliminated. The level of 86Rb+ binding was optimized by vanadate and decreased by ADP and ATP, suggesting that cation binding is stabilized in the E2-like conformation and antagonized in the E1 conformation. The Rb(+)-dependent stabilization of the E2 enzyme conformation was confirmed from the fluorescent quench response of the fluorescein isothiocyanate (FITC)-labeled enzyme, where 86Rb+ bound to the FITC-labeled enzyme with a K0.5 = 0.85 +/- 0.3 mM and a saturable binding capacity of 2121 pmol of 86Rb+/mg of protein and quenched the FITC fluorescence with a K0.5(Rb+) of 3.6 +/- 0.3 mM. The K(+)-competitive inhibitor, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo[3,2-c ]quinoline (MDPQ), also quenched FITC fluorescence with a K0.5(MDPQ) of 24.5 +/- 0.6 microM and competitively inhibited 86Rb+ binding with a K*0.5 = 35.8 microM (MDPQ). The MDPQ-induced quench of FITC fluorescence at Lys517 within the cytoplasmic M4/M5 nucleotide domain and displacement of 86Rb+ from a functionally defined extracytoplasmic binding domain indicate that structural determinants of the E2 conformational state exist within both cytoplasmic and extracytoplasmic domains of the H,K-ATPase and thus provide evidence of concerted conformational changes between the nucleotide and cation binding domains within the FITC-labeled H,K-ATPase. Membrane-bound fragments of the H,K-ATPase were prepared by tryptic hydrolysis in KCl medium. Tryptic digestion rapidly inactivated the phosphoenzyme site with a time course where k = 0.25 +/- 0.04 min-1 but both 86Rb+ binding and MDPQ fluorescence responses were retained. The concentration dependence of 86Rb+ binding and Rb(+)-dependent MDPQ fluorescence responses in the trypsin-digested membranes were described by a single class of binding sites where K0.5 = 1.2 +/- 0.3 and 0.73 +/- 0.09 mM, respectively. The stability of the Rb+ and MDPQ sites suggest their locations are near or within the membrane and are inaccessible to trypsin attack.(ABSTRACT TRUNCATED AT 400 WORDS)
Center for Ulcer Research and Education, Veterans Administration Center, Wadsworth Division, Los Angeles, California 90073.
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D000595:Amino Acid Sequence; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D016650:Fluorescein-5-isothiocyanate; D017506:H(+)-K(+)-Exchanging ATPase / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D054328:Proton Pump Inhibitors; D012044:Regression Analysis; D012413:Rubidium / Q000378:metabolism*; D012414:Rubidium Radioisotopes; D013050:Spectrometry, Fluorescence; D014357:Trypsin
D000634:Aminoquinolines; D008024:Ligands; D010446:Peptide Fragments; D054328:Proton Pump Inhibitors; D012414:Rubidium Radioisotopes; C069657:1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo(3,2-c)quinoline; D014357:Trypsin; D017506:H(+)-K(+)-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D012413:Rubidium