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)
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Center for Ulcer Research and Education, Veterans Administration Center, Wadsworth Division, Los Angeles, California 90073.
Smillie
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Rabon
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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