Abstract
Pig gastric membrane vesicles enriched in (H+ + K+)-ATPase were covalently modified with pyridoxal 5'-phosphate (PLP). The modification resulted in inhibition of K+-dependent ATP hydrolysis, formation of phosphoenzyme and ATP-driven H+-uptake catalyzed by (H+ + K+)-ATPase. ATP, ADP, and adenyl-5'-yl imidodiphosphate were protective ligands, whereas Mg2+ and K+ were not. Specific PLP-binding of about 4.5 nmol/mg membrane protein was necessary for complete inhibition of the enzyme activity, indicating that the stoichiometry of PLP-binding to the enzyme was about 1:1. Limited proteolysis of the enzyme modified with [3H]PLP by trypsin suggests that PLP specifically modifies the lysine residue located in the 16-kDa fragment of the enzyme cleaved by trypsin. These results suggested that PLP binds to a specific lysine residue in the nucleotide-binding site or a region in its vicinity and inhibits the substrate binding or phosphorylation step of (H+ + K+)-ATPase.
Highlights
Masatomo Maeda, MitsuoTagaya, and Masamitsu Futai From the Departmentof Organic Chemistry and Biochemistry, The Institute of Scientific and Industrial Research, Osaka
Since ATP protected the enzyme modifies thelysine residue located in th1e6-kDa frag- from inhibition, PLP was suggestedto modify a specific lysine ment of the enzyme cleaved by trypsin
The catalytic cycle of the enzyme was proposed from kinetic results [7,8,9]: the first step is the binding of ATP to the substrate-binding site,resulting in the replacement of K' by H'
Summary
Gastric (H' + K')-ATPase catalyzes electroneutral exchange of intracellular H' and extracellular K' coupled with the hydrolysis of cytoplasmic ATP [1,2,3]. Gastric (H' + K ")-ATPase cetic acid c o n t & i i gHaPo,.For examination of the chemical nature phate had no protective effect, the latter two comof EP, the membranes labeled with 3*Pwere collected by centrifugation (so00 x g, 5 min) and treatedwith KOH or hydroxylamine [18]. Radioactivity was measured in anMAolodkifaicLuStiCon-7o0f0(liqHu+i+d sKci+n)t-ilAlaTtiPoansceowuinthterP.LR-Vesicles (0.4-1.0 mg of protein/ml) were incubated in 50 mM MOPS-NaOH buffer (pH 8.0) a t 25 "C in the presence of various concentrations of PLP. Modification with PLP resulted in inhibition of H'-uptake (Fig. 3), measured as ATP-dependent fluorescence quenching of acridine orange [19]. Vesicles modified with [3H]PLP (800 pg of protein/l6O pl of 20 mM PIPES-triethanolamine, pH 6.8) were incubated with TPCK-trypsinon ice for 15 min. All other chemicals used were of the highest grade available commercially
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