Abstract

2-Azido-ATP (2-N3-ATP) was investigated as a reagent for the identification of amino acids located within the catalytic ATP binding site of Na,K-ATPase. The enzymatic activity of Na,K-ATPase was inhibited up to 50% by 2-N3-ATP (K0.5 = 5-10 microM) after irradiation with ultra-violet light, and inhibition was prevented by 0.2 mM ATP. The binding of ATP to Na,K-ATPase (KD = 0.1 microM) was inhibited competitively by 2-N3-ATP. [alpha-32P]2-N3-ATP labels the alpha subunit of Na,K-ATPase, and the stoichiometry of covalent ATP-protectable incorporation of the probe into the protein is approximately equal to the stoichiometry of high-affinity binding of ATP to the Na,K-ATPase. 2-N3-ATP is also hydrolyzed by Na,K-ATPase as a substrate. From these data, it is concluded that 2-N3-ATP photochemically labels the Na,K-ATPase from within the catalytic ATP site on the protein. Trypsin digestion of Na,K-ATPase after photochemical labeling with [alpha-32P]2-N3-ATP generated a large 30-kDa fragment containing the radiolabeled nucleotide. This fragment was resistant to further cleavage by trypsin, but it could be digested further after denaturation in urea. High pressure liquid chromatography separation of tryptic peptides from the 30-kDa fragment and subsequent amino acid sequence analysis of the radiolabeled peptides identified the region between His496 and Arg510 of the Na,K-ATPase alpha subunit as the region labeled by [alpha-32P]2-N3-ATP. Gly502 was absent from all sequences of the radiolabeled peptides from this region, consistent with the derivatization of this amino acid by 2-N3-ATP and localization of Gly502 within the ATP binding site.

Highlights

  • 2-Azido-ATP (2-Ns-ATPw) as investigated as a reagent subsequent hydrolysis are associated with conformational for the identification of amino acids located within the changes in the protein that are thought to be linked to the catalytic ATP binding site ofNa,K-ATPase

  • In the firstgroup of amino acids, it has been observed that LysBolis labeled by FITC [2, 3] and SITS [4]L, ys480is labeled by adenosine diphosphopyridoxal[5], pyridoxal phosphate (51, and FITC (61, and aminoacids between 470 and 495 are labeled by 8-N3-ATP[7]

  • Inhibition of Na,K-ATPase peptides from the 30-kDa fragment and subsequent enzymatic activityby each of the reagents ipsrevented by ATP, amino acid sequence analysis of the radiolabeled p e p several of the inhibitors bear little structural resemblance to tides identified the region between His49a6nd A r g B ’ O of the Na,K-ATPase a subunit as the regionlabeled by [a-32P]2-N3-ATPG.lyso2was absent from all sequences of the radiolabeled peptides from this region, consistent with the derivatization of this amino acid by 2-N3-ATP and localization of Gly602 within the ATP binding site

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Summary

Introduction

2-Azido-ATP (2-Ns-ATPw) as investigated as a reagent subsequent hydrolysis are associated with conformational for the identification of amino acids located within the changes in the protein that are thought to be linked to the catalytic ATP binding site ofNa,K-ATPase. 100% inactivation indicates thactomplete inhibition of activity were collected as pool 1, fractions eluting between 58 and 62 occurs at a stoichiometryof incorporation slightlyless than the min were collected as pool 2, and each pool was fractionated binding capacityof 2.4 nmol ATP/mg of protein for this enzyme.

Results
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