Search

The structural stability of the large cytoplasmic domain (H4-H5 loop) of mouseα1 subunit of Na+/K+ ATPase (L354–I777), the number and the location of its binding sites for 2′-3′-O-(trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) andp-nitrophenylphosphate (pNPP) were investigated. C- and N-terminal shortening revealed that neither part of the phosphorylation (P)-domain are necessary for TNP-ATP binding. There is no indication of a second ATP site on the P-domain of the isolated loop, even though others reported previously of its existence by TNP-N3ADP affinity labeling of the full enzyme. Fluorescein isothiocyanate (FITC)-anisotropy measurements reveal a considerable stability of the nucleotide (N)-domain suggesting that it may not undergo a substantial conformational change upon ATP binding. The FITC modified loop showed only slightly diminished phosphatase activity, most likely due to a pNPP site on the N-domain around N398 whose mutation to D reduced the phosphatase activity by 50%. The amino acids forming this pNPP site (M384, L414, W411, S400, S408) are conserved in theα1−4 isoforms of Na+/K+ ATPase, whereas N398 is only conserved in the vertebrates'α1 subunit. The phosphatase activity of the isolated H4-H5 loop was neither inhibited by ATP, nor affected by mutation of D369, which is phosphorylated in native Na+/K+ ATPase. [ABSTRACT FROM AUTHOR]

Mutation of Arg423 at the N-domain of Na+/K+-ATPase resulted in a large decrease of both TNP-ATP and ATP binding. Thus, this residue, localized outside the binding pocket, seems to play a key role in supporting the proper structure and shape of the binding site. In addition, mutation of Glu472 also caused a large decrease of both TNP-ATP and ATP binding. On the basis of our computer model, we hypothesized that a hydrogen bond between Arg423 and Glu472 supports the connection of two opposite halves of the ATP-binding pocket. To verify this hypothesis, we have also prepared the construct containing both these mutations. Binding of neither TNP-ATP nor ATP to this double mutant differed from binding to any of the single mutants. This strongly supported the existence of the hydrogen bond between Arg423 and Glu472. Similarly, the conserved residue Pro489 seems to be substantial for the proper interaction of the third and fourth β-strands of the N-domain, which both contain residues that take part in ATP binding. Mutation of Asp443 affected only ATP, but not TNP-ATP, binding, suggesting that these ligands adopt different positions in the nucleotide-binding pocket. On the basis of a recently published crystal structure [Håkansson, K. O. (2003) J. Mol. Biol. 332, 1175-1182], we improved our model and computed the interaction of these two ligands with the N-domain. This model is in good agreement with all previously reported spectroscopic data and revealed that Asp443 forms a hydrogen bond with the NH2 group of the adenosine moiety of ATP, but not TNP-ATP. [ABSTRACT FROM AUTHOR]

Point mutations of a part of the H[sub 4]-H[sub 5] loop (Leu[sup 354]-Ile[sup 604]) of Na[sup +]/K[sup +]-ATPase have been used to study the ATP and TNP-ATP binding affinities. Besides the previously reported amino acid residues Lys[sup 480], Lys[sup 50l], Gly[sup 502], and Cys[sup 549], we have found four more amino acid residues, viz., Glu[sup 446], Phe[sup 475], Gln[sup 482], and Phe[sup 548], completing the ATP-binding pocket of Na[sup +]/K[sup +]-ATPase. Moreover, mutation of Arg[sup 423] has also resulted in a large decrease in the extent of ATP binding. This residue, localized outside the binding pocket, seems to play a key role in supporting the proper structure and shape of the binding site, probably due to formation of a hydrogen bond with Glu[sup 472]. On the other hand, only some minor effects were caused by mutations of Ile[sup 417], Asn[sup 422], Ser[sup 445], and Glu[sup 505]. [ABSTRACT FROM AUTHOR]

The ATP-binding site of Na+/K+-ATPase is localized on the large cytoplasmic loop of the α-subunit between transmembrane helices H4 and H5. Site-directed mutagenesis was performed to identify residues involved in ATP binding. On the basis of our recently developed model of this loop, Ser445, Glu446, and Phe475 were proposed to be close to the binding pocket. Replacement of Phe475 with Trp and Glu446 with Gln profoundly reduced the binding of ATP, whereas the substitution of Ser445 with Ala did not affect ATP binding. Fluorescence measurements of the fluorescent analog TNP-ATP, however, indicated that Ser445 is close to the binding site, although it does not participate in binding. [Copyright &y& Elsevier]

Focuses on the affinity labelling with Mg adenosines triphosphate (ATP) analogues of Na[sup+]/K[sup +]-adenosines triphosphatase. Revelation of coexisting Na[sup +] and K[sup +] forms; Localization of ATP binding sites on tryptic peptides; Formation of an unlabelled K[sup +]-like N-terminal 40-kDa fragment.

Na+/K+-ATPase from pig kidney is inactivated by protein-reactive N-hydroxysuccinimidyl derivatives of digoxigenin. Like digoxigenin, its protein-reactive derivatives N-hydroxysuccinimidyl digoxigenin-3-methylearbonyl-e-aminocaproate (HDMA), 3-amlno-3-deoxydigoxigenin hemisuccinimide succinimidyl ester (ADHS), 3-iodoacetylamino-3-deoxydigoxigenin (IAD) and digoxigenin-3-O-succinyl-[2-(N-maleimido)]cthylamide (DSME) inhibited the sodium pump in the presence of Na+, Mg2+ and ATP, At 37°C, half-maximal inhibition of Na+/K+-ATPase was seen by HDMA at 0.47 μM, by ADHS at 5.8 μM, by lAD at 8 μM and by DSME at 94 μM. Thus, all compounds bind to the cardiac steroid receptor site of Na+/K+-ATPase. Affinity labeling of the a subunit by ‘front door’ or ‘back door’ phosphorylation was only seen with HDMA or ADHS in the range 0.1 μM. Excess of ouabain protected against affinity labeling. All the other protein-reactive derivatives of digoxigenin labeled the enzyme independent of the formation of a phosphointermediate at much higher concentrations. This labeling was not suppressed by an excess of ouabain. Tryptic hydrolysis of the HDMA-modified Na+/K+-AtPase gave peptides of the apparent molecular masses 20, 12.5 and 11.2 kDa. The 11.2-kDa and 12.5-kDa peptides started amino-terminally with Asp68, and the 20-kDa peptide with Asp24. Thus, the HDMA-labeled peptides originate from the cardioactive steroid-binding site formed by the first and second transmembrane helix. N-Hydroxysuccinimidyl esters such as HDMA are normally thought to modify lysine and arginine residues covalently. Since such residues do not exist in the putative cardiac glycoside-binding site, the possibility of a thioester formation of the digoxigenin derivatives HDMA and ADHS with Cysl04 in the H1 transmembrane domain was tested. In fact, hydroxylaminolysis led to the release of the covalently bound HDMA, and the formation of a free sulfhydryl group. This could be labeled by [2-14C]ICH2COOH. We therefore propose, consistent with a recent conclusion from a site-directed mutagenesis experiment [Canessa, C. M., Horisberger, J.-D., Louvard, D. & Rossier, B. C. (1992) EMBO J. II, 1681–1687], that a cysteine residue (probably Cysl04) participates in the structure and function of the cardiac glycoside binding. [ABSTRACT FROM AUTHOR]

Digital-imaging fluorescence microscopy with fura-2 allows the determination of intracellular calcium concentration ([Ca2+]i) in single cells. At a cell density of 105 cells/petri dish 44% of the chick embryo heart cells had a high [Ca2+]i of 99.4 ± 7.1 nM and 56% of the cells a low [Ca2+]i of 27.8 ± 4.4 nM (mean ± SE). This laboratory previously reported that high-[Ca2+]i and low-[Ca2+]i cells from chick embryo hearts differ in their sensitivity to cardiac glycosides, as shown by measuring the increase in [Ca2+]i to reach a new steady state [Ahlemeyer, B., Weintraut, H., Seibold, G. & Schoner, W. (1991) in The sodium pump; recent developments (Kaplan. J. H. & De Weer, P., eds) pp. 653–656, Rockefeller University Press, New York]. This time we used N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-ϵ-aminocaproate (HDMA) which binds irreversibly to amino groups of the Na+/K+-ATPase, and sheep anti-digoxigenin Fab fragments coupled with fluorescein isothiocyanate to identify different cardiac glycoside-binding sites. Half-maximal labelling of high-[Ca2+]i cells was obtained at 0.36 nM HDMA, and at 12.0 nM with the low-[Ca2+]i cells. Specific labelling of the cells by HDMA was 91 % and 80% in high-[Ca2+]i and low-[Ca2+]i cells, respectively, as revealed by competition experiments with a 1000-fold excess of ouabain. HDMA half-maximally elevated the [Ca2+]i of high-[Ca2+]i cells at a concentration of 50 pM and that of low-[Ca2+]i cells at 8.0 nM. Concentrations higher than 0.1 μM produced signs of intoxication. When the labelled cells were subjected to a SDS/PAGE, a 100-kDa band was found to contain HDMA. The electrophoretic mobility of a protein labelled at 10 nM HDMA was slightly higher than that of a protein labelled at 1.0 nM. The data suggest that different isoforms of the a-subunit of Na+/K+-ATPase may exist in low-[Ca2+]i and high-[Ca2+]i cells of chick embryo heart. [ABSTRACT FROM AUTHOR]