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• W2067937824 abstract "This paper extends our recent report on specific iron-catalyzed oxidative cleavages of renal Na,K-ATPase and effects ofE 1 ↔ E 2conformational transitions (Goldshleger, R., and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596–9601). The experiments indicate that only peptide bonds close to a bound Fe2+ ion are cleaved, and provide evidence on proximity of the different cleavage positions in the native enzyme. A sequence HFIH near trans-membrane segment M3 appears to be involved in Fe2+ binding. Previously we hypothesized that E 2 and E 1conformations are characterized by formation or relaxation of interactions within the α subunit at or near highly conserved sequences, TGES in the minor cytoplasmic loop and CSDK, MVTGD, and VNDSPALKK in the major cytoplasmic loop. This concept has been tested by examining iron-catalyzed cleavage in both non-phosphorylated and phosphorylated conformations and effects of phosphate, vanadate, and ouabain. The results imply that both E 1 ↔E 2 and E 1P ↔E 2P transitions are indeed associated with formation and relaxation of interactions between cytoplasmic domains, comprising the minor loop plus N-terminal tail leading into M1 and major loop, respectively. Furthermore, it appears that either non-covalently or covalently bound phosphate bind near CSDK and MVTGD, and Mg2+ ions may bind to residues within TGES and VNDSPALKK and to bound phosphate. Thus cytoplasmic domain interactions seem to occur within or near the active site. We discuss the relationship between structural changes in the cytoplasmic domain and movements of trans-membrane segments that lead to cation transport. Presumably conformation-dependent formation and relaxation of domain interactions underlie energy transduction in all P-type pumps. This paper extends our recent report on specific iron-catalyzed oxidative cleavages of renal Na,K-ATPase and effects ofE 1 ↔ E 2conformational transitions (Goldshleger, R., and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596–9601). The experiments indicate that only peptide bonds close to a bound Fe2+ ion are cleaved, and provide evidence on proximity of the different cleavage positions in the native enzyme. A sequence HFIH near trans-membrane segment M3 appears to be involved in Fe2+ binding. Previously we hypothesized that E 2 and E 1conformations are characterized by formation or relaxation of interactions within the α subunit at or near highly conserved sequences, TGES in the minor cytoplasmic loop and CSDK, MVTGD, and VNDSPALKK in the major cytoplasmic loop. This concept has been tested by examining iron-catalyzed cleavage in both non-phosphorylated and phosphorylated conformations and effects of phosphate, vanadate, and ouabain. The results imply that both E 1 ↔E 2 and E 1P ↔E 2P transitions are indeed associated with formation and relaxation of interactions between cytoplasmic domains, comprising the minor loop plus N-terminal tail leading into M1 and major loop, respectively. Furthermore, it appears that either non-covalently or covalently bound phosphate bind near CSDK and MVTGD, and Mg2+ ions may bind to residues within TGES and VNDSPALKK and to bound phosphate. Thus cytoplasmic domain interactions seem to occur within or near the active site. We discuss the relationship between structural changes in the cytoplasmic domain and movements of trans-membrane segments that lead to cation transport. Presumably conformation-dependent formation and relaxation of domain interactions underlie energy transduction in all P-type pumps. The molecular mechanism whereby Na,K-ATPase transduces the free energy of hydrolysis of ATP into active transport of Na+and K+ ions is unknown. We have a wealth of knowledge on transport reactions, covalent phosphorylation,E 1/E 2 conformational transitions, and cation occlusion, embodied in the Post-Albers kinetic mechanism (see Ref. 1Glynn I.M. Karlish S.J.D. Annu. Rev. Biochem. 1990; 59: 171-205Crossref PubMed Scopus (177) Google Scholar). Active cation transport involves Nacyt-dependent phosphorylation from ATP, Na+ movement coupled to E 1P →E 2P, Kexc-activated dephosphorylation, K+ movement coupled toE 2(K) → E 1. Knowledge of the structural basis for energy transduction is meager due to lack of information on molecular structure. The best structure of Na,K-ATPase at 20–25-Å resolution reveals only the overall shape of the protein and distribution of mass of α and β subunits (2Maunsbach A.B. Skriver E. Hebert H. Soc. Gen. Physiol. 1991; 46: 159-172PubMed Google Scholar). Recent cryoelectron microscopy studies of Ca-ATPase and H-ATPase demonstrate the overall shape at 8-Å resolution, including “head”, “neck,” and membrane sectors, including 10 trans-membrane α-helical rods, most of which are tilted at an angle to the membrane (3Zhang P. Toyashima C. Yonekura K. Green N.M. Stokes D.L. Nature. 1998; 392: 835-839Crossref PubMed Scopus (265) Google Scholar, 4Auer M. Scarborough G.A. Kühlbrandt W. Nature. 1998; 392: 840-843Crossref PubMed Scopus (185) Google Scholar). The topological organization with 10 trans-membrane segments confirms that shown by a variety of techniques (5Møller J.V. Juul B. Le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (660) Google Scholar). Biochemical and molecular techniques are providing much information on residues involved in cation occlusion within trans-membrane segments (6Karlish S.J.D. Goldshleger R. Stein W.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4566-4570Crossref PubMed Scopus (129) Google Scholar), primarily M4, M5, and M6 (7Jewell-Motz E.A. Lingrel J.B. Biochemistry. 1993; 32: 13523-13530Crossref PubMed Scopus (116) Google Scholar, 8Andersen J.P. Vilsen B. FEBS Lett. 1995; 359: 101-106Crossref PubMed Scopus (115) Google Scholar, 9Nielsen J.M. Pedersen P.A. Karlish S.J.D. Jørgensen P.L. Biochemistry. 1998; 37: 1961-1968Crossref PubMed Scopus (78) Google Scholar), or ATP binding within the large cytoplasmic loop (see Ref. 5Møller J.V. Juul B. Le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (660) Google Scholar). These studies indicate the necessity for interactions between the ATP sites and cation occlusion sites. These interactions are mediated by E 1 ↔E 2 conformational transitions, which have been studied extensively using proteolytic digestion, fluorescent probes, ligand binding, etc. (see Ref. 10Robinson J.D. Pratap P.R. Biochim. Biophys. Acta. 1993; 1154: 83-104Crossref PubMed Scopus (41) Google Scholar for a review and references). The fact that probes bound at different sites report theE 1 ↔ E 2 transition implies that substantial structural changes must occur. However, the nature of those changes has been largely obscure. All P-type pumps contain the conserved cytoplasmic sequences TGES in the minor loop between M2 and M3, MVTGD in the major loop, and TGDGVNDSPALKK in the so-called “hinge region” before M5 (5Møller J.V. Juul B. Le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (660) Google Scholar). Proteolytic cleavage and site-directed mutagenesis in these sequences usually stabilizeE 1 conformations, implying an involvement in the conformational transitions (see Refs. 5Møller J.V. Juul B. Le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (660) Google Scholar and 8Andersen J.P. Vilsen B. FEBS Lett. 1995; 359: 101-106Crossref PubMed Scopus (115) Google Scholar for full references). Based on functional effects of mutations and proteolytic cleavages in the β-strand minor cytoplasmic loop of sarcoplasmic reticulum Ca-ATPase, an interaction between the minor and major cytoplasmic loop near the phosphorylation site was proposed earlier (11Green N.M. Stokes D.L. Acta Physiol. Scand. 1992; 152: 59-68Google Scholar). For yeast H-ATPase, mutations within the minor loop suggested a similar conclusion (12Harris S.L. Perlin D.S. Seto-Young D. Haber J.E. J. Biol. Chem. 1991; 266: 24439-24445Abstract Full Text PDF PubMed Google Scholar). Recently, we have described specific iron-catalyzed or copper-catalyzed oxidative cleavage of renal Na,K-ATPase (13Goldshleger R. Karlish S.J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9596-9601Crossref PubMed Scopus (68) Google Scholar, 14Karlish S.J.D. Acta Physiol. Scand. 1998; 163: 89-98Google Scholar, 15Bar Shimon M. Goldshleger R. Karlish S.J.D. J. Biol. Chem. 1998; 273: 34190-34195Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The process seems to involve a site-specific mechanism in which peptide bonds close to the bound metals are cleaved, presumably by OH radicals generated locally by the Fenton reaction or a reactive metal-peroxyl derivative (16Stadtman E.R. Annu. Rev. Biochem. 1993; 62: 797-821Crossref PubMed Scopus (1270) Google Scholar, 17Rana T.M. Meares C.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10578-10582Crossref PubMed Scopus (201) Google Scholar, 18Platis I.E. Ermacora M.R. Fox R.A. Biochemistry. 1993; 32: 12761-12767Crossref PubMed Scopus (83) Google Scholar, 19Berlett B.S. Stadtman E.R. J. Biol. Chem. 1997; 272: 20313-20316Abstract Full Text Full Text PDF PubMed Scopus (2809) Google Scholar). Because more than one peptide bond can be cleaved from the same metal site, this technique provides information on interacting segments of individual subunits or neighboring subunits (unlike proteolytic cleavage). Incubation of Na,K-ATPase with Fe2+/ascorbate/H2O2 induces specific cleavage of the α subunit at the cytoplasmic surface without cleaving the β subunit (13Goldshleger R. Karlish S.J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9596-9601Crossref PubMed Scopus (68) Google Scholar). Copper-catalyzed oxidative cleavage occurs at the extracellular surface and both α and β subunits are cleaved (15Bar Shimon M. Goldshleger R. Karlish S.J.D. J. Biol. Chem. 1998; 273: 34190-34195Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Iron-catalyzed cleavages are very sensitive to the conformational state (13Goldshleger R. Karlish S.J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9596-9601Crossref PubMed Scopus (68) Google Scholar). In (E 2) or E 2(Rb) conformations, we observed four major and two minor fragments of the α subunit. In E 1 orE 1Na conformations, cleavage was much slower and only one major cleavage was observed. Positions of cleavages were either identified exactly by N-terminal sequencing or, for fragments with blocked N termini, approximately using sequence-specific antibodies. In E 2 conformations only, cleavages (identified by short sequences at or near the N termini) were found at214ESE in the minor loop between M2 and M3, near367CSDK after M4 which includes the phosphorylated Asp369, near 608MVTGD in the major loop, and at712VNDS in the “hinge” region before M5. In eitherE 2 or E 1 conformations, a cleavage was seen near 263IATL, before M3. The observations suggested strongly that peptide bonds are cleaved with a probability depending on their proximity to a bound Fe3+ or Fe2+ ion, and thus imply that the different cleavage points are also in proximity to each other. Several cleavages lie in or near the highly conserved cytoplasmic sequences, suggesting that these sequences mediate mutual interactions. We proposed that, inE 2 conformations, the minor and major loops interact near the conserved sequences while, inE 1 conformations, the loops separate (13Goldshleger R. Karlish S.J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9596-9601Crossref PubMed Scopus (68) Google Scholar). This paper extends our observations on iron-catalyzed cleavages in two ways. First, we have obtained further evidence for the site-specific mechanism. Second, we have looked at iron-catalyzed cleavages in both phosphorylated and non-phosphorylated conformations and effects of inhibitors. The results provide novel information on the energy transduction mechanism. Na,K-ATPase, with specific activities of 12–17 units/mg protein, was prepared from pig kidney (20Jørgensen P.L. Methods Enzymol. 1988; 156: 29-43Crossref PubMed Scopus (167) Google Scholar) and was stored at −70 °C in a solution of 250 mm sucrose, 25 mmhistidine, pH 7.2, and 1 mm EDTA (Tris). Rat axolemma microsomes (2–3 units/mg enzyme) prepared as in Ref. 21Sweadner K.J. Methods Enzymol. 1988; 156: 65-71Crossref PubMed Scopus (25) Google Scholar or rat kidney microsomes (3–4 units/mg protein) were prepared as in Ref. 20Jørgensen P.L. Methods Enzymol. 1988; 156: 29-43Crossref PubMed Scopus (167) Google Scholar. Prior to incubation with Fe2+/ascorbate/H2O2, the microsomal membrane preparations (1.5 mg/ml) were pretreated with sodium deoxycholate 1.2 mg/ml for 15 min at 20 °C, washed twice, and resuspended in a solution containing 10 mm Tris·HCl, pH 7.2. Suspensions of pig kidney Na,K-ATPase (0.1–1 mg/ml) or rat microsomal preparations (1 mg/ml) were incubated at 20 °C with freshly prepared solutions of 5 mmascorbate (Tris) plus 5 mm H2O2, without or with added FeSO4. To arrest the reaction, 5–10 mm EDTA or 5-fold concentrated gel sample buffer with 5 mm EDTA was added. Samples were assayed for Na,K-ATPase activity or applied to gels, respectively. Procedures for running of 7.5% Tricine SDS-PAGE, including precautions prior to sequencing, electroblotting to PVDF paper, immunoblots, and microsequencing of fragments have been described in detail (22Capasso J.M. Hoving S. Tal D.M. Goldshleger R. Karlish S.J.D. J. Biol. Chem. 1992; 267: 1150-1158Abstract Full Text PDF PubMed Google Scholar, 23Goldshleger R. Tal D.M. Karlish S.J.D. Biochemistry. 1995; 34: 8668-8679Crossref PubMed Scopus (54) Google Scholar). Anti-K1012–Y1016, referred to as “anti-KETYY,” was used to detect fragments of the α subunit. Immunoblots were stained with diaminobenzidine with metal ion enhancement (15–20 μg of protein/lane) (24Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 509Google Scholar) or developed by enhanced chemiluminescence (ECL; 3–5 μg of protein/lane) using anti-rabbit IgG horseradish peroxidase conjugate and the protocol supplied with ECL reagents from the 1998 Amersham Pharmacia Biotech catalogue. For quantification of bands, the stained PVDF paper or developed x-ray films were scanned with a Bio-Rad GS-690 imaging densitometer and analyzed with Bio-Rad Multi-Analyst software (version 1.01). For quantification of Coomassie-stained α subunit the band was cut out of the gel, and optical density of Coomassie stain extracted into 1% SDS solution was measured at 595 nm (13Goldshleger R. Karlish S.J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9596-9601Crossref PubMed Scopus (68) Google Scholar). Non-linear curve-fitting was performed using Enzfitter (Elsevier-Biosoft). For SDS-PAGE, all reagents were electrophoresis-grade from Bio-Rad. Tris (ultra pure) was from Bio-Lab, Jerusalem. l-(+)-ascorbic acid (catalogue no. 100127), 30% H2O2 (catalogue no. 822287), and α-chymotrypsin (catalogue no. 2307) were from Merck. Phosphocreatine, P6502, creatine phosphokinase P3755. and oligomycin O4876 were from Sigma. All other reagents were of analytical grade. Fig.1 presents an immunoblot using anti-KETYY to detect fragments produced in media containing different proportions of K+ and Na+ ions (sum 150 mm). In 150 mm K+ (E 2(K),left), we observed five major fragments referred to as:a, near M1; b, at 214ESE;c, near 283HFIH (previously referred to as near IATL, see below); d, near 608MVTGD; ande, at 712VNDS. ApparentM r values are 91.3, 80.6, 73.4, 38.2, and 26.3 kDa, respectively. By comparison with previous experiments done in low ionic strength media (13Goldshleger R. Karlish S.J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9596-9601Crossref PubMed Scopus (68) Google Scholar), the cleavage near 367CSDK was largely suppressed, while that near M1 was more prominent. About half the α subunit was cleaved in these conditions. As K+ was replaced by Na+ ions, cleavage at ESE, near MVTGD, and at VNDS was progressively suppressed, essentially completely at 150 mm Na+ (E 1Na,right), while in parallel, the cleavages near M1 and HFIH were amplified. A similar change of pattern was observed upon transfer of the enzyme from low ionic strength (10 mm Tris, pH 7,E 2) to high ionic strength media (10 mm Tris, pH 7, 300 mm choline chloride, E 1). Bound Fe2+ ion is predicted to be in contact with more residues in E 2 than in E 1forms (see Ref. 13Goldshleger R. Karlish S.J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9596-9601Crossref PubMed Scopus (68) Google Scholar and model in Fig. 11), and thus the Fe2+should bind more tightly in E 2 forms. Fig.2 depicts the relationship between Fe2+ ion concentration and cleavage of the α subunit and inactivation of Na,K-ATPase activity, in Rb+- and Na+-containing media (E 2(Rb) orE 1Na, respectively). The curves are fitted well by simple hyperbolas with K 0.5 values of 0.57 and 3.49 μm for cleavage of the α subunit and 0.20 and 1.16 μm for inactivation of Na,K-ATPase, respectively. As discussed previously (13Goldshleger R. Karlish S.J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9596-9601Crossref PubMed Scopus (68) Google Scholar, 15Bar Shimon M. Goldshleger R. Karlish S.J.D. J. Biol. Chem. 1998; 273: 34190-34195Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), the 2–3-fold lowerK 0.5 value for inactivation of Na,K-ATPase compared with cleavage could imply that oxidative reactions occur in addition to chain cleavage and inactivate the enzyme. In any event, the 5–6-fold lower values of K 0.5 in theE 2(Rb) compared with theE 1Na conformation, by either measure, are compatible with the prediction.Figure 2Fe2+ concentration dependence of cleavage of the α subunit and inactivation of Na,K-ATPase activity in Rb- or Na+-containing media. Na,K-ATPase (0.1 mg/ml) suspended in a medium containing 10 mm Tris·HCl, pH 7.2, and 30 mm RbCl or NaCl, and was incubated with Fe2+ at varying concentrations and 5 mm ascorbate and H2O2 for 2 min at 20 °C. Samples were either taken for determination of the Na,K-ATPase activity or applied to a gel (45 μg of protein/lane), and the α subunit was quantified as described under “Experimental Procedures.”View Large Image Figure ViewerDownload (PPT) Fe2+ ions can replace Mg2+ ions in catalysis of Na-dependent phosphorylation (25Fukushima Y. Post R.L. J. Biol. Chem. 1978; 253: 6853-6872Abstract Full Text PDF PubMed Google Scholar) and thus the question arose whether Fe2+ ions bind to the Mg2+ site and catalyze cleavage from this site. Fig.3 depicts the cleavages at three concentrations of Fe2+ (0, 1.5, and 15 μmadded in addition to the 0.05 μm contaminant in the solutions), and Mg2+ from 0 to 7 mm. As the Mg2+ ion concentration was raised, the cleavages at ESE, near CSDK, near MVTGD, and at VNDS were progressively suppressed, while cleavages near M1 and near HFIH were amplified (Fig. 3 A). Thus, Mg2+ ions stabilized an E 1conformation, noticeable especially at 7 mm. However, quantification of amounts of the fragments, by scanning the immunoblots, do not reveal any systematic influence of Fe2+concentration on effects of Mg2+ ions, as seen in the examples in Fig. 3 B. Hence, Fe2+ and Mg2+ ions do not compete and must bind at different sites. In Ref. 13Goldshleger R. Karlish S.J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9596-9601Crossref PubMed Scopus (68) Google Scholar, we speculated that histidine residues in the sequence HFIH near M3 are involved in Fe2+ binding. To test this hypothesis, and also the site-specific mechanism, we compared cleavage of rat axolemma and rat kidney enzymes. Rat axolemma enzyme consists of about 65% α3, 25% α2, and only 10% α1 isoforms, while kidney enzyme is essentially all α1 isoform (26Urayama O. Sweadner K.J. Biochem. Biophys. Res. Commun. 1988; 156: 796-800Crossref PubMed Scopus (83) Google Scholar, 27Sweadner K.J. Biochim. Biophys Acta. 1989; 988: 185-220Crossref PubMed Scopus (868) Google Scholar, 28Munzer J.S. Daly S.E. Jewell-Motz E.A. Lingrel J.B. Blostein R. J. Biol. Chem. 1994; 269: 16668-16676Abstract Full Text PDF PubMed Google Scholar). In α3 and α2 isoforms, the second histidine is replaced by a glutamine,i.e. HFIQ. The experiment in Fig.4 A compared the time course of cleavage of axolemma and kidney enzymes. For axolemma enzyme, the cleavage near HFIH was largely absent and that at VNDS was less prominent, while the cleavages at ESE and near MVTGD were similar to those of the kidney enzyme. For axolemma, a small amount of the fragment with N terminus near CSDK also appeared, although this is less certain because the control itself contains a minor fragment with the same mobility. (The axolemma fragments cleaved near MVTGD and at VNDS have slightly lower mobility than equivalent fragments of kidney (41.8versus 39.1 and 29 versus 27 kDa, respectively). However, this cannot be taken to indicate that the cleavage sites are different, since intact α3 and α2 also have a slightly lower mobility than the α1 isoform, even though theM r value of rat α1 (112, 566) is slightly higher than α2 (111, 580) or α3 (111, 735) (27Sweadner K.J. Biochim. Biophys Acta. 1989; 988: 185-220Crossref PubMed Scopus (868) Google Scholar, 29Shull G.E. Greeb J. Lingrel J.B. Biochemistry. 1986; 25: 8125-8132Crossref PubMed Scopus (549) Google Scholar).) Fig.4 B depicts cleavage of axolemma enzyme in Rb+- or Na+-containing media at different concentrations of added Fe2+ ions. The two major fragments, with N termini ESE and near MVTGD, increased in amount as Fe2+ was raised progressively to 10 μm, as did the minor fragments. In parallel experiments with axolemma and kidney enzyme (data not shown), it was found that cleavage of axolemma enzyme required significantly higher concentrations of added Fe2+ ions. For example, theK 0.5 values for appearance of the fragment with N terminus near MVTGD were 1 ± 0.2 μm for kidney and 1.95 ± 0.2 μm for axolemma, respectively. In the sodium-containing medium, cleavage of both the axolemma and kidney enzymes was suppressed and the fragment with N terminus ESE and the fragment near MVTGD were not observed. In summary, axolemma and kidney enzymes differ in specificity of the cleavages and affinity for Fe2+ ions, whereas the E 2(Rb) →E 1Na transition affects cleavage essentially similarly. Fig. 5 depicts iron-catalyzed cleavage of the pig kidney Na,K-ATPase in conditions of Na-dependent phosphorylation from ATP. A low concentration of ATP (5 μm) was used together with a regenerating system. In the presence of 140 mm Na+ ions, 0.5 mm Mg2+ ions, and the regenerating system, we observed cleavages typical for the E 1Na form (N termini near M1 and HFIH). Upon addition of ATP and oligomycin, the pump should be phosphorylated and, due to block of theE 1P → E 2P conformational transition (30Fahn S. Koval G.J. Albers W. J. Biol. Chem. 1966; 241: 1882-1889Abstract Full Text PDF PubMed Google Scholar), the predominant form should beE 1P. This condition indeed produced cleavages typical of an E 1 conformation. In the absence of oligomycin, the combination of ATP/Na+/Mg2+phosphorylates the pump, but the predominant form should now beE 2P. In this condition, we observed two additional cleavages typical of an E 2 form (N termini ESE and VNDS), but, strikingly, the prominent cleavage near MVTGD normally seen for unphosphorylated E 2 orE 2(K) forms did not appear. The cleavage near CSDK was not seen, but this was expected in the high ionic strength medium. Addition of a low concentration of Rb+ (2 mm) to the medium containing ATP/Na+/Mg2+ accelerates dephosphorylation from high affinity extracellular sites, leading toE 2(Rb) as the predominant form. In this condition, the fragments with N termini ESE and VNDS were somewhat amplified, but again the cleavage near MVTGD did not appear. The latter result was surprising because the cleavage near MVTGD is very prominent in the E 2(Rb) form generated directly by adding Rb+ (K+) ions to the enzyme (see Ref. 13Goldshleger R. Karlish S.J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9596-9601Crossref PubMed Scopus (68) Google Scholar and this paper). The cleavage pattern in the medium containing 2 mm Rb+, 150 mm Na+, and 0.5 mm Mg2+, without ATP, shows that the enzyme remained in the E 1 form. Thus, suppression of the cleavage near MVTGD in the presence of Rb+ ions and ATP/Na+/Mg2+ was associated with formation of the E 2(Rb) form via dephosphorylation ofE 2P. It seemed possible that ATP itself or the products of its hydrolysis, ADP and Pi, were responsible for suppressing the cleavage near MVTGD, and thus a number of control experiments were performed. In low ionic strength media (E 2), the cleavage near MVTGD was prominent, but neither ATP nor ADP at 5 μm had any effect (data not shown). We calculated that, during incubation with ATP/Mg2+/Na+/Rb+ in Fig. 5, 200–250 μm Pi could accumulate. Therefore, we looked for effects on cleavages of Pi(Tris), 0.1–1 mm, without or with Mg2+ and Rb+ions (Fig. 6). Addition of Pito a medium of low ionic strength indeed suppressed the cleavages near CSDK and MVTGD, without significantly affecting other cleavages (Fig.6 A). In this condition, one could expect the enzyme to be in an E 2·P form, with Pi bound non-covalently. In combination with 1 mm Mg2+ions, addition of 2 mm Pi also partially suppressed the cleavages at ESE and VNDS (Fig. 6 B, see legend for quantification based on scans). When added alone, Mg2+ ions at 1 mm or lower concentrations had little or no effect (see also Fig. 3). The presence of 1 mmRb+ ions did not alter the effect of Pi but prevented partial suppression of cleavages at ESE and VNDS by the combination of Pi and Mg2+ ions (Fig.6 C, see legend for quantification). Another effect of the combination of Pi and Mg2+ ions was observed in experiments that examined Pi concentration dependence of suppression of the cleavage near MVTGD at 0–1 mmMg2+. Data from scans of gels (Fig.7) show that lower concentrations of Pi were required in the presence of Mg2+ ions. In the presence of 1 mm Mg2+ and 2 mm Pi, a substantial fraction of the enzyme should be phosphorylated as a form referred to asE 2i-P, which is insensitive to Rb+(K+) ions (31Post R.L. Toda G. Rogers F.N. J. Biol. Chem. 1975; 250: 691-701Abstract Full Text PDF PubMed Google Scholar). In the presence of Mg2+, Pi, and Rb+, a major fraction should not be phosphorylated (E 2(Rb)·Pi) and a minor faction could be phosphorylated in a Rb+(K+)-bound form (E 2-P·Rb) (31Post R.L. Toda G. Rogers F.N. J. Biol. Chem. 1975; 250: 691-701Abstract Full Text PDF PubMed Google Scholar). An economical explanation of the results in Figs. Figure 5, Figure 6, Figure 7 is that either non-covalently bound phosphate or covalently bound phosphate, derived from ATP or Pi, directly interfere with the cleavages near CSDK and MVTGD. In addition the cleavages at ESE and VNDS are somewhat suppressed in the phosphorylated forms (see “Discussion” and Fig. 11).Figure 7Co-operative effect of Pi and Mg2+ ions in suppression of the specific cleavage near MVTGD. Na,K-ATPase (1 mg/ml) was suspended in a medium containing 10 mm Tris·HCl, pH 7.2, and 0, 0.1, 1, or 5 mm Pi(Tris) and 0, 0.1, or 1 mmMgCl2. The ionic strength was maintained constant by addition of choline chloride as necessary. The suspension was incubated with 5 μm FeSO4 and 5 mmascorbate/H2O2 for 2 min at 20 °C. The diaminobenzidine-stained PVDF paper was scanned and analyzed as described under “Experimental Procedures.” The figure depicts the quantity of the fragment near MVTGD at different Piconcentrations as a percentage of that without Pi, at the MgCl2 concentration of 0, 0.1, and 1 mm.View Large Image Figure ViewerDownload (PPT) Figs. 8 and9 present some paradoxical observations. As seen in Fig. 8, the presence of Mg2+/ouabain somewhat suppressed cleavages near MVTGD and at VNDS, while in the presence of Pi/Mg2+/ouabain all cleavages were suppressed except the two near M1 and HFIH. Similarly, with vanadate (2 μm)/Mg2+, only the two cleavages near M1 and HFIH were observed. Thus, the cleavage pattern with Pi/Mg2+/ouabain and vanadate/Mg2+was characteristic of E 1 forms, although the enzyme should be in an E 2 form in both conditions (see also Fig. 10). Fig. 9presents another surprising finding with Pi. With the usual order of addition of the components, first equilibration of enzyme with Pi and then incubation with Fe2+/ascorbate/H2O2, the effect of Pi was as described in Fig. 6 A. However, if the enzyme was preincubated with both Pi and Fe2+prior to addition of ascorbate/H2O2, the cleavages at ESE and VNDS were also largely suppressed, although those near M1 and HFIH were largely unchanged. The development of this effect is seen in Fig. 9. After 20 min of preincubation of Pi with Fe2+, prior to addition of ascorbate/H2O2, the cleavage pattern was similar to that with Pi/Mg2+/ouabain or vanadate/Mg2+.Figure 9Effect of preincubation of Piwith Fe2+ ions on iron-catalyzed cleavage of the α subunit. Na,K-ATPase (1 mg/ml) was suspended in a medium containing 10 mm Tris·HCl, pH 7.2, 1 mm Pi (Tris), and 5 μmFeSO4 and was incubated for 0–20 min. Then 5 mm ascorbate/H2O2 was added and the suspension incubated for 2 min at 20 °C.View Large Image Figure ViewerDownload (PPT)Figure 10Chymotryptic digestion of Na,K-ATPase.Na,K-ATPase (1 mg/ml) was suspended in a medium containing 5 mm Tris·HCl, pH 7.2, 20 mm NaCl, or 20 mm RbCl or 7 mm MgCl2(A) or 1 mm Pi, 1 mmMgCl2, and 1 mm ouabain (B). α-Chymotrypsin was added at a ratio of 1:20 (w/w) with respect to Na,K-ATPase and incubated together at 37 °C for 10 min. 150 mm RbCl was added and then 1 mm PMSF, and the mixture was incubated for 15 min at room temperature before centrifu" @default.
• W2067937824 created "2016-06-24" @default.
• W2067937824 creator A5005190748 @default.
• W2067937824 creator A5082141342 @default.
• W2067937824 date "1999-06-01" @default.
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