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Triphosphate and Its Cr(H20)4 and Co(NH 3)4 Complex Derivatives Are New Fluorescent Tools for Labelling ATP Binding Sites of Na+/K +-Atpase

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Triphosphate and Its Cr(H20)4 and Co(NH 3)4 Complex Derivatives Are New Fluorescent Tools for Labelling ATP Binding Sites of Na+/K +-Atpase Physiol. Res. 46: 345-349, 1997 2’(3’)-0-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5’-triphosphate and its Cr(H20)4 and Co(NH 3)4 Complex Derivatives are New Fluorescent Tools for Labelling ATP Binding Sites of Na+/K +-ATPase H. LINNERTZ, I. LASTRES BECKER, R. KRUMSCHEID1, E. AMLER2, D. THOENGES, W. SCHONER Institute of Biochemistry and Endocrinology, ‘Biochemical Institute, Justus-Liebig-University Giessen, Giessen, Germany and 2 Institute of Physiology, Czech Academy o f Sciences, Prague, Czech Republic Receh’ed March 5, 1997 Accepted May 14, 1997 Summary 2’(3’)-0-[N - [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5’-triphosphate (FEDA-ATP), a spectroscopic tool used for studying skeletal muscle myosin ATPase subfragment 1, was applied to Na+/K +- ATPase (EC 3.6.1.37). In contrast to the myosin subfragment, we found that FEDA-ATP is not a substrate for Na + /K + -ATPase. On the other hand, FEDA-ATP showed an affinity for both the low (E2, K<j = 2001MM) and the high (Ei, Kd = 22,«M) affinity ATP-binding sites. When the microscopic affinities of FEDA-ATP were used for calculating the macroscopic affinity in the overall reaction according to Kj = (KdEl*KdE2)1/2, the experimentally measured inhibition constant of 6 6,wM was obtained. To evoke irreversible binding inhibitors, FEDA-ATP was transferred in its chromium (III) and cobalt(III) complex analogs, which are suitable tools for labelling the ATP binding sites of Na + /K + -ATPase in a specific way. Key words Na + /K +-ATPase - Complex derivatives of ATP - Low-affinity ATP-binding site - High-affinity ATP binding site Abbreviations Cr(H2 0 )4ATP, /?, y bidentate complex of chromium (III)-tetraaqua-adenosine-5’-triphosphate Cr(H2 0 )4FEDA-ATP, (3, y bidentate complex of chromium (Ill)-tetraaqua- 2’(3’)-0-[N- [2- [3- [5- fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine-5’-triphosphate Co(NH 3)4ATP, ¡3, y bidentate complex of cobalt (III)-tetramino-adenosine-5’-triphosphate Co(NH3)4FEDA-ATP, (3,7 bidentate complex of cobalt (Ill)-tetramino- 2’(3’)-0-[N- [2- [3- [5- fluoresceinyl] thioureido] ethyl] carbamoyl]- adenosine-5’-triphosphate Co(NH 3)4P 0 4, tetramine cobalt(III)phosphate FITC, fluorescein 5’-isothiocyanate FEDA-ATP, 2’(3’)-0-[N- [2- [3- [5- fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5’-triphosphate EDA-ATP, 2’(3’)-0-[amino ethyl carbamoyl] adenosine 5’-triphosphate EjATP site, nucleotide-binding site of Na+/K +-ATPase with high affinity for ATP E2ATP site, nucleotide-binding site of Na+/K +-ATPase with low affinity for ATP 346 Linnertz et al. Vol. 46 Introduction E. Merck (Darmstadt, Germany) and Molecular Probes (Eugene, USA). The Lab-Trol protein standard Na+/K +-ATPase (EC 3.6.1.37) is the was a product of Merz & Dade (Munich, Germany). biochemical equivalent of the sodium pump of [y32p]ATP was from Amersham Buchler mammalian plasma membranes (Skou and Esmann (Braunschweig, Germany). 1992). The enzyme probably works as a functional («/02-diprotomer (Schoner et al. 1994). Kinetic studies Synthesis of FEDA-ATP, MgATP- and MgFEDA-ATP- on ATP hydrolysis revealed that phosphorylation of the complex analogs catalytic a-subunit at Asp-369 occurs in the presence of Ethylenediamine was first coupled to ATP Na+ from a high affinity ATP site (Ku^l/^M, EiATP according to Cremo et al. (1990) to yield 2’(3’)-0- site). This process is followed by K+-activated [amino ethyl carbamoyl] adenosine 5’-triphosphate hydrolysis of the phospho-intermediate. Na+- (EDA-ATP). This intermediate product was then dependent phosphorylation of the a-subunit leads to made to react with fluorescein 5’-isothiocyanate the occlusion of three Na+ ions. These ions are (isomer 1) according to Sowerbyet al. (1993) to yield released into the external medium during the reaction 2’(3’)-0-[N- [2- [3- [5- fluoresceinyl] thioureido] ethyl] cycle. Occlusion of two K+ ions, however, takes place carbamoyl] adenosine 5’-triphosphate (FEDA-ATP). in the nonphosphorylated a subunit. To release K+ at The concentration of FEDA-ATP was determined the inner membrane face, ATP has to bind to a low- assuming an absorbance coefficient at pH 9.0, A495 of affinity ATP site (E2ATP site), which hydrolyzes 75 000 M " 1 cm-1. Cr(H20 )4ATP, Co(NH3)4ATP, p-nitrophenylphosphate and supports the binding of Co(NH 3)4FEDA-ATP and Cr(H20 ) 4FEDA-ATP ouabain in the presence of Pi (Linnertz et al. 1994). complex analogs were prepared by the aniline Studies with chromium(III) and cobalt(III) complex procedure of Cleland and coworkers (De Pamphilis analogs of ATP revealed that the EiATP and E2ATP and Cleland 1973, Cornelius et al. 1977). sites coexist and that these MgATP complex analogs Isolation of Na+ /K +-ATPase can be used to differentiate between them. Na + /K +-ATPase was isolated from pig kidney Cr(H2 0 )4ATP is an almost irreversible specific according to the method of Jorgensen (1974) and inhibitor of the EiATP and Co(NH3)4ATP of the assayed as described earlier (Schoner et al. 1994, E2ATP-binding site (Schoneret al. 1994, Linnertz et al. Linnertz et al. 1995). 1995, Linnertz and Schoner 1996). 2’(3’)-0-[N- [2- [3- [5-fluoresccinyl] Inhibition of ATP hydrolysis by FEDA-ATP thioureido] ethyl] carbamoyl] adenosine 5’-triphosphate Various concentrations of FEDA-ATP (0-100 (FEDA-ATP) known from studies of skeletal muscle /zM) were used in 1 ml of the optical assay mixture myosin subfragment 1 (SI) (Conibaret al. 1996) makes (Schoner et al. 1967) and ATP concentration range it possible to determine the activity of single myosin from 25 to 500 /<M. filaments. FEDA-ATP as substrate is hydrolyzed toPj and FEDA-ADP, which is released with the same rate Protective action of FEDA-ATP against the inactivation constant as ADP. As a result of fluorescence changes, by MgA TP analogs it was possible to follow turnover of this ATP analog by Affinities of FEDA-ATP for the EiATP and fluorescence microscopy (Conibaret al. 1996). E2ATP binding sites were determined from the The aim of the present work was to ascertain protective effect of FEDA-ATP as a function of its whether FEDA-ATP is a substrate of Na + /K + - concentration (0— 500/¿M). Na + /K +-ATPase ATPase and whether it and its MgATP complex (0.5 units, 20 units/mg protein) was incubated in a total analogs provide information about the EiATP and/or volume of 250/¿I at 37 °C, 15 mM Tris/HCl buffer E2ATP sites. We therefore studied the interaction of (pH 7.5) and various concentrations of Cr(H30 ) 4ATP FEDA-ATP with the high-affinity EiATP site, which is (O-IOO^M) or Co(NH 3)4ATP (0 — 500 ^M). Residual inactivated by Cr(H2 0 )4ATP, and with the low-affinity Na+/K +-ATPase activity was measured at the time E2ATP site, which is inactivated by Co(NH3)4ATP. indicated by transferring aliquots of 30ju\ to the optical Furthermore, FEDA-ATP was transferred in its assay (Schoner et al. 1967). chromium(III) and cobalt (III) complex analogs, which are suitable tools for labelling the ATP binding sites of Inactivation with Cr(H20)4FEDA-ATP and Na+/K +-ATPase in a specific way. Co (NH3) 4FEDA-A TP One unit of Na+/K +-ATPase was incubated Materials and Methods in a total volume of 1 ml at 37 °C with 20 mM Tris/HCl buffer at pH 7.25, 15 mM NaCl and different All chemicals were of highest available purity concentrations of the inhibitor. Residual activity was and were obtained from Bio-Rad (Munich, Germany), measured by transferring 50p\ aliquots to the optical Boehringer-Mannheim (Mannheim, Germany), assay mixture (Schoneret al. 1967). 1997 Labelling of ATP Binding Sites 347 (lU /m l) was incubated in a mixture of 20 mM Tris/HCl (pH 7.25) and 15 mM NaCl at 37 °C with 0 //M, 50 mM and 100 mM Cr(H20 )4ATP and the enzyme inhibition curves were measured (Fig. 2). Fig. 2. Protective effect of different FEDA-ATP concentrations against the inactivation by Cr(H2 0 )4ATP. One unit N a+ /K + -ATPase was C FEDA-ATP (MM) incubated in a total volume of 250 pi with different concentrations of Cr(H 2 0 )4ATP (0, 20, 40, 60, 80 and 100 pM) and FEDA-ATP (full square 0 pM, open circle Fig. 1.Inhibition of ATP hydrolysis by FEDA-ATP. (A) 50 pM and full rhombus 100 pM). kapp are the apparent ATP hydrolysis was measured in 1 ml of the optical velocity constants. assay mixture (Schoner et al. 1967) containing 25 -500 pM ATP and various concentrations of FEDA- A TP (full square 0 pM, open square 20 pM, full circle 40 pM, open circle 60 pM and full rhombus 100 pM). We detected a reduction of the maximum velocity of ATP-hydrolysis. (B) The inhibition of ATP-hydrolysis by FEDA-ATP results in an inhibition constant of 6 6 pM and a maximum velocity of 26 .7 U/ml. Results Pig kidney Na + /K +-ATPase (1 U/m l) was incubated with 0, 20, 40, 60 and 100 /¿M FEDA-ATP and rates of ATP hydrolysis were measured by the optical assay. Hydrolysis of ATP was inhibited by —T ------- f ------1------------1--------- 1--------- 1 -0.002 0.000 0.002 0.004 FEDA-ATP (Fig. 1A), at 100p U FEDA-ATP, being about 25 % of the control. The inhibition constant Kj ^ C CoATP 0 / p M ) was found to be 66 pM (Fig. IB). However, this macroscopic affinity of FEDA-ATP of 66 pM in the Fig. 3. Protective effect of different FEDA-ATP overall reaction is the result of the microscopic concentrations against the inactivation by affinities of the substance for the EiATP and E2ATP Co(NH3)4ATP.
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