ЖУРНАЛ ФИЗИЧЕСКОЙ ХИМИИ, 2009, том 83, № 9, с. 1778-1784
BIOPHYSICAL CHEMISTRY =
УДК 541.64
Na+/K+ -ATPASE - ACTIVITY AND INHIBITION
© 2009 M. Colovic*, D. Krstic**, K. Krinulovic*, T. Momic*, J. Savic*,
A. Vujacic*, and V. \&sic*
*Department of Physical Chemistry, Institute of Nuclear Sciences Vinca, P.O. Box 522, 11001 Belgrade, Serbia **Institute of Medicinal Chemistry, University School of Medicine, University of Belgrade, Visegradska 26, Belgrade, Republic of Serbia Corresponding author. Tel.: +381-11-2453-967; fax: + 381-11-2447-207 E-mail: evasic@vin.bg.ac.yu
Abstract — The aim of the study was to give an overview of the mechanism of inhibition of Na+/K+-ATPase activity induced by some specific and non specific inhibitors. For this purpose, the effects of some ouabain like compounds (digoxin, gitoxin), noble metals complexes ([PtCl2DMSO2], [AuCl4]-, [PdCl4]2-, [PdCl(di-en)]+, [PdCl(Me4dien)]+), transition metal ions (Cu2+, Zn2+, Fe2+, Co2+) and heavy metal ions (Hg2+, Pb2+,
Cd2+) on the activity of Na+/K+-ATPase from rat synaptic plasma membranes (SPM), porcine cerebral cortex and human erythrocytes were discussed.
INTRODUCTION
Na+/K+-ATPase (EC 3.6.1.3) is a cell membrane located enzyme, which plays a key role in the active transport of monovalent cations (Na+ and K+) across the cell membrane [1, 2]. The enzyme is composed of a a subunit, which contains the adenosinetriphos-phate (ATP) - Na+, K+ and ouabain-binding sites, as well as the site for phosphorylation and a p-subunit, which stabilizes the K+ binding cage. Na+/K+-ATPase acts as a dimer (ap—pa). The most widely accepted view related to such a dimmer to act is a "flip-flop" model, in which both subunits show complementary conformation:
E1E2 E2Eb where E; is the conformation of each a subunit. The activity of this enzyme is very sensitive to the presence of some metal ions [3, 4] and organic compounds of various structures, especially some drugs and pesticides [5, 6]. Beside its transporter function, Na+/K+-ATPase acts as the receptor for cardiac glycosides such as ouabain like compounds [7], which are the specific inhibitors of the enzyme. Ouabain binds to the extracellular part of the protein with very high affinity, leading to the inhibition of enzymatic activity. The non specific inhibitors, e.g. metal ions and complexes, interfere with the enzyme due to the complex formation with sulfhydril groups of cysteine residues. Furthermore, nephrotoxicity, ototoxicity etc. of platinum anticancer drugs, such as cisplatin and chloroplatinic acid, is related to inhibition of Na+/K+-ATPase activity. Since Pd(II) complexes have great affinity for binding with —SH containing ligands [8, 9] and react about 105 times faster, these complexes represent an excellent model for investigation of the reaction between
Na+/K+-ATPase and platinum group anticancer drugs. The similar kind of interaction with Na+/K+-ATPase was also observed for heavy and transition metal ions [10].
Our study was undertaken with the aim to make an overview concerning the mechanism of interaction between ouabain-like compounds, noble metals complexes, metal ions and Na+/K+-ATPase. The extensive kinetic analysis was done in order to determine kinetic parameters and type of Na+/K+-ATPase inhibition. In addition, the ability of sulphur-donor ligands (L-cys-teine and glutathione) to prevent metal ions and complexes induced inhibition of Na+/K+-ATPase and to recover enzymatic activity was investigated. Finally, development of highly sensitive and selective analytical tools using the immobilized enzyme is discussed in this paper.
EXPERIMENTAL
Chemicals. All chemicals were of analytical grade. Na+/K+-ATPase from porcine cerebral cortex and ATP were purchased from Sigma Co., as well as some chemicals for medium assay (magnesium chloride and Tris-HCl). The specific Na+/K+-ATPase activity was 25.8 ^mol Pj/(h mg protein), Pj is the inorganic orthophosphate). Other medium assay chemicals (sodium chloride, potassium chloride) and chemicals for determination of Pi (stannous chloride and ammonium molybdate) were from Merck (Darmstadt, Germany).
Red cells membranes preparation. Red cells membranes were prepared according to the method of Post et al. [11] with certain modifications. Whole blood, 5 ml, was suspended in 0.5 ml of 0.2 M Na4EDTA and 30 ml
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of 0.15 M NaCl. The cells were packed by centrifuga-tion at 12000 rpm for 10 minutes and were washed three times in 0.15 M NaCl. Cell lyses was performed with 5 mM Tris-HCl (pH 7.4) and freezing on -20°C. The haemoglobin free membranes were sedimented at 3000 rpm for 30 minutes and washed three times in this buffer. All procedures were carried out at 0—2°C. Protein concentration measurements were made according to the method of Bradford using crystalline bovine serum albumin (BSA) as a standard. The specific Na+/K+-ATPase activity was 0.288 |mol Pj/(h mg protein).
Synaptic plasma membranes (SPM) preparation. SPM were isolated from the whole brain of 3-month-old male Wistar albino rats from the local colony. Animals were kept under controlled illumination (lights on: 5:00am—5:00pm) and temperature (23 ± 2°C), and had free access to food and water. The "Guiding Principles for the Care and Use of Animals" based upon Helsinki Declaration (1964) and "Protocol of the "Vinc a" Institute on care and treatment of laboratory animals" were strictly followed. After decapitation with a guillotine (Harvard Apparatus), brains were rapidly excised and pooled (six per pool) for immediate preparation of synaptic plasma membranes. The SPM were isolated according to the method of Cohen [12] and were stored at —70°C until used.
ATPase assay. The enzyme activity was determined in a standard incubation medium (200 |l), containing 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 20 mM KCl, 5 mM MgCl2, 2 mM adenosinetriphosphate (ATP) and erythrocyte membrane fragments (100 |g protein) or 25 |g SPM proteins (i.e.58 |g commercial porcine cerebral cortex proteins in the presence or absence (control) of the desired concentration of inhibitor. Incubation mixtures were preincubated at 37°C in the presence of inhibitor or distilled water (control). The reaction was started by the addition of ATP, and allowed to proceed until stopped by ice cold HClO4. The activity obtained in the presence of 2 mM ouabain (without the NaCl and KCl) was attributed to Mg2+-ATPase. Na+/K+-ATPase activity was calculated as a difference between the total ATPase and Mg2+-ATPase activity. The inorganic ortho-phosphate (Pj) liberated due to the hydrolysis ofATP was measured using modified spectrophotometric procedure based on the stan-nous chloride method [3, 4], by reading the absor-bance at 690 nm. In addition, the concentration of liberated Pi was compared to the concentration of adenosinediphosphate (ADP) measured by slightly modified ultra performance liquid chromatography (UPLC) method [13]. Excellent agreement was obtained, confirming that only orthophosphates liberated from enzyme catalyzed ATP hydrolysis were detected.
Kinetic experiments were carried out according to the slightly modified method of Philips [14] using the commercial porcine cerebral cortex Na+/K+-ATPase. The initial velocities were measured in the same incu-
bation medium as a function of rising concentrations of MgATP2-(0.1—5.0 mmol / l). The measurements were performed in the absence and presence of inhibitor, while maintaining the concentrations of other ions (Na+, K+ and Mg2+) constant. The experimental data were fitted to the Michaelis—Menten equation by nonlinear regression analysis using EZ FIT [15]. Vmax and Km values were derived from a Lineweaver—Burk plot and expressed in |mol Pi/(h mg protein) and mmol l-1 of ATP. Results are given as means ±S.E.M.
Apparatus. The spectrophotometric measurements were performed on Beckman 5260 UV VIS spectro-photometer. The chromatographic experiments were performed on Waters ACQUITY Ultra Performance Liquid Chromatography (UPLC) system, using an ACQUITY UPLC™ BEH C18, 1.7 |im, 50 mm x x 2.1 mm column as a stationary phase. The mobile phase was composed of 4 mM tetrabutylammonium hydroxide (TBAH) in 4 mM phosphate buffer (phase A) and methanol (phase B) in the ratio 75:25. Flow rate was 0.25 ml/min, injection volume 10 |l and the column temperature 40°C. The separation was monitored in a single wavelength mode at 254 nm.
RESULTS AND DISCUSSION
Inhibition of Na+/K+-ATPase activity by digitalis
Some widely used digitalis glycosides (synthetic drugs and medical plants isolates) strongly modulate enzyme activity in a concentration dependent manner. The basic cardenolide structure of specific Na+/K+-ATPase inhibitors is schematically presented in Fig. 1.
Recently, it was confirmed that basicity, i.e. a strong ionic interaction between one of carboxylate residues present in the a subunit of the Na+/K+-ATPase and the cationic form of some digitalis like derivatives is relevant for interference with enzyme activity [16]. In naturally occurring digitalis glycosides the unsaturated y- and 8-lactones present in 17p-position of the steroi-dal skeleton are associated with high affinity for the Na+/K+-ATPase receptor. The presence of -OH groups at different positions of the steroidal skeleton reduces, in general, the interaction energy, though it depends on
Fig. 1. Basic cardenolide structure of specific Na+/K+-ATPase inhibitors.
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A, %
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Fig. 2. Inhibition of Na+,K+-ATPase activity (A) by digoxin in cerebral cortex of (1) pig, (2) human erythrocyte membranes and (3) rat brain.
the location and spatial disposition of such —OH groups.
Digitoxin, one of the most frequently used drugs to improve cardiac contractility, undergoes a complex metabolic degradation generating digitoxigenin, digi-toxigenin mono-digitoxoside and gitoxin [16]. The structural difference between digoxin and its isomer gitoxin, that usually appears as a resu
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