L-cysteine is used as effective oral chelating agent due to property of its sulfhydryl
group to bind heavy metal ions. The aim of this study was to investigate ability of
L-cysteine to prevent mercury (II) and cadmium (II) induced ecto-ATPase activity
inhibition of rat uterus plasma membranes. Results show that 10 mmol/l L-cysteine
have protective effect on enzyme activity in the presence of cadmium and mercury
ions

Drakulić, Dunja R.

Horvat, Anica

Milošević, Maja

Petrović, Snježana

Stanojević, Ivana

Veličković, Nataša

Repository of the Vinča Nuclear Institute (VinaR)

       PHYSICAL CHEMISTRY 2010  10th International Conference on Fundamental and Applied Aspects of Physical Chemistry  Proceedings      The Conference is dedicated to the 100th Anniversary of the academician Pavle Savić birthday and 20th Anniversary of the Society of Physical Chemists of Serbia         21-24 September 2010 B E L G R A D E                            ISBN 978-86-82475-17-0 Title: Physical Chemistry 2010. (Proceedings) Editors: S. Anić and Ž. Čupić Published by: Society of Physical Chemists of Serbia, Studentski trg 12-16 P.O.Box 47, 11158 Beograd, 218, Srbija Publisher: Society of Physical Chemists of Serbia For Publisher: S. Anić, President of Society of Physical Chemists of Serbia Printed by: “Jovan” Printing and Publishing Company; 200 Copies; Number of pages 16 + 388, Format: B5; Printing finished in September 2010.  Text and Layout: “Jovan”  200 - Copy printing    II   The Society of Physical Chemists of Serbia  in co-operation with   Institute of Catalysis, Bulgarian Academy of Sciences  Boreskov Institute of Catalysis, Siberian Branch of  the Russian Academy of Sciences  Faculty of Physical Chemistry, University of Belgrade, Serbia  Institute of Chemistry Technology and Metallurgy, University of Belgrade, Serbia  Vinča Institute, University of Belgrade, Serbia   Institute of General and Physical Chemistry, Serbia                         IV  International Organizing Committee  Chairman:             S. Anić (Serbia) Vice-chairman:     N. Stepanov (Russia)                                B. Adnađević (Serbia)  Members: M. Gabrovska (Bulgaria), N. Cvijetićanin (Serbia), T. Grozdić (Serbia),  D. Jovanović (Serbia), M. Lalić (BiH), D. Marković (Serbia), B. Milosavljević (USA), N. Miljević (Serbia), M. Mojović (Serbia), N. Ostrovski (Serbia), C. Pona (Italy), B. Simonović (Serbia), D. Stanisavljev (Serbia), A. G. Stepanov (Russia), V. Vasić (Serbia), N. Vukelić (Serbia), V. Vukojević (Sweden)   International Scientific Committee  Chairman:              Ž. Čupić (Serbia) Vice-chairmans:     V. N. Parmon (Russia)                                 M. Franko (Slovenia)  V. Vasić (Serbia)  Members: A. Antić-Jovanović (Serbia), G. Bačic (Serbia), R. Cervellati (Italy), R. Compton (United Kingdom), V. Gaspar (Hungary),  M. Jeremić (Serbia), A. L. Kawczyñski (Poland), Lj. Kolar-Anić (Serbia), S. Kuchanov (Russia), R. Leblanc (USA),  S. Mentus (Serbia), S. Milonjić (Serbia), Lj. Morozova-Roche (Sweden),  D. Moscone (Italy), J. Nedeljković (Serbia), Z. Noszticzius (Hungary), M. Perić (Serbia), V. Petruševski (Macedonia), M. Plavšić (Serbia), G. Smulevich (Italy),  G. Schmitz (Belgium), I. Schreiber (Czech), P. Ševčik (Slovakia), N. Stepanov (Russia), M. Trtica (Serbia), D. Veselinović (Serbia)  Local Executive Committee  Chairman:              B. Adnadjević Vice-chairmans:    S. Blagojević                                 A. Ivanović  Members: A. Abu Rabi-Stanković, P. Banković, N. Begović, S. N. Blagojević,  N. Cvjetičanin, M. Daković, A. Đerić, A. Ignjatović, Lj. Ignjatović, A. Jović,  J. Krstić, S. Kuprešak, D. Lončarević, J. Maksimović, V. Marković, M. Milenković, M. Milojević, Z. Mojović, B. Nedić, I. Pašti, N. Pejić, A. Popović-Bjelić, M. Petković, N. Potkonjak, D. Ranković, R. Ranković, M. Stević,  I. Stojković, B. Šljukić, M. Vujković    VL-CYSTEINE MODULATES THE ecto-ATPase ACTIVITY INHIBITION IN PRESENCE OF CADMIUM (II) AND MERCURY (II) IONS  M. Milošević, D. Drakulić, S. Petrović, I. Stanojević,  N. Veličković and A. Horvat  Laboratory of Molecular Biology and Endocrinology, Institute of Nuclear Sciences “ VINČA”, University of Belgrade, P.O. Box 522, 11001 Belgrade, Serbia  Abstract L-cysteine is used as effective oral chelating agent due to property of its sulfhydryl group to bind heavy metal ions. The aim of this study was to investigate ability of L-cysteine to prevent mercury (II) and cadmium (II) induced ecto-ATPase activity inhibition of rat uterus plasma membranes. Results show that 10 mmol/l L-cysteine have protective effect on enzyme activity in the presence of cadmium and mercury ions.  Introduction Heavy metals could be accumulated in the body during low, chronic exposure, originate from various sources such as drinking water, cigarette smoke or even from tooth fillings made of silver-mercury amalgam. According to numerous data cadmium and mercury are classified as reproductive toxicants [1] and their effects on uterine plasma membrane ecto-ATPase activity were reported previously [2]. During the years, the importance of purinergic signaling in mammalian reproductive tract has been emphasized [3]. Extracellular nucleotides and/or their degradation products affect via purinoceptors various functions of uterine tissue such as contraction, growth, remodeling and immunity. In uterine tract, ATP is co-released from autonomic nerves with neurotransmitters, but physiological sources of extracellular nucleotides are also platelets and semen. Ecto-ATPase is a member of E-NTPDases family (EC 3.6.1.5), the most abundant enzyme family among ectonucleotidases. Ectonucleotidases are surface plasma membrane bound enzymes that modulate purinergic signaling by hydrolyzing nucleotide di- and tri phosphates.   Experimental procedure Animals: Experiments were performed on 3-month-old female Wistar albino rats obtained from the local colony. Animals were maintained under standard conditions with ad libidum access to food and water. Myometrial plasma membranes (MPM) were isolated as previously described [4].  334ATP assay: Incubation medium contained: 50 mmol/l Tris-HCl buffer (pH 7.4), 5 mmol/l MgCl2, increasing concentration of metal ions in absence or presence of 10 mmol/l L-cysteine and 7μg MPM protein. The enzyme reaction was started by addition of 2 mmol/l ATP, allowed to proceed for additional 10 min and stopped with 22μl of the ice cold 3 mol/l perchloric acid. The inorganic phosphate (Pi) liberated from the ATP hydrolysis was determined by the spectrophotometric method. All measurements were performed in triplicate.  Results and discussion Cadmium chloride (CdCl2) and mercury chloride (HgCl2) were added to the reaction mixture in concentration ranges from 1x10-7 to 1x10-1 mol/l. The effects of increasing metal ion concentrations were measured in the absence and in the presence of 10 mmol/l L-cysteine.   Fig.1. Inhibition of ecto-ATPase activity by CdCl2 in the absence (open symbol) and in the presence (solid symbols) of 1×10-2 mol/l  L-cysteine.  The values given are the mean ±S.E.M of tree independent MPM isolation done in triplicate.  Fig.2. Inhibition of ecto-ATPase activity by HgCl2 in the absence (open symbol) and in the presence (solid symbols) of 1×10-2 mol/l  L-cysteine.  The values given are the mean ±S.E.M of three independent MPM isolation done in triplicate.   Compared to the control samples, in the absence of L-cysteine and in the presence of 0.01 mol/l of CdCl2 (Fig 1.) ecto-ATPase activity showed up to 90% inhibition, while total enzyme inhibition was detected in presence of 0.1 mol/l HgCl2 (Fig 2.). The half-maximum inhibitory activities (IC50) determined by Hill 335analysis of experimental curves in the absence or presence of L-cysteine were: 3.49×10-4 and 1.34×10-3 mol/l for cadmium (II) (Fig.1 inset) and 1.74×10-3 and 2.06×10-3 for mercury (II) (Fig.2 inset), respectively. The IC50 value for mercury (II) ions in the presence of 10 mmol/l L-cysteine was determined directly from inhibition curve. When cysteine and mercuric chloride mole ratio approached 2:1, white precipitation was formed and enzyme activity disappeared. Precipitation occurs due to formation of Hg(Cys)2 and Hg(Cys)2x2HgCl2 [5].  L-cysteine is an important constituent of proteins, largely by virtue of its sulfhydryl group to form disulfide bonds and stabilize protein molecular configuration. Cadmium and mercury have high affinity for sulfur and sulfhydryls and therefore potential of binding to proteins throughout the body, disrupting their structure and function. Binding tightly to glutathione (GSH), they might deplete cell’s antioxidant capacity. At the same time, cadmium and mercury promote formation of reactive oxygen species that alter plasma membrane structure as a result of lipid peroxides formation.  Ecto-ATPase contains 10 cysteine residues on its extracellular domain but has no free sulpfhidryl groups, so this might explain relatively high IC50 value for cadmium and mercury in the absence of chelator.  Conclusion According to the results presented in this study, 10 mmol/l L-cysteine increased by one order of magnitude CdCl2 concentration for half-maximal inhibition (IC50), as a consequence of decreasing metal ions concentrations capable to interact with enzyme amino acid residues. In the presence of mercuric ions, L-cysteine was less efficient in prevention of enzyme activity, probably because of complex compounds formation at higher concentrations of metal ions.  Acknowledgements This study was supported by the Serbian Ministry of Sciences, Project No. 143044. References [1]  P. Kovačić, J. D. Jacintho, Curr. Med. Chem., 2001, 8, 863-892. [2]  M. Milošević, M. Demajo, A. Horvat, Physical Chemistry 2006, A. Antić-Jovanović (ed.), SPCS, Belgrade, 2006, 1, 395-397. [3]  M. Martín-Satué, E.G. Lavoie, M. Fausther, J. Lecka, E. Aliagas, F. Kukulski, J. Sévigny, Histochem. Cell Biol., 2009, 131, 615-628. [4]  M. Milošević, S. Petrović, M. Demajo, A. Horvat, Ann. N .Y.Acad. Sci., 2005, 1048, 445-448. [5]  W. Stricks and I. M. Kolhoff, J. Am. Chem. Soc., 1953, 75, 5673–5681. 336

L-cysteine modulates the ecto-atpase activity inhibition in presence of cadmium (ii) and mercury (ii) ions

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L-cysteine modulates the ecto-atpase activity inhibition in presence of cadmium (ii) and mercury (ii) ions

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