BIOCHEMISTRY andmolecular BIOLOGY INTERNATIONAL pages 227-232 EFFECTS OF SULFHYDRYL COMPOUNDS ON THE INHIBITION OF ERYTHROCYTE MEMBRANE Na+-K + ATPase BY OZONE Rmnazan Bilgin, Sermin Gill, S. Seyhan Ttikel The University of ~ukurova, Art & Science Faculty, Department of Chemistry, 01330 BaIcali, Adana-TURKEY Received June 22, 1998 Accepted September 29, 1998 SUMMARY Exposure of human erythrocyte membranes to ozone (5 gmol/10 min) resulted in the inhibition of erythrocyte membrane Na+-K + ATPase (EC.3.6.1.39). It was determined that, the degree of enzyme inhibition in the directly ozone exposed membranes was greater than that of membranes obtained from ozone exposed intact erythrocytes. In the presence of varying concentrations (0-1.0 ram) of dithiotrethiol or mercaptoethanol Na+-K + ATPase activities of both types of ozone exposed membranes were increased almost proportionally with the concentration of dithiotrethiol or mercaptoethanol however, the activities were still lower than the normal Na+-K + ATPase value. The results indicate that, dithiotrethiol or mercaptoethanol prevent the enzyme inhibition by ozone in vitro. This suggests that the membrane thiol groups are primary targets for ozone and thereby preventing the oxidation of essential functional groups of enzyme,protein. Key words: Ozone, erythrocyte, sulfhydryl compounds, Na+-K + ATPase INTRODUCTION Because ozone is a very reactive agent, every biomolecule may be a potential target for reaction with ozone. There are many in vitro and in vivo studies on the oxidative damage of ozone to biological systems (1, 2, 3, 4). In studies, in vitro erythrocytes have been used as a well-characterized model cell. It has been shown that the membrane constituents, lipids, enzymes and other essential proteins were susceptible to oxidation by ozone. The relationship between polyunsaturated fatty acid peroxidation and protein oxidation is further complicated by the fact that the maintenence of the tertiary structures of cellular membrane proteins is dependent on their associated lipids. Alterations in lipids surrounding the embedded proteins may result in structural alterations and changes in membrane function (5). Earlier reports (6,7) showed that exposure of erythrocytes or their membranes to low lewels of ozone 227 Copyright 9 1999 1UBMB 1039-9712/99 $12.00 + 0.00
BIOCHEMISTRY andmolecular BIOLOGY INTERNATIONAL lead to a loss of the Na+-K + ATPase activity as a result of a) destruction of phospholipids, b) cross linking of the membrane proteins by lipid peroxidation products, c) oxidation of essential sulphydryl groups and d) destruction of other essential components of enzyme complex. Several plasma ezymes such as lactate dehydrogenase, alkaline phosphatase, gamma glutamyl transferase and aspartate transaminase were studied in ozone exposed blood samples. The results suggested that exposure to ozone may cause oxidative damage to proteins in human body fluids (8). This study was aimed at determining the effects of dithiothreitol and mercaptoethanol on the inhibition of erythrocyte membrane Na+-K + ATPase by ozone. MATERIALS AND METHODS Materials: All reagents were of highest purity available and were obtained from BDH (England), Merck (Germany) or Sigma Chemical Company (U.S.A.). Blood samples were obtained from the BloOd Bank of ~ukurova University Medical Faculty. Preparation of ozone exposed erythrocytes and preparation of membranes from ozone exposed erythrocytes: Erythrocytes were washed four times with 10 mm potassium phosphate buffer, (ph 7.4) containing 135 mm NaC1. The cells were suspended to 10% by volume in the same buffer solution. A series of 50 ml cell suspension having the final dithiothreitol or mercaptoethanol concentrations of 0.0, 0.1, 0.3, 0.5, 0.7, 1.0 mm were prepared. Each of the samples were then immediately exposed to a flow of 5 gmolo3/min. The ozone exposed samples were centrifuged at 5000 g for 5 rain to sediment the elythrocytes which were rewashed four times with 0.9 % NaC1 solution. Then, membranes of these ozone exposed eltthrocytes were prepared according to the procedure described by (9) with some modifications. Briefly, ozone exposed cells were hemolyzed in an ice bath by adding them into 5 volumes of 10 mm Tris buffer (ph 7.4) containing 1 mm EDTA and centrifuged at 15000 g for 10 min. The membranes were washed with the same buffer until the supernatant fluid was colorless. The membrane fragments were finally suspended in the same buffer at a concentration of 3.0 mg of protein/ml in a series of tubes and were stored at -20~ until used. Preparation of membranes from intact erythrocytes and exposure of these membranes to ozone: Erythrocytes were washed four times with 10 mm potassium phosphate buffer, (ph 7.4) containing 135 mm NaCt. The cells were suspended to 10% by volume in the same buffer solution. Membranes of washed intact erythrocytes were prepared according to the procedure described above. An aliquot of this membrane suspension was used to detmrnine the Na+-K ATPase activity which was designated the control value (0.420 + 0.020 gmol Pi/mg prot./h). 10 ml portions of this membrane suspension were immediately exposed to ozone again in the presence of (0.0, 0.1, 0.3, 0.5, 0.7, 1.0) mm dithiothreitol or the same concentration range of mercaptoethanol. Ozone generation: Ozone was generated by a Fisher-Ozone Generator Model 50I at an ozone flow rate 5 gmol O3/min. Ozone concentration was measured by titration with KI- Na2S203 - starch in 5 mm sodium phosphate (ph 7.0). 228
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Measurement of Na + ATPase activity: Na+-K + ATPase was assayed as the release of inorganic phosphate (Pi) from ATP according to the method described by (10). Assays were carried out in a final volume of 2.5 ml containing 0.3 mg membrane protein as the enzyme sotu-ce, 120 mm NaC1, 10 mm KCI, 35 mm Tris HC1 buffer (ph 7.4), 3 mm NazATP and the presence or absence of 0.2 mm ouabain. Membranes were preincubated in the mixture for 10 min at 37~ before starting the reaction by adding the substrate, Na2ATP. After 30 rain incubation with substrate reaction was stopped by adding 0.5 ml of 30 % trichloroacetic acid. The released Pi was assayed by the method of (11) and protein was measured by the method of (12). Na+-K + ATPase activity was calculated by subtracting the activity in the presence of ouabain from the activity in its absence. Enzyme activity is expressed as gmol Pi/mg prot./h. RESULTS AND DISCUSSION The Na+-K + ATPase activity of membranes prepared from normal erythrocytes was 0.420 + 0.020 gmol Pi/mg prot./h(n:10). This value was considered as the control value. The Na+-K + ATPase activities of the membranes prepared from the erythrocytes exposed to ozone in the presence of varying concentrations (0-1.0 mm) of dithiothretiol or mercaptoethanol and the membranes prepared from the erythrocytes and then exposed to ozone in the presence of varying concentrations (0-1.0 ram) of dithiothretiol or mercaptoethanol are presented in Figures 1, 2. ~" 0.30 e~ "-- 0.25.~ 0.20 "7..< [,,,,, < o.15 0.10 0.0 i i i i 0.2 0.4 0.6 0.8 1.0 Concentration (ram) 1.2 Fig 1. Na+-K + ATPase activities of membranes prepared from erythrocytes, which had prior exposure to ozone for 10 rain, in the presence of varying concentrations of mercaptoethanol and dithiothreitol. --13-- mercaptoethanol --A-- dithiothreitol 229
BIOCHEMISTRY andmolecular BIOLOGY INTERNATIONAL Ozone exposure time was chosen as 10 min, because a previous study (13) showed that the Na+-K + ATPase activity was inhibited by approximately 70 % in membrane preparation from ozone exposed erythrocytes or membranes exposed to ozone for 10 rain. Figures 1, 2 Show that the presence of dithiothretiol or mercaptoethanol prevents the ozone inhibition of Na+-K + ATPase almost in the same range. The Na+-K + ATPase activities of the membranes prepared from the erythrocytes exposed to ozone in the presence of 1.0 mm dithiothreitol (0.27 + 0.012 gmol Pi/mg prot./h ) was found to be 2.25 times greater than that of the Na+-K + ATPase activity of the membranes prepared from the erythrocytes exposed to ozone in the absence of dithiothreitol (0.120 _+ 0.006 lamol Pi/mg prot./h ). Similar results were obtained in the presence of mercaptoethanol. Na+-K + ATPase activity of the membranes prepared from the erythrocytes exposed to ozone in the presence of 1.0 mm mercaptoethanol (0.25 + 0.008 gmol Pi/mg prot./h) was 2.1 times greater than that of (0.12 + 0.006 gmol Pi/mg prot./h) the corresponding membrane preparation in the absence of mercaptoethanol. '~. 0.30 0.25 ;;,.. 0.20 '~. 'i 0.15 & 0.10 I., 9 ; 9 ~ 9,, ; Z 0.0 0.2 0.4 0.6 0.8 1.0 Concentration (mm) 1.2 Fig 2, Na+-K + ATPase activities of isolated erythrocyte membranes which were then exposed to ozone for t0 rain in the presence of varying concentrations of mercaptoethanol and dithioth~eitol. --D-- --A-- mercaptoethanol dithiothreitol 230
BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL The Na+-K + ATPase activities of the membranes prepared from the intact erythrocytes and then exposed to ozone in the presence of 1.0 mm dithiothreitol (0.27 _+ 0.012 lamol Pi/mg prot./h) or 1 mm mercaptoethanol (0.26 + 0.010 gmol Pi/mg prot./h) were 2.1 or 2.0 times greater than that of the Na+-K + ATPase activity of the membranes treated with ozone in the absence of these reducing agents. In earlier studies, it was reported that exposure of erythrocytes in vitro, or their membranes to ozone lead to a loss of the Na+-K + ATPase activity of the erythrocyte membranes (3, 13) and it is possible that ozone oxidized plazma proteins directly by attacking histidine and tyrosine residues in proteins (14, 15). Oxidative damage to proteins as well as to lipids should be a consideration in attempting to understand the mechanism of ozone toxicity and in the design of suitable protective agents. The results suggest that, in vitro, dithiothreitol and mercaptoethanol prevent the inhibition of Na+-K + ATPase by ozone. This may be due to the fact that their thiol groups are preferentially oxidized by ozone thereby limiting the oxidation of some essential functional groups of the enzyme protein. The actions of dithiothreitol, and mercaptoethanol were similar in their opposition to the inhibitory effects of ozone, although the protective effect of mercaptoethanol was marginally greater than that of dithiothreitol in all cases. Acknowledgement This work supported by Research Grant (DTP-TBAG/49) fi'om Turkish Reseaxch and Scientific Council. REFERENCES 1. Moore, R. B., Brummitt, L. M. and Mankad, V. N. (1984) Arch. Biochem. Biophys. 273 (2), 527-534. 2. Van, D. Z., Christanse, J. T., Dubbelman, K. and Steveninok, V. J. (t987) Biochim. Biophys. Acta. 924, 111-118. 3. Kesner, L., Kindya, R. J. and Chan, P. C. (1979) J. Biol. Chem. 254 (9), 2705-2709. 4. Goldstein, B. D. (1979) Oxygen Free Radicals Tissue Damage, pp.295-319, Elsevier, NewYork. 5. Borek, C. and Mehlman, M. A. (1983) In The Biomedical Effects of Ozone and Related Photochemical Oxidants (Lee S. D., Mustafa, M. G. and Mehlman. M. A., Eds.), pp. 325-361, Princeton Scientific Publishers, Princeton. N. J. 6. Kindya, R. J and Chan, P. C (1976) Biochim. Biophys. Acta. 429, 608-615. 8. Cross, E. C., Reznick, A. Z., Packer, L., Davis, A. P., Suziki, J. Y. and Halliwell, B. (1992) Free Radical Res. Com. 15, 347-352. 9. Hanahan, U. J. and Eckholm, J. E. (1974) Method Enzymol. 31,168-172. 10. Luly, P., Baldini, P., Incerpi, S. and Tria, E. (1981) Experienta 37,431-433. 11. Atkinson, A., Gatenby, A. D. and Lowe, A. G. (1973) Biocbem. Biophys. Acta. 320, 195-204 231
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