INT . J . RADIAT . BIOL .,
1979,
VOL .
36,
NO .
4, 3 2 5 -334
A spin-label study of the effect of gamma radiation on erythrocyte membrane. Influence of lipid peroxidation on membrane structure-
Int J Radiat Biol Downloaded from informahealthcare.com by Cornell University on 05/31/12 For personal use only.
E . GRZELINSKA, G . BARTOSZ, K . GWO7,DZINSKI and W . LEYKO Department of Biophysics, Institute of Biochemistry and Biophysics, University of Lodz, 90-237 Lodz, Poland (Received 28 July 1978 ; accepted 27 December 1978)
Gamma-irradiation of bovine erythrocyte membranes (0 . 1-4Mrad) resulted in a decrease in the degree of order of membrane lipids, as measured by spin-labelled fatty acid esters, at the depth of C 12 but not at the depth of C 5 . Dose dependence of this phenomenon corresponded to dose dependence of malondialdehyde formation in the membranes . On this basis a mechanism for the effect of lipid peroxidation on the membrance structure is proposed . Membrane proteins underwent radiation-induced conformational transitions revealed bv_ maleimide spin label which could be also connected with lipid peroxidation . 1.
Introduction Previous studies on the effect of ionizing radiation on the erythrocyte membrane have mainly dealt with functional changes of the membrane such as increase in permeability for sodium and potassium (Bianchi, Boccacci, Misiti-Dorello and Quintiliani 1964, Bresciani, Auricchio and Fiore 1964 a, Kankura, Nakamura, Eto and Nakao 1969), increase in osmotic fragility (Simmons and Naftalin 1976) and autohaemolysis (Kollmann, Shapiro and Martin 1969), disturbances in lipid metabolism (Hansen, Karle and Stender 1978) or inactivation of red cell membrane enzymes, mainly ATPases (Bresciani, Auricchio and Fiore 1964b) . Structural changes observed after irradiation have been mainly confined to such chemical events as decrease in the membrane -SH groups (Sutherland and Pihl 1968) or lipid peroxidation (Myers and Bide 1966) . However, changes in the physical parameters of the red cell membrane can be even more directly related to functional alterations and could be more promising for revealing structural-functional relationships by means of radiobiological experiments . For instance, fluidity of the lipid phase of the membrane is critical in determining the rate of permeation of the membrane by permeants moving in the lipid phase (Deuticke and Ruska 1976) ; radiation inactivation of enzymes is thought to be due directly to conformational changes of enzyme molecules rather than to chemical transformation of groups not involved in catalysis (Okada 1970) . In this study, we wanted to investigate the effect of ionizing radiation on the physical state of the erythrocyte membrane, using the versatile technique of spinlabelling which has the advantage of enabling independent probing of the lipid and the protein parts of the membrance . In particular,' we aimed at determining the influence of radiation-induced lipid peroxidation on the state of erythrocyte membrane lipids . t This paper is partly based on work performed under the contract
No . R .III .13 .2 .4 .
326
E . Grzelinska et al.
Int J Radiat Biol Downloaded from informahealthcare.com by Cornell University on 05/31/12 For personal use only.
2.
Material and methods Bovine blood was taken into citrate in an abattoir . Erythrocyte membranes were isolated according to Dodge, Mitchell and Hanahan (1963) . Membrane preparations in 10 mM sodium phosphate buffer, pH 7.4 were irradiated from a 60 Co gamma source at room temperature, the dose-rate amounting to 0. 8 or 1 .OMrad h-1 for experiments in the higher dose range and to 0 . 1 Mrad h -1 for experiments in the dose range of 0.1-0 •8 Mrad . The protein concentration in membrane preparations was adjusted to 5 mg cm - 3 as estimated by the method of Lowry, Rosenbrough, Farr and Randall (1951) in the modification of Lees and Paxman (1972) . The maleimide spin label (4-maleimido-2, 2, 6, 6-tetramethyl piperidino-loxyl, MSL) was synthesized according to the method of Misharin and Polanovskij (1974) . The lipid spin probes, methyl 12-doxylstearate (M12NS) and methyl 5doxylpalmitate (M5NP) were synthesized after Waggoner, Kingzett, Rottschaefer, Griffith and Keith (1969) and after Hubbell and McConnell (1971), respectively, in the latter case the synthesis being completed at the ester step . For spin labelling, ethanol solutions of appropriate labels were added to membrance suspensions so that final label concentrations amounted to 5 x 10 -4 M and final ethanol concentrations were lower than or equal to 0 . 5 per cent (v/v) . In the case of lipid spin labels, the membranes were shaken for 30min at room temperature . In the case of MSL, the membranes were incubated with the label for 12-14 hours at a temperature of 4 °C and the unbound label was removed by 3-4 washings of the membranes with phosphate buffer prior to measurement . The measurements were performed at room temperature (21-22°C) in a SE/X-28 e .s .r . spectrometer (Wroclaw Technical University, Poland), operating at the X-band . Radiation-induced lipid peroxidation was estimated by measurement of malondialdehyde formation in membrane preparations following the procedure of Brownlee, Huttner, Panganamala and Cornwell (1977) . All the experiments were repeated at least 4-7 times on different membrane preparations . Results are presented as mean ±S .D . Calculations of statistical significance of differences were made using the two-tailed Student's t-test . For clarity of comparisons, the radiation-induced changes in e .s .r . spectral parameters are expressed as a percentage of appropriate control values taken as 100 per cent . 3. Results 3 .1 . Protein spin label The maleimide spin label binds covalently mainly to sulphydryl groups of erythrocyte membrane proteins . The population of membrane -SH groups is heterogeneous and, as far as the interaction with MSL is concerned, is composed of two subpopulations, causing weak and strong immobilization respectively, of the bound spin label residues . In the low-field part of e .s .r . spectra of this label bound to the erythrocyte membrane (figure 1), peaks W and S can be ascribed to weakly and strongly immobilized spin label residues, respectively (Butterfield 1977) . Conformational changes of the spin label-binding proteins may result in alterations of the microenvironment of the bound label residues, thus changing the ratio of the two subpopulations of -SH groups and its rough estimate, the ratio of the heights of peaks W and S (ham,+,/hs+l) (Butterfield 1977, Kirkpatrick and Sandberg 1973) . We observed a monotonous increase in the hr,,+l/hs+l ratio with increasing doses of gamma radiation (figure 2) . From the spectra obtained we also calculated the
Effect of radiation on erythrocyte membrane
327 0
O-N -
NJ
M-SL
0
H
Int J Radiat Biol Downloaded from informahealthcare.com by Cornell University on 05/31/12 For personal use only.
hs,f l hw1
A HN*
f
Figure 1 . E .s .r . spectrum of MSL bound to control bovine erythrocyte membranes .
Figure 2 . Effect of radiation on the hW+i/hs+i ratio of MSL bound to bovine erythrocyte membranes . Absolute control values : 3 . 97±0 . 25 . For all differences with respect to the control value, P
1979,
VOL .
36,
NO .
4, 3 2 5 -334
A spin-label study of the effect of gamma radiation on erythrocyte membrane. Influence of lipid peroxidation on membrane structure-
Int J Radiat Biol Downloaded from informahealthcare.com by Cornell University on 05/31/12 For personal use only.
E . GRZELINSKA, G . BARTOSZ, K . GWO7,DZINSKI and W . LEYKO Department of Biophysics, Institute of Biochemistry and Biophysics, University of Lodz, 90-237 Lodz, Poland (Received 28 July 1978 ; accepted 27 December 1978)
Gamma-irradiation of bovine erythrocyte membranes (0 . 1-4Mrad) resulted in a decrease in the degree of order of membrane lipids, as measured by spin-labelled fatty acid esters, at the depth of C 12 but not at the depth of C 5 . Dose dependence of this phenomenon corresponded to dose dependence of malondialdehyde formation in the membranes . On this basis a mechanism for the effect of lipid peroxidation on the membrance structure is proposed . Membrane proteins underwent radiation-induced conformational transitions revealed bv_ maleimide spin label which could be also connected with lipid peroxidation . 1.
Introduction Previous studies on the effect of ionizing radiation on the erythrocyte membrane have mainly dealt with functional changes of the membrane such as increase in permeability for sodium and potassium (Bianchi, Boccacci, Misiti-Dorello and Quintiliani 1964, Bresciani, Auricchio and Fiore 1964 a, Kankura, Nakamura, Eto and Nakao 1969), increase in osmotic fragility (Simmons and Naftalin 1976) and autohaemolysis (Kollmann, Shapiro and Martin 1969), disturbances in lipid metabolism (Hansen, Karle and Stender 1978) or inactivation of red cell membrane enzymes, mainly ATPases (Bresciani, Auricchio and Fiore 1964b) . Structural changes observed after irradiation have been mainly confined to such chemical events as decrease in the membrane -SH groups (Sutherland and Pihl 1968) or lipid peroxidation (Myers and Bide 1966) . However, changes in the physical parameters of the red cell membrane can be even more directly related to functional alterations and could be more promising for revealing structural-functional relationships by means of radiobiological experiments . For instance, fluidity of the lipid phase of the membrane is critical in determining the rate of permeation of the membrane by permeants moving in the lipid phase (Deuticke and Ruska 1976) ; radiation inactivation of enzymes is thought to be due directly to conformational changes of enzyme molecules rather than to chemical transformation of groups not involved in catalysis (Okada 1970) . In this study, we wanted to investigate the effect of ionizing radiation on the physical state of the erythrocyte membrane, using the versatile technique of spinlabelling which has the advantage of enabling independent probing of the lipid and the protein parts of the membrance . In particular,' we aimed at determining the influence of radiation-induced lipid peroxidation on the state of erythrocyte membrane lipids . t This paper is partly based on work performed under the contract
No . R .III .13 .2 .4 .
326
E . Grzelinska et al.
Int J Radiat Biol Downloaded from informahealthcare.com by Cornell University on 05/31/12 For personal use only.
2.
Material and methods Bovine blood was taken into citrate in an abattoir . Erythrocyte membranes were isolated according to Dodge, Mitchell and Hanahan (1963) . Membrane preparations in 10 mM sodium phosphate buffer, pH 7.4 were irradiated from a 60 Co gamma source at room temperature, the dose-rate amounting to 0. 8 or 1 .OMrad h-1 for experiments in the higher dose range and to 0 . 1 Mrad h -1 for experiments in the dose range of 0.1-0 •8 Mrad . The protein concentration in membrane preparations was adjusted to 5 mg cm - 3 as estimated by the method of Lowry, Rosenbrough, Farr and Randall (1951) in the modification of Lees and Paxman (1972) . The maleimide spin label (4-maleimido-2, 2, 6, 6-tetramethyl piperidino-loxyl, MSL) was synthesized according to the method of Misharin and Polanovskij (1974) . The lipid spin probes, methyl 12-doxylstearate (M12NS) and methyl 5doxylpalmitate (M5NP) were synthesized after Waggoner, Kingzett, Rottschaefer, Griffith and Keith (1969) and after Hubbell and McConnell (1971), respectively, in the latter case the synthesis being completed at the ester step . For spin labelling, ethanol solutions of appropriate labels were added to membrance suspensions so that final label concentrations amounted to 5 x 10 -4 M and final ethanol concentrations were lower than or equal to 0 . 5 per cent (v/v) . In the case of lipid spin labels, the membranes were shaken for 30min at room temperature . In the case of MSL, the membranes were incubated with the label for 12-14 hours at a temperature of 4 °C and the unbound label was removed by 3-4 washings of the membranes with phosphate buffer prior to measurement . The measurements were performed at room temperature (21-22°C) in a SE/X-28 e .s .r . spectrometer (Wroclaw Technical University, Poland), operating at the X-band . Radiation-induced lipid peroxidation was estimated by measurement of malondialdehyde formation in membrane preparations following the procedure of Brownlee, Huttner, Panganamala and Cornwell (1977) . All the experiments were repeated at least 4-7 times on different membrane preparations . Results are presented as mean ±S .D . Calculations of statistical significance of differences were made using the two-tailed Student's t-test . For clarity of comparisons, the radiation-induced changes in e .s .r . spectral parameters are expressed as a percentage of appropriate control values taken as 100 per cent . 3. Results 3 .1 . Protein spin label The maleimide spin label binds covalently mainly to sulphydryl groups of erythrocyte membrane proteins . The population of membrane -SH groups is heterogeneous and, as far as the interaction with MSL is concerned, is composed of two subpopulations, causing weak and strong immobilization respectively, of the bound spin label residues . In the low-field part of e .s .r . spectra of this label bound to the erythrocyte membrane (figure 1), peaks W and S can be ascribed to weakly and strongly immobilized spin label residues, respectively (Butterfield 1977) . Conformational changes of the spin label-binding proteins may result in alterations of the microenvironment of the bound label residues, thus changing the ratio of the two subpopulations of -SH groups and its rough estimate, the ratio of the heights of peaks W and S (ham,+,/hs+l) (Butterfield 1977, Kirkpatrick and Sandberg 1973) . We observed a monotonous increase in the hr,,+l/hs+l ratio with increasing doses of gamma radiation (figure 2) . From the spectra obtained we also calculated the
Effect of radiation on erythrocyte membrane
327 0
O-N -
NJ
M-SL
0
H
Int J Radiat Biol Downloaded from informahealthcare.com by Cornell University on 05/31/12 For personal use only.
hs,f l hw1
A HN*
f
Figure 1 . E .s .r . spectrum of MSL bound to control bovine erythrocyte membranes .
Figure 2 . Effect of radiation on the hW+i/hs+i ratio of MSL bound to bovine erythrocyte membranes . Absolute control values : 3 . 97±0 . 25 . For all differences with respect to the control value, P
