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Effect of hypocrellin A sensitization on the lateral mobility of cell membrane proteins
Journal of Photochemistry
and Photobiology,
B: Biology,
2 (1988)
395 - 398
395
Preliminary Note
Effect of hypocrellin A sensitization on the lateral mobility of cell membrane proteins CHENG LONGSHENG’, Department
WANG JIAZHEN and FU SHIM1
of Radiobiophysics,
(Received October 26,1987;
Keywords. Hypocrellin, mobility.
Institute
of Biophysics,
Academia
Sinica, Beuing (China)
accepted May 5,1988)
photosensitization,
membrane
proteins,
lateral
Hypocrellin A (HA) is a photosensitizer derived from perylenequinone which can be isolated from a kind of parasitic fungi, Hypocrelka bambusue Sacc., growing in southwest China. Hypocrellin A photosensitizes the crosslinking of erythrocyte membrane protein and the photo-oxidation of some amino acid residues and membrane lipids [ 1, 21. Antioxidizing agents such as butylated hydroxytoluene (BHT) inhibit the hypocrellin-A-photosensitized peroxidation of lipids. In the present work, the fluorescence photobleaching recovery technique was used to examine the effect of HA and BHT on the lateral mobility in the membrane of L,, cells. Fluorescein isothiocyanate (FITC) was dissolved in phosphate-buffered saline (PBS, pH 7.4). BHT was added to 200 ~1 of Tween 80, and the mixture diluted with ethanol to 5 ml. The final concentration of BHT was 0.1 M. HA was kindly provided by the Institute of Microbiology, Yunnan, China, and was purified before use. The experiments were carried out with L,, cells, i.e. C&l mouse fibroblast cells cultured in Eagle’s medium supplemented with 10% calf serum and kept in a humidified atmosphere of 5% CO2 and 95% air at 37 “C. The cells were cultured for 24 h as a monolayer attached on a flask wall. The adherent cells were washed three times with PBS, and then HA (final concentration 8 PM) and 2 ml PBS were added. After 30 min incubation at 37 “C, the mixture was irradiated for 5 or 10 s with a tungsten lamp (light fluence 1.2 W mw2). Irradiated attached cells were washed three times with PBS, and then eluted with a jet stream of PBS. After addition of 0.1 ml FITC (1 mg ml-i, pH 7.4) and incubation for 20 min at 20 - 25 “C, the suspensions were centrifuged and washed to remove excess FITC. The labelled cells were cultured in Eagle’s medium for 2 h, and centrifuged to remove the supernatant. *Author to whom correspondence loll-1344/88/$3.50
should be addressed. @ Elsevier Sequoia/Printed
in The Netherlands
396
FITC-labelled cells thus prepared were used for photobleaching recovery measurements. In a set of experiments, BHT (1 PM or 3 PM) was added to cultured L,,, cells and the suspension kept for 3 min at room temperature. Cells containing BHT in the presence of HA were incubated in the dark for 20 min. The procedures for irradiation and FITC labelling were the same as described above, except that cells remained attached to the flask wall. After FITClabelled cells were cultured for 2 h in Eagle’s medium, the cells were eluted from the flask wall, centrifuged and used for fluorescence photobleaching recovery measurements. Three control groups were used: (A) Lgz9 cells; (B) Lgz9 cells with HA and without light; (C) Lsz9 cells with HA and BHT in the absence of light. Since the excitation wavelength of FITC is 485 nm, the photobleaching was performed with an argon laser operated at 488 nm. The power was maintained at 28 mW, and the laser light passed through a neutral density filter whose attenuation was 103. The bleaching time of the laser light was 50 ns and the radius of the light beam was 2 pm. Each cell was bleached at one spot. Each sample consisted of 10 - 20 cells. The diffusion coefficient (D) and fluorescence recovery rate (R) in the cell membranes were calculated from the fluorescence recovery curve according to the method of Axehod et al. [ 31. The diffusion coefficient is given by D=
rW2 4t l/2
where W is the ee2 radius of the beam, r is the coefficient for the unbleached fraction (F(0)) and t,,2 is the half-time for recovery. The fluorescence recovery rate is given by I(o) -I(O) R = I(NO)-I(O) where I(t < 0) is the fluorescence intensity before bleaching, I(0) is the fluorescence intensity immediately after bleaching, and I(o) is the fluorescence intensity at the maximum height of the recovery curve. In the case of Lg2s cells cultured with HA, ring-shaped fluorescence of the cell membrane is observed under a fluorescent microscope, indicating that HA attaches to the cell membrane. Fluorescence photobleaching recovery curves for lateral diffusion in membrane protein are shown in Fig. 1. The lateral diffusion coefficients (D) obtained from the curves are given in Table 1. The value of D for membrane proteins of L,, cells is 10.4 X 10e9 cm2 s-l. When cells are incubated with HA for 20 min, D is 10.4 X 10e9 cm2 s-l. Thus, it appears that the chemical effect of HA on L929 cells is small. When HA-treated L929 cells are illuminated, the value of D decreases, although the process is of limited importance. Thus, D is 4.1 X lo-’ cm2 s-* and 3.7 X 10d9 cm2 s-i for illumination doses of 6 J m-’ and 12 J mP2 respectively. This suggests that HA can induce the photosensitization of L,, cells,
391
0.5 0.0
I-_::
0
2
,
b
8
IO t (sect
a
Fig. 1. Fluorescence photobleaching recovery curves for lateral diffusion in membrane proteins. (a) Lms cells; (b) Lass cells + HA; (c) L 92s cells + HA, illumination dose 6 J rn-?; (d) Lss cells + HA, illumination dose 12 J m- 2. Z(t) and Z(0) are the fluorescence intensity before and after photobleaching. TABLE 1 Effect of HA on lateral diffusion in membrane proteins of La9 cells Condition
D X 10M9 (cm2 s-r)
E (s)
L929 cells Lm9 cells + HA La9 cells + HA, dose 6 J rnd2 L-9 cells + HA, dose 12 J mm2
10.4 10.4 4.1 3.7
36.4 32.0 37.2 36.7
+ f f +
1.6 1.9 0.8 0.6
zk6.6 f 8.2 f 6.7 f 6.4
but no simple correlation between illumination dose and photosensitization is detected. After photobleaching of L9z9 cells, the fluorescence recovery rate is in the range 23% - 43% with an average rate of about 35%. The results were obtained with a covalently attached FITC label. Covalent lables are in principle non-specific but in practice they may react with only one or a few
393
exposed membrane proteins. Thus, these fluctuations could be due to the differences between the structures of different domains. The fluorescence recovery in the bleached region is less than 100%. These observations indicate that, under the influence of intense laser pulses, certain proteins in the cell membranes are immobilized owing to cross-linking with each other. Since BHT inhibits HA-sensitized peroxidation of unsaturated lipids [2], it was interesting to investigate its effect on lateral diffusion. As shown in Table 2, the lateral diffusion coefficient of the controls is 10.2 X 10e9 cm2 s-l, while that of cells with 1 NM BHT is 5.7 X 10e9 cm2 s-l, i.e. greater than the value of D (4.1 X lob9 cm2 s-l) found in the experiment without BHT. A further increase in BHT concentration to 3 PM leads to the even higher D value of 8.5 X 10h9 cm2 s-l. Thus, it is clear that BHT can partially protect the photodamage due to HA photosensitization; in particular, the decrease in the lateral diffusion coefficient in membrane protein is less important. TABLE 2 Effect of BHT on lateral diffusion in membrane proteins of La9 cells Condition
D X 10mg (cm2 s-l)
R 6)
L929 cells Lwg cells + HA Lwg cells + HA + 1 @no1 1-l BHT LNg cells + HA, dose 12 J rnw2 Lag cells + HA + 1 pmoll-l BHT, dose 12 J me2 Lng cells + HA + 3 /.nnol 1-l BHT, dose 12 J mm2
10.6 10.1 10.2 4.1 5.7
25.3 31.4 17.3 25.7 30.9
+ + f * f
1.4 1.9 1.1 1.0 0.9
8.5 + 1.7
?: 8.7 rt 11.8 + 3.4 + 8.9 f 10.4
20.7 f 6.6
Acknowledgments This work was supported by the Science Foundation of the Chinese Academy of Sciences. The authors are grateful to Professor Lijin Jiang for her careful reading of the manuscript.
References 1 L. S. Cheng and J. Z. Wang, Photodamage in human erythrocyte membranes, induced by a new photosensitizer-hypocrellin A, Acta Biol. Exp. Sinica, 18 (1985) 89 - 97. 2 L. S. Cheng and X. Liu, Effect of lipid peroxidation on hypocrellin A photosensitization cross-linking of membrane protein, Acta Biol., Exp. Sin&, 20 (1987) 373 - 380. 3 D. Axelrod, D. E. Koppel, J. Schlessinger, E. L. Elson and W. W. Webb, Mobility measurement by analysis of fluorescence photobleaching recovery kinetics, Biophys. J., 16 (1976) 1055 - 1069.
and Photobiology,
B: Biology,
2 (1988)
395 - 398
395
Preliminary Note
Effect of hypocrellin A sensitization on the lateral mobility of cell membrane proteins CHENG LONGSHENG’, Department
WANG JIAZHEN and FU SHIM1
of Radiobiophysics,
(Received October 26,1987;
Keywords. Hypocrellin, mobility.
Institute
of Biophysics,
Academia
Sinica, Beuing (China)
accepted May 5,1988)
photosensitization,
membrane
proteins,
lateral
Hypocrellin A (HA) is a photosensitizer derived from perylenequinone which can be isolated from a kind of parasitic fungi, Hypocrelka bambusue Sacc., growing in southwest China. Hypocrellin A photosensitizes the crosslinking of erythrocyte membrane protein and the photo-oxidation of some amino acid residues and membrane lipids [ 1, 21. Antioxidizing agents such as butylated hydroxytoluene (BHT) inhibit the hypocrellin-A-photosensitized peroxidation of lipids. In the present work, the fluorescence photobleaching recovery technique was used to examine the effect of HA and BHT on the lateral mobility in the membrane of L,, cells. Fluorescein isothiocyanate (FITC) was dissolved in phosphate-buffered saline (PBS, pH 7.4). BHT was added to 200 ~1 of Tween 80, and the mixture diluted with ethanol to 5 ml. The final concentration of BHT was 0.1 M. HA was kindly provided by the Institute of Microbiology, Yunnan, China, and was purified before use. The experiments were carried out with L,, cells, i.e. C&l mouse fibroblast cells cultured in Eagle’s medium supplemented with 10% calf serum and kept in a humidified atmosphere of 5% CO2 and 95% air at 37 “C. The cells were cultured for 24 h as a monolayer attached on a flask wall. The adherent cells were washed three times with PBS, and then HA (final concentration 8 PM) and 2 ml PBS were added. After 30 min incubation at 37 “C, the mixture was irradiated for 5 or 10 s with a tungsten lamp (light fluence 1.2 W mw2). Irradiated attached cells were washed three times with PBS, and then eluted with a jet stream of PBS. After addition of 0.1 ml FITC (1 mg ml-i, pH 7.4) and incubation for 20 min at 20 - 25 “C, the suspensions were centrifuged and washed to remove excess FITC. The labelled cells were cultured in Eagle’s medium for 2 h, and centrifuged to remove the supernatant. *Author to whom correspondence loll-1344/88/$3.50
should be addressed. @ Elsevier Sequoia/Printed
in The Netherlands
396
FITC-labelled cells thus prepared were used for photobleaching recovery measurements. In a set of experiments, BHT (1 PM or 3 PM) was added to cultured L,,, cells and the suspension kept for 3 min at room temperature. Cells containing BHT in the presence of HA were incubated in the dark for 20 min. The procedures for irradiation and FITC labelling were the same as described above, except that cells remained attached to the flask wall. After FITClabelled cells were cultured for 2 h in Eagle’s medium, the cells were eluted from the flask wall, centrifuged and used for fluorescence photobleaching recovery measurements. Three control groups were used: (A) Lgz9 cells; (B) Lgz9 cells with HA and without light; (C) Lsz9 cells with HA and BHT in the absence of light. Since the excitation wavelength of FITC is 485 nm, the photobleaching was performed with an argon laser operated at 488 nm. The power was maintained at 28 mW, and the laser light passed through a neutral density filter whose attenuation was 103. The bleaching time of the laser light was 50 ns and the radius of the light beam was 2 pm. Each cell was bleached at one spot. Each sample consisted of 10 - 20 cells. The diffusion coefficient (D) and fluorescence recovery rate (R) in the cell membranes were calculated from the fluorescence recovery curve according to the method of Axehod et al. [ 31. The diffusion coefficient is given by D=
rW2 4t l/2
where W is the ee2 radius of the beam, r is the coefficient for the unbleached fraction (F(0)) and t,,2 is the half-time for recovery. The fluorescence recovery rate is given by I(o) -I(O) R = I(NO)-I(O) where I(t < 0) is the fluorescence intensity before bleaching, I(0) is the fluorescence intensity immediately after bleaching, and I(o) is the fluorescence intensity at the maximum height of the recovery curve. In the case of Lg2s cells cultured with HA, ring-shaped fluorescence of the cell membrane is observed under a fluorescent microscope, indicating that HA attaches to the cell membrane. Fluorescence photobleaching recovery curves for lateral diffusion in membrane protein are shown in Fig. 1. The lateral diffusion coefficients (D) obtained from the curves are given in Table 1. The value of D for membrane proteins of L,, cells is 10.4 X 10e9 cm2 s-l. When cells are incubated with HA for 20 min, D is 10.4 X 10e9 cm2 s-l. Thus, it appears that the chemical effect of HA on L929 cells is small. When HA-treated L929 cells are illuminated, the value of D decreases, although the process is of limited importance. Thus, D is 4.1 X lo-’ cm2 s-* and 3.7 X 10d9 cm2 s-i for illumination doses of 6 J m-’ and 12 J mP2 respectively. This suggests that HA can induce the photosensitization of L,, cells,
391
0.5 0.0
I-_::
0
2
,
b
8
IO t (sect
a
Fig. 1. Fluorescence photobleaching recovery curves for lateral diffusion in membrane proteins. (a) Lms cells; (b) Lass cells + HA; (c) L 92s cells + HA, illumination dose 6 J rn-?; (d) Lss cells + HA, illumination dose 12 J m- 2. Z(t) and Z(0) are the fluorescence intensity before and after photobleaching. TABLE 1 Effect of HA on lateral diffusion in membrane proteins of La9 cells Condition
D X 10M9 (cm2 s-r)
E (s)
L929 cells Lm9 cells + HA La9 cells + HA, dose 6 J rnd2 L-9 cells + HA, dose 12 J mm2
10.4 10.4 4.1 3.7
36.4 32.0 37.2 36.7
+ f f +
1.6 1.9 0.8 0.6
zk6.6 f 8.2 f 6.7 f 6.4
but no simple correlation between illumination dose and photosensitization is detected. After photobleaching of L9z9 cells, the fluorescence recovery rate is in the range 23% - 43% with an average rate of about 35%. The results were obtained with a covalently attached FITC label. Covalent lables are in principle non-specific but in practice they may react with only one or a few
393
exposed membrane proteins. Thus, these fluctuations could be due to the differences between the structures of different domains. The fluorescence recovery in the bleached region is less than 100%. These observations indicate that, under the influence of intense laser pulses, certain proteins in the cell membranes are immobilized owing to cross-linking with each other. Since BHT inhibits HA-sensitized peroxidation of unsaturated lipids [2], it was interesting to investigate its effect on lateral diffusion. As shown in Table 2, the lateral diffusion coefficient of the controls is 10.2 X 10e9 cm2 s-l, while that of cells with 1 NM BHT is 5.7 X 10e9 cm2 s-l, i.e. greater than the value of D (4.1 X lob9 cm2 s-l) found in the experiment without BHT. A further increase in BHT concentration to 3 PM leads to the even higher D value of 8.5 X 10h9 cm2 s-l. Thus, it is clear that BHT can partially protect the photodamage due to HA photosensitization; in particular, the decrease in the lateral diffusion coefficient in membrane protein is less important. TABLE 2 Effect of BHT on lateral diffusion in membrane proteins of La9 cells Condition
D X 10mg (cm2 s-l)
R 6)
L929 cells Lwg cells + HA Lwg cells + HA + 1 @no1 1-l BHT LNg cells + HA, dose 12 J rnw2 Lag cells + HA + 1 pmoll-l BHT, dose 12 J me2 Lng cells + HA + 3 /.nnol 1-l BHT, dose 12 J mm2
10.6 10.1 10.2 4.1 5.7
25.3 31.4 17.3 25.7 30.9
+ + f * f
1.4 1.9 1.1 1.0 0.9
8.5 + 1.7
?: 8.7 rt 11.8 + 3.4 + 8.9 f 10.4
20.7 f 6.6
Acknowledgments This work was supported by the Science Foundation of the Chinese Academy of Sciences. The authors are grateful to Professor Lijin Jiang for her careful reading of the manuscript.
References 1 L. S. Cheng and J. Z. Wang, Photodamage in human erythrocyte membranes, induced by a new photosensitizer-hypocrellin A, Acta Biol. Exp. Sinica, 18 (1985) 89 - 97. 2 L. S. Cheng and X. Liu, Effect of lipid peroxidation on hypocrellin A photosensitization cross-linking of membrane protein, Acta Biol., Exp. Sin&, 20 (1987) 373 - 380. 3 D. Axelrod, D. E. Koppel, J. Schlessinger, E. L. Elson and W. W. Webb, Mobility measurement by analysis of fluorescence photobleaching recovery kinetics, Biophys. J., 16 (1976) 1055 - 1069.