Volume 96, number 2 FEBS LETTERS ADP, ATP TRANSLOCATOR MITOCHONDRIA PROTEIN EXHIBITS December OF RAT HEART, IMMUNOLOGICAL 1978 zyxwvutsr LIVER AND HEPATOMA CROSS-REACTIVITY Jordan KOLAROV, Stefan KUZELA, Vladimir KREMPASKY, Jan LAKOTA and Wham UJHAZY zyxwvutsrqpon Cancer Research Institute, Slovak Academy of Sciences, ul. &. armridy 21, 880 32 Bratislava, Czechoslovakia Received 1. Introduction Differences in the content and kinetic properties of the ADP, ATP translocator in mitochondria from various sources have been demonstrated (reviewed [ 1,2] ). Recently the translocator protein was isolated from several organs [3-51 and data were reported suggesting differences between immunological properties of this protein isolated from heart, liver and kidney mitochondria [5]. In this study a comparison of immunological properties of translocator protein of rat heart, liver and hepatoma by a radioimmunological technique revealed that the same antigenic determinant(s) related to ADP, ATP translocator protein are present in the mitochondria of all the above tissues. In addition, the data obtained suggest that antigenic properties of translocator protein isolated from different sources are unequally modified during the isolation. 2. Materials and methods The maintenance and propagation of Zajdela hepatoma and the isolation of mitochondria from the tumor and rat liver were done as detailed in [6] . Rat heart mitochondria were isolated by a procedure similar to that used for the preparation of liver mitochondria with the exception that the isolation medium was supplemented with 10 mM KCl. Mitochondria from respective sources were loaded with carboxyatractylate (CAT) and then extracted with Triton X-100 to solubilize ADP, ATP translocator protein as CAT-protein complex [7]. The extracts ElsevierjNorth- Holland Biomedical Press 10 October 1978 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM obtained after centrifugation of the Triton X-100 treated mitochondria at 140 000 X g for 40 min were used in the radioimmunoassay. CAT-proteins were purified from these supernatant fractions by hydroxylapatite chromatography and gel filtration on Sepharose 6B as in [3,.5] . The binding of 35S-labeled CAT to mitochondria was measured according to [8]. SDS-polyacrylamide gel electrophoresis was performed as in [9] in the slab gels with 12-20% linear acrylamide gradient. The distribution of radioactivity in the gels was detected by autoradiography of dried [lo] gel slabs. Rabbit antisera against rat heart or rat liver CATprotein were obtained using the immunization scheme in [5]. Immunodiffusion was done by the double diffusion agar plate technique of Ouchterlony in 0.9% agar plates. Second antibody (swine antirabbit IgG) was kindly supplied by Dr G. Russ. CAT-protein isolated from rat heart mitochondria was radioiodinated to a spec. radioact. 2 X lo3 cpm/ng protein using N-succinimidyl-3/Qhydroxy phenyl/ propionate (11). In competitive inhibition radioimmunoassay a limited amount of antiserum against rat heart CAT-protein (final dilution 2 X 106), a constant amount of radioiodinated CAT-protein from heart, and then varying amounts of unlabeled CAT-proteins or Triton X-100 extracts of mitochondria from different sources were added to 0.25 ml RIA buffer (0.8% NaCl, 0.02% KCl, 0.02% KH2P04, 0.92% NazHP04, 0.5% Triton X-100, 5 mM EDTA, 0.05% NaN3, 0.1% bovine serum albumin, pH 7.4). Final volume of the mixture was 0.275 ml. The mixture was incubated 1 h at 37°C and 4 days at 4°C. Appropriate amounts of nonimmune serum and 373 Volume 96, number 2 FEBS LETTERS of precipitating second antibody were then introduced into the samples and incubated 3 h at 37°C. The precipitates were washed 3-times with RIA buffer and counted in a gamma-counter. Protein concentration was estimated according to [13] and in the presence of Triton X-100 as in [14] . 3. Results and discussion The translocator protein isolated from rat heart, liver and hepatoma mitochondria as a CAT-protein complex exhibited identical electrophoretic mobility in SDS-polyacrylamide gel electrophoresis corresponding to mol. wt 30 000. Two different antisera against heart and liver CAT-proteins were prepared. In accordance with [S] , no crossreaction was detected between CAT-protein from rat liver and antibody against heart CAT-protein (fig.1) and vice versa in Ouchterlony double diffusion test. In addition, neither of the two antisera reacted with the CATprotein isolated from rat hepatoma mitochondria 3 Fig.1. Immunodiffusion reaction of CAT-protein from different sources with antiserum against heart CAT-protein. Centre well, antiserum against rat heart CAT-protein. Other wells, equal concentrations of isolated CAT-protein from (1) rat heart, (2) liver and (3) hepatoma. 374 December 1978 when tested by the same technique. The data obtained by this technique with CAT-protein isolated from heart, liver and kidney mitochondria and antibody against heart CAT-protein had been interpreted in terms of an immunological organ specificity of this protein [5]. However, such an interpretation would require a corroboration by additional immunological technique, since in several studies [15,16] a crossreaction of antigenic determinants could be demonstrated by radioimmunoassay in case when it hadnot been detected by conventional immunological methods. To examine whether the CAT-proteins from heart, liver and hepatoma mitochondria share common antigenic determinant(s) a competitive inhibition radioimmunoassay has been employed in this study. In the assay, heart CAT-protein as a labeled antigen and homologous heart CAT-protein antiserum were used. The isolated heart CAT-protein was electrophoretically pure (fig.ZA), gave a single precipitin line in Ouchterlony double diffusion test (see fig.1). and after iodination retained its integrity and ability to interact with homologous antiserum (fig.ZB). In the competitive inhibition radioimmunoassay it was found that the CAT-protein isolated from hepatoma was able to inhibit the binding of labeled heart CATprotein with homologous antibody almost completely, whereas only partial (25%) inhibition was obtained with the CAT-protein isolated from liver mitochondria even at the highest concentration used (fig.3A). One of the reasons why high concentrations of isolated hepatoma and liver CAT-protein are required to demonstrate a crossreaction with CAT-protein isolated from heart might be a modification of antigenic structure of these proteins during the isolation. To restrain such a modification, Triton X-100 solubilized CAT-loaded mitochondria were used instead of isolated CAT-proteins in Ouchterlony double diffusion and in the competitive inhibition radioimmunoassay. Triton X-lOO-solubilized mitochondria from all the sources examined gave visible precipitin line with heart CAT-protein antibody. Yet, the use of detergent-solubilized membranes in immunodiffusion may lead to artifactual results [ 171. More conclusive data can be obtained using the competitive inhibition radioimmunoassay. In this assay almost complete inhibition of binding of the labeled CAT-protein isolated from heart with Volume 96, number M.W. x 2 EEBS LETTERS December 1978 xi3 LO- 68 - 80 - 60 - 21 13 - Fig.2. SDS-polyacrylamide gel eiectrophoresis of isolated and immunoprecipitated rat heart CAT-protein. (A) 15 ~crg CAT-protein isolated from rat heart mitochondria were Log COMPETING PROTEIN CONCENTRATION (ng) electrophoresed and stained. (B) ‘*‘I-labeled heart CATprotein (2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA X lo6 cpm) in RIA buffer was incubated with Fig.3, immunological cross-reactivity between CAT-proteins 5 ~1 homologous antiserum for 5 min at room temperature. from rat liver, heart and hepatoma mitochondria. Competitive The precipitate formed after addition of second antibody was washed 5 times with RIA buffer, dissolved in 2’Z SDS, 8 M inhibition radioimmunoassay was performed as in section 2 (5 X 10” cpm/tube) and urea, 4% p-mercaptoethanol, 1070 glycerol, and heated for with ‘251-labeled heart CAT-protein 2 min at 100°C The sample was electrophoresed in the antiserum against heart CAT-protein (final dilution 2 X 10’). same slab as that to be stained, dried and autoradiographed. In the competition isolated CAT-protein trom (-o-) rat The position of molecular weight standards is indicated; heart, (-A-) liver and (-0 -> hepatoma mitochondrra or cytochrome c (13 000), soybean trypsin inhibitor (21 000), Triton X-100-solubilized mitochondria from (-•-) rat heart, chymotrypsinogen (26 OOO), bovine serum albumin (68 000). (-A-) liver and t-m-) hepatoma were used. isolated CAT-protein in the assay resulted not only homologous antibody was obtained with detergentin the complete inhibition of binding but also in the solubilized mitochondria of all the sources examined mutual approaching of competition curves for (fig.3B). The extent of inhibition and the slope of the respective competing antigens. Lateral displacement competition curves for purified heart CAT-protein of identical competition curves reflects a modification and for solubilized mitochondria of heart, hepatoma of antigenicity of all antigenic determinants involved and liver were almost equal. This indicates that all in the assay [ 181. Thus the differences in the heart CAT-protein specific antigenic determinants antigenicity of the CAT-proteins present in the involved in the radioimmunoassay under the condirespective solubilized mitochondria are smaller than tions used, are present in the solubilized mitochondria of heart, liver and hepatoma. those of corresponding isolated CAT-protein The use of solubilized mitochondria instead of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA preparations. While the ratio of the competing protein 375 Volume 96, number 2 FEBS LETTERS concentration required for 50% inhibition was 1 : 40 : >lOOO for isolated CAT-proteins, the ratio of 1 : 5 : 50 was found for solubilized mitochondria of heart, hepatoma, and liver respectively. The amounts of CAT-protein in these mitochondria which bind to antibody raised against heart CATprotein are proportional to the capacities of the mitochondria to bind 35S-labeled CAT. It was found that Zajdela hepatoma mitochondria bind 0.35 pmol, rat liver - 0.15 pmol, and rat heart mitochondria 1.1 pmol 35S-labeled CAT/gram protein. Thus the differences in the antigenicity of the competing antigens present in the respective mitochondria after Triton X-l 00 solubilization are actually smaller than those indicated by the mutual distances between the competition curves. The differences in the ratios of the competing protein concentrations required for 50% inhibition using isolated antigens and solubilized mitochondria demonstrate an unequal attenuation of CAT-protein antigenicity during the purification of the antigen (hydroxylapatite chromatography and gel filtration) from respective solubilized mitochondria. It can be extrapolated that a similar, through less pronounced effect takes place also in the course of the solubilization of mitochondria. A lack of cross-reactivity between CAT-proteins isolated from different organs in Ouchterlony double diffusion test thus probably reflects an unequal modification of CAT-protein antigenicity by the solubilization and isolation. Acknowledgements We are indebted to Dr G. Russ for valuable advice and for gift of swine anti-rabbit IgG and to Dr L. Kovac’ for continuous interest and critical reading of the manuscript. The technical assistance of Mrs H. Ferancova and Mrs D. siffelova is gratefully acknowledged. This work was partially supported by a grant from WHO. 376 December 1978 References Klingenberg, M. (1970) in: Essays in Biochemistry (Campbell, P. N. and Dickens, F. eds) vol. 6, pp. 119-160. Academic Press, New York. [2] Vignais, P. V. (1976) Biochim. Biophys. Acta 456, l-38. [ 31 Riccio, P., Aquila, H. and Klingenberg, M. (1975) FEBS Lett. 56. 133-138. [4] Brandolin, G., Meyer, C.. Defaye, C., Vignais, P. M. and Vignais, P. V. (1974) FEBS Lett. 46,149-153. 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(1978) Proc. Natl. Acad. Sci. USA, 75, 198991993. 1171 Clausen. J. (1969) in: Laboratory Techniques in Biochemistry and Molecular Biology (Work. T. S. and Work, E. eds) vol. 1. pp. 397-557. North-Holland, Amsterdam. 12, 365-372. 1181 Venning, M. M. (1975) Immunochemistry [l]