Summary
Malate synthase was investigated by the small-angle X-ray scattering technique in aqueous solution. Measurements extending for several hours revealed a continuous increase of the intensity in the innermost portion of the scattering curve. There is clear evidence that this increase was caused by an X-ray induced aggregation of enzyme particles during the performance of the small-angle X-ray scattering experiment. The monitoring of the aggregation process in situ by means of small-angle X-ray scattering led to a model of the way how the aggregation might proceed. The analysis of the scattering curves of malate synthase taken at various stages of aggregation established the retention of the thickness factor of the native enzyme and the occurrence of one and later on of two cross-section factors. The process of aggregation was also reflected by the increase of extension of the distance distribution function. According to these results, the first step of aggregation might be a linear side-by-side association of the oblate enzyme particles, a process which is followed by a twodimensional aggregation. An aggregation in the third dimension was not observed during the time covered by our experiment. The predominance of aggregation in only one or two dimensions was corroborated by comparison of appropriate theoretical scattering curves with the experimental curves. The theoretical scattering curves for this comparison were obtained by averaging over the properly weighted scattering curves calculated for various species of hypothetical aggregates. The time dependence of the apparent mean radius of gyration was used to compare the aggregation of enzyme samples that were irradiated under different experimental conditions. It turned out that by addition of dithiothreitol to the enzyme solutions as well as in the presence of the substrates (acetyl-CoA, glyoxylate) or of a substrate analogue (pyruvate) or of ethanol the rate of aggregation is reduced. Enzymic activity was found to decrease about exponentially with increasing X-ray dose. The presence of dithiothreitol or of the substrate glyoxylate or of the substrate analogue pyruvate protects the enzyme against X-ray induced inactivation. The substrate acetyl-CoA does not exhibit a comparable protective effect against inactivation. Measurements of enzymic activity and small-angle X-ray scattering on samples, which had been X-irradiated with a defined dose prior to the measurements, established two different series of efficiency for the protection of the enzyme against aggregation (pyruvate > glyoxylate > acetyl-CoA) and inactivation (glyoxylate > pyruvate\( > > \) acetyl-CoA). The results showed that there is no direct relation between the extent of aggregation and the loss of enzymic activity.
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References
Armstrong, D. A., Buchanan, J. D.: Reactions of O −2 , H2O2 and other oxidants with sulfhydryl enzymes. Photochem. Photobiol.28, 743–755 (1978)
Bick, Y. A. E., Jackson, W. D.: Chemical protection against X-irradiation by a new reducing agent, 1,4-dithiothreitol in marsupial leucocytes in culture. Nature217, 479–480 (1968)
Buchanan, J. D., Armstrong, D. A.: Free radical inactivation of lactate dehydrogenase. Int. J. Radiat. Biol.30, 115–127 (1976)
Buchanan, J. D., Armstrong, D. A.: The radiolysis of glyceraldehyde-3-phosphate dehydrogenase. Int. J. Radiat. Biol.33, 409–418 (1978)
Chan, P. C., Bielski, B. H. J.: Pulse radiolysis study of optical absorption and kinetic properties of dithiothreitol free radical. J. Am. Chem. Soc.95, 5504–5508 (1973)
Delincée, H., Jakubick, V.: The effect of carbohydrates and lipids on the radiation-induced aggregation of proteins. Int. J. Appl. Radiat. Isot.28, 939–945 (1977)
Dertinger, H., Jung, H.: Molekulare Strahlenbiologie. Berlin, Heidelberg, New York: Springer 1969
Dixon, G. H., Kornberg, H. L., Lund, P.: Purification and properties of malate synthetase. Biochim. Biophys. Acta41, 217–233 (1960)
Draganić, I. G., Draganić, Z. D.: The radiation chemistry of water. New York, London: Academic Press 1971
Durchschlag, H., Zipper, P.: Electrophoretic studies on X-irradiated and H2O2-treated malate synthase. Hoppe-Seyler's Z. Physiol. Chem.361, 239 (1980)
Durchschlag, H., Biedermann, G., Durchschlag, G., Eggerer, H.: Isolation and characterization of malate synthase from baker's yeast. Abstracts of the 10th International Congress of Biochemistry, Hamburg 1976. p. 205
Durchschlag, H., Goldmann, K., Wenzl, S., Durchschlag, G., Jaenicke, R.: Ultracentrifugal and spectroscopic investigations on malate synthase from baker's yeast. FEBS Lett.73, 247–250 (1977)
Durchschlag, H., Bogner, F., Wilhelm, D., Jaenicke, R., Zipper, P., Mayer, F.: The subunit structure of malate synthase from baker's yeast. Hoppe-Seyler's Z. Physiol. Chem.359, 1077 (1978)
Eggerer, H., Klette, A.: Über das Katalyseprinzip der Malatsynthase. Eur. J. Biochem.1, 447–475 (1967)
Fricke, H., Hart, E. J.: Chemical dosimetry. In: Radiation dosimetry. Attix, F. H., Roesch, W. C. (eds.), Vol. II, pp. 167–239. New York, London: Academic Press 1966
Glatter, O.: A new iterative method for collimation correction in small-angle scattering. J. Appl. Cryst.7, 147–153 (1974)
Greening, J. R.: Dosimetry of low energy X-rays. In: Topics in radiation dosimetry. Radiation dosimetry. Attix, F. H. (ed.), Suppl. 1, pp. 261–315. New York, London: Academic Press 1972
Grossweiner, L. L: Photochemical inactivation of enzymes. Curr. Top. Radiat. Res. Q.11, 141–199 (1976)
Guinier, A.: La diffraction des rayons X aux très petits angles: application a l'ètude de phénomènes ultramicroscopiques. Ann. Phys. (Paris)12, 161–237 (1939)
Habeeb, A. F. S. A.: Reaction of protein sulfhydryl groups with Ellman's reagent. In: Methods in enzymology. Hirs, C. H. W., Timasheff, S. N. (eds.), Vol. 25, pp. 457–464. New York, London: Academic Press 1972
Jakubick, V., Delincée, H.: Strahleninduzierte Aggregatbildung von Proteinen: Bindung von Aminosäuren an Myoglobin. Z. Naturforsch. [C]33, 203–209 (1978)
Kratky, 0.: Neues Verfahren zur Herstellung von blendenstreuungsfreien Röntgen-Kleinwinkelaufnahmen, IV. Z. Elektrochem.62, 66–73 (1958)
Kratky, 0.: X-ray small angle scattering with substances of biological interest in diluted solutions. Prog. Biophys. Mol. Biol.13, 105–173 (1963)
Kratky, O., Pilz, I.: Recent advances and applications of diffuse X-ray small-angle scattering on biopolymers in dilute solutions. Q. Rev. Biophys.5, 481–537 (1972)
Kratky, O., Skala, Z.: Neues Verfahren zur Herstellung von blendenstreuungsfreien Röntgen-Kleinwinkelaufnahmen, V. Z. Elektrochem.62, 73–77 (1958)
Kratky, O., Pilz, I., Schmitz, P. J.: Absolute intensity measurement of small angle X-ray scattering by means of a standard sample. J. Colloid Interface Sci.21, 24–34 (1966)
Lal, M., Lin, W. S., Gaucher, G. M., Armstrong, D. A.: The repair, protection and sensitization of papain with respect to inactivation by H2O2 and OH: Effects of dithiothreitol, penicillamine, cystine and penicillamine disulphide. Int. J. Radiat. Biol.28, 549–564 (1975)
Lin, W. S., Clement, J. R., Gaucher, G. M., Armstrong, D. A.: Repairable and nonrepairable inactivation of irradiated aqueous papain: Effects of OH, O −2 , e −aq , and H2O2. Radiat. Res.62, 438–455 (1975a)
Lin, W. S., Armstrong, D. A., Gaucher, G. M.: Formation and repair of papain sulfenic acid. Can. J. Biochem.53, 298–307 (1975b)
Lin, W. S., Armstrong, D. A., Lal, M.: Effects of superoxide dismutase, dithiothreitol and formate ion on the inactivation of papain by hydroxyl and superoxide radicals in aerated solutions. Int. J. Radiat. Biol.33, 231–243 (1978)
Lynn, K. R.: Cross linking in the radiolysis of some enzymes and related proteins. In: Advances in experimental medicine and biology. Protein crosslinking. Biochemical and molecular aspects. Friedman, M. (ed.), Vol. 86A, pp. 557–570. New York, London: Plenum Press 1977
Mee, L. K., Adelstein, S. J., Stein, G.: Inactivation of ribonuclease by the primary aqueous radicals. Radiat. Res.52, 588–602 (1972)
Pilz, I., Glatter, O., Kratky, O.: Small-angle X-ray scattering. In: Methods in enzymology. Hirs, C. H. W., Timasheff, S. N. (eds.), Vol. 61, pp. 148–249. New York, San Francisco, London: Academic Press 1979
Porod, G.: Die Röntgenkleinwinkelstreuung von dichtgepackten kolloiden Systemen, I. Teil. Kolloid-Z.124, 83–114 (1951)
Redpath, J. L.: Pulse radiolysis of dithiothreitol. Radiat. Res.54, 364–374 (1973a)
Redpath, J. L.: Radioprotection of enzyme and bacterial systems by dithiothreitol. Radiat. Res.55, 109–117 (1973b)
Sanner, T., Pihl, A.: Identification of the water radicals involved in X-ray inactivation of enzymes in solution and determination of their rate of interaction with the enzyme. Biochim. Biophys. Acta146, 298–301 (1967)
Schmid, G., Durchschlag, H., Biedermann, G., Eggerer, H., Jaenicke, R.: Molecular structure of malate synthase and structural changes upon ligand binding to the enzyme. Biochem. Biophys. Res. Commun.58, 419–426 (1974)
Schüßler, H.: Effect of ethanol on the radiolysis of ribonuclease. Int. J. Radiat. Biol.27, 171–180 (1975)
Schüßler, H., Mee, L. K., Adelstein, S. J.: Reaction of ethanol radicals with ribonuclease. Int. J. Radiat. Biol.30, 467–472 (1976)
Schüßler, H.: Einwirkung ionisierender Strahlung auf Proteine in wäßriger Lösung. Biophysik9, 315–324 (1973)
Yamamoto, O.: Ionizing radiation-induced crosslinking in proteins. In: Advances in experimental medicine and biology. Protein crosslinking. Biochemical and molecular aspects. Friedman, M. (ed.), Vol. 86A, pp. 509–547. New York, London: Plenum Press 1977
Yost, H. T., Goetzl, E. J.: Substrate protection of lactic acid dehydrogenase against inactivation by ionizing radiation. Radiat. Res.33, 293–302 (1968)
Zipper, P.: Ein einfaches Verfahren zur Monochromatisierung von Streukurven. Acta Phys. Austriaca30, 143–151 (1969)
Zipper, P.: The evaluation of small-angle X-ray scattering data by a computer. Acta Phys. Austriaca36, 27–38 (1972)
Zipper, P., Durchschlag, H.: The molecular structure of malate synthase. Abstracts of the Xth International Congress of Crystallography, Amsterdam 1975. Acta Crystallogr. Sect.A31, S 59 (1975)
Zipper, P., Durchschlag, H.: Small-angle X-ray studies on malate synthase from baker's yeast. Biochem. Biophys. Res. Commun.75, 394–400 (1977)
Zipper, P., Durchschlag, H.: Small-angle X-ray scattering on malate synthase from baker's yeast. The native substrate-free enzyme and enzyme-substrate complexes. Eur. J. Biochem.87, 85–99 (1978a)
Zipper, P., Durchschlag, H.: Small-angle X-ray scattering on malate synthase from baker's yeast. Considerations on the effects of bound ligands. Z. Naturforsch. [C]33, 504–510 (1978b)
Zipper, P., Durchschlag, H.: Radiobiological aspects of small-angle X-ray scattering. Abstracts of the 12th FEBS-Meeting, Dresden 1978. Abstr. Nr. 2545 (1978c)
Zipper, P., Durchschlag, H.: Small-angle X-ray scattering studies on the X-ray induced aggregation of malate synthase. I. Structural and kinetic studies. Monatsh. Chem. (In press, 1980a)
Zipper, P., Durchschlag, H.: Small-angle X-ray scattering studies on the X-ray induced aggregation of malate synthase. II. Inactivation and aggregation experiments. Monatsh. Chem. (In press, 1980b)
Zipper, P., Durchschlag, H.: Small-angle X-ray scattering studies on the X-ray induced aggregation of malate synthase. Computer simulations and models. Z. Naturforsch. [C] (Submitted, 1980c)
Zipper, P., Glatter, O.: The Graz program system for the evaluation of scattering data. Abstracts of the 4th International Conference on Small-Angle Scattering of X-Rays and Neutrons. Gatlinburg (USA) 1977. p. 49
Zipper, P., Gatterer, H. G., Schurz, J., Durchschlag, H.: Röntgenkleinwinkeluntersuchungen der strahleninduzierten Aggregation von Ribonuclease, Lactat-Dehydrogenase, Glycerinaldehyd-3-phosphat-Dehydrogenase and Serumalbumin. Ein Vergleich mit Malatsynthase. Monatsh. Chem. (In press, 1980)
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Zipper, P., Durchschlag, H. Small-angle X-ray scattering studies on the X-ray induced aggregation of malate synthase. Radiat Environ Biophys 18, 99–121 (1980). https://doi.org/10.1007/BF01326049
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DOI: https://doi.org/10.1007/BF01326049