Summary
We have identified cytoplasmic and membrane-associated proteinases from Micrococcus lysodeikticus (M. luteus) by the use of 125I-labeled casein and insulin as substrates. The membrane-associated activities were released by shock washing. Proteolytic activities showed pH optima at slightly alkaline values and we have concentrated on the activities at pH 8.0. The total units of both proteolytic activities were higher in the cytoplasmic than in any other fractions but the situation was different when the results were expressed in terms of specific activity. The activities against casein and insulin were differentiated by the action of inhibitors, divalent metal ions, Arrhenius plots and dependence on ionic strength. On these grounds, it is proposed that the membrane-associated enzyme acting on insulin is a single thiol proteinase while the proteolysis of casein reflects the action of, at least, two enzymes (thiol proteinase and serine proteinase). The distinction between the casein and insulin degrading activities was confirmed by crossed-inhibition experiments and by their behaviour on gel chromatography and concentration-dependent experiments.
The aggregating properties have hampered the purification of the enzymes. The present results raise doubts about the significance of preventing membrane damage and degradation of membrane proteins by the addition of indiscriminated proteinase inhibitors during membrane isolation and manipulation.
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References
Morré, D. J., 1975. Ann. Rev. Plant Physiol. 26: 441–481.
Taylor, J. M., Dehlinger, P. J., Dice, J. F. & Schimke, R. T., 1973. Drug Metab. Dispos 1: 84–91.
Matile, P., 1974. In: Dynamic Aspects of Plant Ultrastructure (Robards, A. W., ed.), McGraw-Hill, London.
Goldberg, A. L., Voellmy, R. & Sreedhara Swany, K. H., 1979. In: Biological Functions of Proteinases (Holzer, H. & Tschesche, H., eds.) Springer-Verlag, Berlin, Heidelberg, New York, pp. 35–48.
Pacaud, M. & Uril, J., 1971. Eur. J. Biochem. 23: 435–442.
Pacaud, M. & Richaud, C., 1975. J. Biol. Chem. 250: 7771–7779.
Regnier, P. & Thang, M. N., 1975. Eur. J. Biochem. 54: 445–451.
Lazdunski, C., Busuttil, J. & Lazdunski, A., 1975. Eur. J. Biochem. 60:363–369.
McGregor, C. H., Bishop, C. N. & Blech, J. E., 1979. J. Bacteriol. 137: 574–583.
Mantsala, P. & Zalkin, H., 1980. J. Bacteriol. 141: 493–501.
Nieto, M., Muñoz, E., Carreira, J. & Andreu, J. M., 1975. Biochem. Biophys. Acta 413: 394–414.
Carreira, J., Andreu, J. M., Nieto, M. & Muñoz, E., 1976. Mol. Cell. Biochem. 10: 67–76.
Andreu, J. M., & Muñoz, E., 1979. Biochemistry 18: 1836–1844.
Huberman, M. & Salton, M. R. J., 1979. Biochem. Biophys. Acta 547: 230–240.
Risi, S., Höckel, M., Hulla, F. W. & Dose, K., 1977. Eur. J. Biochem. 81: 103–109.
Salton, M. R. J., 1980. In: Subcell. Biochem(Roodyn, D. B., ed.) vol. 7, Plenum Press, New York and London, pp. 309–373.
Andreu, J. M., Albendea, J. A. & Muñoz, E., 1973. Eur. J. Biochem. 37: 505–515.
Muñoz, E., Salton, M. R. J., Ng, M. H. & Schor, M. T., 1969. Eur. J. Biochem. 7: 490–501.
Greenwood, F. C., Hunter, W. M. & Glover, J. S., 1963. Biochem. J. 39: 114–123.
Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J., 1951. J. Biol. Chem. 193: 265–275.
Muñoz, E., Nachbar, M. S., Schor, M. T. & Salton, M. R. J., 1968. Biochem. Biophys. Res. Commun. 32: 539–546.
Downie, J. A., Gibson, F. & Cox, G. B., 1979. Ann. Rev. Biochem. 48: 103–131.
Gorini, L. & Fromageot, C., 1950. Biochem. Biophys. Acta 5: 524–534.
Matsubara, H. & Feder, J., 1971. In: The Enzymes 3rd edition (Boyer, P. D., ed.) vol 111, Academic Press, New York and London, pp.721–797.
Muñoz, E. & Estrugo, S. F., 1979. Ferns Microbiol. Lett. 6: 235–239.
Cox, G. B., Downie, J. A., Fayle, D. R. M., Gibson, F. & Radik, J., 1978. J. Bacteriol. 133: 287–282.
Downie, J. A., Senior, A. E., Cox, G. B. & Gibson, F., 1979. J. Bacteriol. 138: 87–91.
Ryrie, I. J., 1977. Arch. Biochem. Biophys. 184: 464–475.
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Rivas, L., Marquet, A. & Muñoz, E. Partial characterization of membrane-associated proteinases from Micrococcus lysodeikticus . Mol Cell Biochem 43, 27–34 (1982). https://doi.org/10.1007/BF00229536
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DOI: https://doi.org/10.1007/BF00229536