1887

Abstract

Proteinases secreted by myxamoebae during growth and starvation were analysed using three arginine-containing peptide nitroanilides as substrates. The enzymes were secreted differentially, both during growth and after resuspension of the myxamoebae in phosphate buffer. Secretion was stimulated by the addition of sucrose to starvation buffer, but this did not affect the type of proteinase released. Activity towards -benzoyl--prolyl--phenylalanyl--arginine 4-nitroanilide (BzPFRase activity) was released in large quantities, and over 80% of the total activity in starved suspensions was extracellular within 6 h of transfer to buffer containing sucrose. A smaller fraction of the total activity towards -carbobenzoxy--arginyl--arginine 4-nitroanilide (ZRRase activity) was secreted. Extracellular activity towards -carbobenzoxy--tyrosyl--lysyl--arginine 4-nitroanilide (ZYKRase) was either too low to be detected or present in quantities which were small enough to be accounted for by cell lysis. BzPFRase and ZRRase activities were both inactivated by E-64 and two peptidyldiazomethanes, specific inhibitors of cysteine proteinases. ZYKRase activity was also inhibited by peptidyldiazomethanes but E-64 was effective only at high concentrations. When electrophoresis in polyacrylamide gels containing gelatin (gelatin-SDS-PAGE) was used to analyse the proteinases involved, one cysteine proteinase, ddCP42, correlated exactly with the secreted BzPFRase activity under all conditions, although contributions to this activity from other proteinases are also likely. Overall, the results demonstrate significant differences between individual proteinases with respect to secretion which suggests that their physiological roles may differ.

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1990-05-01
2021-08-05
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References

  1. Barrett AJ., Buttle D.J., Mason R.W. 1988; Lysosomal cysteine proteinases. ISI Atlas of Science: Biochemistry 1:256–260
    [Google Scholar]
  2. Bradford M.M. 1976; A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
    [Google Scholar]
  3. Cardelli J.A., Golumbeski G.S., Woychik N.A., Mierendorf R.C., Dimond R.L. 1987; Defining the intracellular localization pathways followed by lysosomal enzymes in Dictyostelium discoideum. Methods in Cell Biology 28:139–155
    [Google Scholar]
  4. Crean E.V., Rossomando E.F. 1979; Effects of sugars on glycosidase secretion in Dictyostelium discoideum. Journal of General Microbiology 110:315–322
    [Google Scholar]
  5. Dimond R.L., Burns R.A., Jordan K.B. 1981; Secretion of lysosomal enzymes in the cellular slime mold Dictyostelium discoideum. Journal of Biological Chemistry 256:6565–6572
    [Google Scholar]
  6. Kirschke H., Barrett A.J. 1987; Chemistry of lysosomal proteases. In Lysosomes: Their Role in Protein Breakdown, pp 193–238 Glaumann H., Ballard F. J. Edited by London: Academic Press;
    [Google Scholar]
  7. Mason R.W., Wilcox D., Wikstrom P., Shaw E.N. 1989; The identification of active forms of cysteine proteinases in Kirsten-virus-transformed mouse fibroblasts by use of a specific radiolabelled inhibitor. Biochemical Journal 257:125–129
    [Google Scholar]
  8. Mizuno K., Nakamura T., Ohshima T., Tanaka S., Matsuo H. 1988; Yeast KEX2 gene encodes an endopeptidase homologous to subtilisin-like serine proteases. Biochemical and Biophysical Research Communications 156:246–254
    [Google Scholar]
  9. Mizuno K., Nakamura T., Ohshima T., Tanaka S., Matsuo H. 1989; Characterization of KEX2-encodedendopeptidase from yeast Saccharomyces cerevisiae. Biochemical and Biophysical Research Communications 159:305–311
    [Google Scholar]
  10. North M.J. 1982a; Proteolytic activities in Dictyostelium discoideum detected with chromogenic peptide substrates. Experimental Mycology 6:345–352
    [Google Scholar]
  11. North M.J. 1982b; A study of the proteinase activity released by Dictyostelium discoideum during starvation. Journal of General Microbiology 128:1653–1659
    [Google Scholar]
  12. North M.J. 1985; Cysteine proteinases of cellular slime moulds. Biochemical Society Transactions 13:288–290
    [Google Scholar]
  13. North M.J. 1988; A bacterial factor induces changes in cysteine proteinase forms in the cellular slime mouldDictyostelium discoideum. Biochemical Journal 254:269–275
    [Google Scholar]
  14. North M.J., Whyte A. 1984; Purification and characterization of two acid proteinases from Dictyostelium discoideum. Journal of General Microbiology 130:123–134
    [Google Scholar]
  15. North M.J., Coombs G.H., Barry J.D. 1983; A comparative study of the proteolytic enzymes of Trypanosomabrucei, T. equiperdum, T. evansi, T. vivax, Leishmaniatarentolae and Crithidia fasciculata. Molecular and Biochemical Parasitology 9:161–180
    [Google Scholar]
  16. North M.J., Scott K.I., Lockwood B.C. 1988; Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic analysis. Biochemical Journal 254:261–268
    [Google Scholar]
  17. North M.J., Cotter D.A., Franek K.J. 1990; Dictyostelium discoideum spore germination: increases in proteinase activity are not directly coupled to the emergence of myxamoebae. Journal of General Microbiology 136:000–000
    [Google Scholar]
  18. Schechter I., Berger A. 1967; On the size of the active site in proteases. I. Papain. Biochemical and Biophysical Research Communications 27:157–162
    [Google Scholar]
  19. Seshadri J., Cotter D.A., Dimond R.L. 1986; The characterization and secretion pattern of the lysosomaltrehalases of Dictyostelium discoideum. Experimental Mycology 10:131–143
    [Google Scholar]
  20. Wagner J.-C., Escher C., Wolf D.H. 1987; Some characteristics of hormone (pheromone) processing enzymes in yeast. FEBS tetters 218:31–34
    [Google Scholar]
  21. Watts D.J., Ashworth J.M. 1970; Growth of myxamoebae of the cellular slime mouldDictyostelium discoideum in axenic culture. Biochemical Journal 119:171–174
    [Google Scholar]
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