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Antonie van Leeuwenhoek

, Volume 64, Issue 1, pp 67–74 | Cite as

Effect of digitonin on membrane-bound and chitosomal chitin synthetase activity in protoplasts from yeast cells ofCandida albicans

  • Daniel Gozalbo
  • Francisco Dubón
  • Rafael Sentandreu
Research Articles

Abstract

The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) fromC. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen,in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processesin vivo andin vitro or, alternatively, that both enzyme activities (activein vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml−1 of digitonin and 64 µg ml−1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 µg ml−1 of protease, whereas activity strongly decreases in the presence of 64 µg ml−1 of trypsin. Digitonin does not produce zymogen activationper se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.

Key words

Candida albicans chitin synthetase digitonin proenzyme activation protoplasts solubilization zymogen 

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References

  1. Au-Young J & Robbins PW (1990) Isolation of chitin synthase gene (CHS1) fromCandida albicans by expression inSaccharomyces cerevisiae. Mol. Microbiol. 4: 197–207Google Scholar
  2. Bartnicki-Garcia S, Bracker CE, Reyes E & Ruiz-Herrera J (1978) Isolation of chitosomes from taxonomically diverse fungi and synthesis of chitin microfibrilsin vitro. Exp. Mycol. 2: 173–192Google Scholar
  3. Bowen AR, Chen-Wu JL, Momany M, Young R, Szaniszlo PJ & Robbins PW (1992) Classification of fungal chitin synthases. Proc. Natl. Acad. Sci. USA 89: 519–523Google Scholar
  4. Braun PC & Calderone RA (1978) Chitin synthesis inCandida albicans: comparison of yeast and hyphal forms. J. Bacteriol. 135: 1472–1477Google Scholar
  5. —— (1979) Regulation and solubilization ofCandida albicans chitin synthetase. J. Bacteriol. 140: 666–670Google Scholar
  6. Bulawa CE & Osmond BC (1990) Chitin synthase I and chitin synthase II are not required for chitin synthesisin vivo inSaccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 87: 7424–7428Google Scholar
  7. Cabib E, Sburlati A, Bowers B & Silverman J (1989) Chitin synthase 1, an auxiliary enzyme for chitin synthesis inSaccharomyces cerevisiae. J. Cell Biol. 108: 1665–1672Google Scholar
  8. Chen-Wu JL, Zwicker J, Bowen AR & Robbins PW (1992) Expression of chitin synthase genes during yeast and hyphal growth phases ofCandida albicans. Mol. Microbiol. 6: 497–502Google Scholar
  9. Duran A & Cabib E (1978) Solubilization and partial purification of yeast chitin synthetase. Confirmation of the zymogenic nature of the enzyme. J. Biol. Chem. 253: 4419–4425Google Scholar
  10. Elorza MV, Rico H, Gozalbo D & Sentandreu R (1983) Cell wall composition and protoplasts regeneration inCandida albicans. Antonie van Leeuwenhoek 49: 457–469Google Scholar
  11. Fernandez MP, Correa JV & Cabib E (1982) Activation of chitin synthetase in permeabilized cells of aSaccharomyces cerevisiae mutant lacking proteinase B. J. Bacteriol. 130: 1193–1199Google Scholar
  12. Hardy JC & Gooday GW (1983) Stability and zymogenic nature of chitin synthase fromCandida albicans. Curr. Microbiol. 9: 51–54Google Scholar
  13. Gögelein H & Hüby A (1984) Interaction of saponin and digitonin with black lipid membranes and lipid monolayers. Biochim. Biophys. Acta 773: 32–38Google Scholar
  14. Gozalbo D, Dubon F & Sentandreu R. (1985) Studies on zymogenicity and solubilization of chitin synthetase fromCandida albicans. FEMS Microbiol. Letters 26: 59–63Google Scholar
  15. Gozalbo D, Dubon F, Schwencke J & Sentandreu R (1987) Characterization of chitosomes inCandida albicans protoplasts. Exp. Mycol. 11: 331–338Google Scholar
  16. Gozalbo D, Dubon F & Sentandreu R (1991a) Subcellular distribution of chitin synthetase inCandida albicans yeast cells and protoplasts. Mycol. Res. 95: 513–520Google Scholar
  17. —— (1991b) Evidence for the involvement of acylglycerides on chitin synthetase activity inCandida albicans. FEMS Microbiol. Letters 82: 79–82Google Scholar
  18. Hänseler E, Nyhlen LE & Rast DM (1983a) Isolation and properties of chitin synthase fromAgaricus bisporus. Exp. Mycol. 7: 7–30Google Scholar
  19. —— (1983b) Dissociation and reconstitution of chitosomes. Biochim. Biophys. Acta 745: 121–133Google Scholar
  20. Hernandez J, Lopez-Romero E, Cerbon J & Ruiz-Herrera J (1981) Lipid analysis of chitosomes, chitin synthesizing microvesicles fromMucor rouxii. Exp. Mycol. 5: 349–356Google Scholar
  21. Lowry OH, Rosebrough NJ, Farr AL & Randall RJ (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275Google Scholar
  22. Marriot MS (1975) Isolation and chemical characterization of plasma membranes from the yeast and mycelial forms ofCandida albicans. J. Gen. Microbiol. 86: 115–132Google Scholar
  23. Orlean P (1987) Two chitin synthases inSaccharomyces cerevisiae. J. Biol. Chem. 262: 5732–5739Google Scholar
  24. Pesti M, Campbell JM & Peberdy JF (1981) Alteration of ergosterol content and chitin synthase activity inCandida albicans. Curr. Microbiol. 5: 187–190Google Scholar
  25. Ruiz-Herrera J, Bartnicki-Garcia S & Bracker CE (1980) Dissociation of chitosomes by digitonin into 16S subunits with chitin synthetase activity. Biochim. Biophys. Acta 629: 201–216Google Scholar
  26. Ruiz-Herrera J & Sentandreu R (1989) Fungal cell wall synthesis and assembly. In: McGinnis MR & Borgers M (Eds) Current Topics in MedicalMycology, Vol. 3 (pp. 168–217) Springer-Verlag, New YorkGoogle Scholar
  27. Ruiz-Herrera J, Sentandreu R & Martinez JP (1992) Chitin biosynthesis in fungi. In: Arura KD, Elander RP & Mukerji KG (Eds) Handbook of Applied Mycology, Vol. 4, Fungal Biotechnology (pp. 281–312) Marcel Dekker Inc. New York, Basel & Hong KongGoogle Scholar
  28. Shaw JA, Mol PC, Bowers B, Silverman SJ, Valdivieso MH, Duran A & Cabib E (1991) The function of chitin synthases 2 and 3 in theSaccharomyces cerevisiae cell cycle. J. Cell Biol. 114: 111–123Google Scholar
  29. Silverman SJ (1989) Similar and different domains of chitin synthases 1 and 2 ofSaccharomyces cerevisiae: two isoenzymes with distinct functions. Yeast 5: 459–467Google Scholar
  30. Silverman SJ, Sburlati A, Slater ML & Cabib E (1988) Chitin synthase 2 is essential for septum formation and cell division inSaccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 84: 4735–4739Google Scholar
  31. Sundaram MS, Sullivan PA & Shepherd MG (1981) Changes in lipid composition during starvation and germ tube formation inCandida albicans. Exp. Mycol. 5: 140–147Google Scholar
  32. Vermeulen CA & Wessels JGH (1983) Evidence for a phospholipid requirement of chitin synthase inSchizophyllum commune. Curr. Microbiol. 8: 67–71Google Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Daniel Gozalbo
    • 1
  • Francisco Dubón
    • 1
  • Rafael Sentandreu
    • 1
  1. 1.Department de Microbiologia, Facultat de FarmàciaUniversitat de València, Avgda. Vicent Andrés Estellés s.n.Burjassot, ValènciaSpain

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