Skip to main content
SpringerLink
Log in

(1,3)-β-Glucan synthase fromSaccharomyces cerevisiae: In vitro activation byβ-lactoglobulin or Brij-35, and photoaffinity labeling of enriched microsomal fractions with 5-azido-UDP-Glc and 8-azido-GTP

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

Two new activators of (1,3)-β-glucan synthase fromSaccharomyces cerevisiae were identified, and a procedure for preparing enriched enzyme fractions by removal of peripheral membrane proteins and entrapped soluble proteins was developed. Microsomal enzyme activity, known to be enhanced by bovine serum albumin (BSA), was stimulated threefold by both β-lactoglobulin and Brij-35. Both apparently substituted for BSA, since no synergistic effects were observed with activators added in combination. Successive washings of microsomal fractions with the detergents Brij-35 and Tergitol NP-40 to remove peripheral and vesicle-entrapped proteins yielded particulate fractions five-fold enriched in glucan synthase activity. GTP, an important effector of glucan synthase, improved purification of the enzyme during detergent extractions. Various membrane fractions were photolabeled with 5[32P]N3UDP-Glc or 8N3[32P]GTP, and potential UDP-Glc and GTP-binding polypeptides were identified. However, further enrichment will be required to determine which of these might represent subunits of the yeast glucan synthase complex.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Literature Cited

  1. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Google Scholar 

  2. Czarnecki J, Geahlen R, Haley B (1979) Synthesis and use of azido photoaffinity analogs of adenine and guanine nucleotides. Methods Enzymol 56:642–653

    Google Scholar 

  3. Drake RR, Evans RK, Wolf JK, Haley BE (1989) Synthesis and properties of 5-azido-UDP-glucose. J Biol Chem 264:11928–11933

    Google Scholar 

  4. Espinel-Ingroff A, Shadomy S (1989) In vitro and in vivo evaluation of antifungal agents. Eur J Clin Microbiol Infect Dis 8:353–361

    Google Scholar 

  5. Evans RK, Haley BE (1987) Synthesis and biological properties of 5-azido-2′-deoxyuridine 5′-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA. Biochemistry 26:269–276

    Google Scholar 

  6. Fevre M (1979) Digitonin solubilization and protease stimulation of β-glucan synthetase ofSaprolegnia. Z Pflanzenphysiol 95:129–140

    Google Scholar 

  7. Frost DJ, Read SM, Drake RR, Haley BE, Wasserman BP (1990) Identification of the UDP-glucose-binding polypeptide of callose synthase fromBeta vulgaris L. by photoaffinity labeling with 5-azido-UDP-glucose. J Biol Chem 265:2162–2167

    Google Scholar 

  8. Kang MS, Cabib E (1986) Regulation of fungal cell wall growth: a guanine nucleotide-binding, proteinaceous component required for activity of (1–3)-β-D-glucan synthase. Proc Natl Acad Sci USA 83:5808–5812

    Google Scholar 

  9. Matsumoto K, Kaibuchi K, Arai K, Nakafuku M, Kaziro Y (1989) Signal transduction by GTP-binding proteins inSaccharomyces cerevisiae. In: Walton EF, Yarranton GT (eds) Molecular and cell biology of yeast. New York: Van Nostrand Reinhold, pp 201–220

    Google Scholar 

  10. Palamarczyk G, Drake R, Haley B, Lennarz W (1990) Evidence that the synthesis of glucosylphosphodolichol in yeast involves a 35-kDa membrane protein. J Biol Chem 87:2666–2670

    Google Scholar 

  11. Perberdy JF (1990) Fungal cell walls—a review. In: Kuhn PJ, Trinci APJ, Jung MJ, Gosey MW, Copping LG (eds) Biochemistry of cell walls and membranes in fungi. New York, Heidelberg, Berlin: Springer-Verlag, pp 5–30

    Google Scholar 

  12. Quigley DR, Hrmova M, Selitrennikoff CP (1988) β-(1,3)Glucan synthase ofNeurospora crassa: solubilization and partial characterization. Exp Mycol 12:141–150

    Google Scholar 

  13. Rothman-Denes LB, Cabib E (1970) Two forms of yeast glycogen synthetase and their role in glycogen accumulation. Proc Natl Acad Sci USA 66:967–974

    Google Scholar 

  14. San-Blas G, San-Blas F (1982) Effect of detergents on membrane-associated glucan synthase fromParacoccidioides brasiliensis. J Bacteriol 152:563–566

    Google Scholar 

  15. Shematek EM, Cabib E (1980) Biosynthesis of the yeast cell wall. II. Regulation of β-(1-3)glucan synthetase by ATP and GTP. J Biol Chem 255:895–902

    Google Scholar 

  16. Shematek EM, Braatz JA, Cabib E (1980) Biosynthesis of the yeast cell wall. I. Preparation and properties of β-(1-3)glucan synthetase. J Biol Chem 255:888–894

    Google Scholar 

  17. St. Georgiv V (1988) Fungal infections and the search for novel antifungal agents. In: St. Georgiev V (ed) Antifungal drugs, vol. 544, New York: Ann NY Acad Sci, pp 1–3

    Google Scholar 

  18. Szaniszlo PJ, Kang MS, Cabib E (1985) Stimulation of β-(1-3)glucan synthetase of various fungi by nucleoside triphophates: generalized regulatory mechanism for cell wall biosynthesis. J Bacteriol 161:1188–1194

    Google Scholar 

  19. Wasserman BP, Frost DJ, Lawson SG, Mason TL, Rodis P, Sabin RD, Sloan ME (1989) Biosynthesis of cell-wall polysacharides: membrane isolation, in vitro glycosyl transferase assay and enzyme solubilization. In: Linskens HF, Jackson JK (eds) Modern methods of plant analysis, vol 10. Berlin, Heidelberg, New York: Springer-Verlag, pp 1–11

    Google Scholar 

Download references

Author information

Author notes

  1. David J. Frost

    Present address: Pharmaceutical Products Division, Department 47M, Abbott Laboratories, Building AP9A, One Abbott Park Road, 60064-3500, Abbott Park, Il, USA

  2. Rick R. Drake

    Present address: Department of Biochemistry, University of Arkansas for Medical Sciences, 4301 West Markham, 72212, Little Rock, AR, USA

Authors and Affiliations

  1. Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, 08903-0231, New Brunswick, NJ, USA

    David J. Frost &  Bruce P. Wasserman

  2. Department of Biochemistry, University of Kentucky, Lexington, Kentucky, USA

    Rick R. Drake

Authors
  1. David J. Frost
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rick R. Drake
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bruce P. Wasserman
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Frost, D.J., Drake, R.R. & Wasserman, B.P. (1,3)-β-Glucan synthase fromSaccharomyces cerevisiae: In vitro activation byβ-lactoglobulin or Brij-35, and photoaffinity labeling of enriched microsomal fractions with 5-azido-UDP-Glc and 8-azido-GTP. Current Microbiology 24, 295–300 (1992). https://doi.org/10.1007/BF01577336

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01577336

Keywords

Search

Navigation