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A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure.
Anal Biochem. 2002 Feb 01; 301(1):136-50.AB

Abstract

In yeast and other fungi, cell division, cell shape, and growth depend on the coordinated synthesis and degradation of cell wall polymers. We have developed a reliable and efficient micro method to determine Saccharomyces cerevisiae cell wall composition that distinguishes between beta1,3- and beta1,6-glucan. The method is based on the sequential treatment of cell walls with specific hydrolytic enzymes followed by dialysis. The low molecular weight (MW) products thus separated account for each particular cell wall polymer. The method can be applied to as little as 50-100 mg (wet wt) of radioactively labeled cells. A combination of chitinase and recombinant beta-1,3-glucanase is initially used, releasing all of the chitin and 60-65% of the beta1,3-glucan from the cell walls. Next, recombinant endo-beta-1,6-glucanase from Trichoderma harzianum is utilized to release all the beta-1,6-glucan present in the wall. The chromatographic pattern of endoglucanase digested beta-1,6-glucan provides a characteristic "fingerprint" of beta-1,6-glucan and the fine structure of the oligosaccharides in this pattern was determined by 1H NMR and electrospray ionization mass spectroscopy. The final enzymatic step uses laminarinase and beta-glucosidase to release the remaining beta-1,3-glucan. The cell wall mannan remains as a high MW fraction at the end of the fractionation procedure. Good sensitivity and correlation with cell wall composition determined by traditional methods were observed for wild-type and several cell wall mutants.

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Authors+Show Affiliations

Magnelli P
Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118, USA.
Cipollo JF
No affiliation info available
Abeijon C
No affiliation info available

MeSH

Cell WallChemical FractionationChromatography, GelGlucansGlycoside HydrolasesHydrolysisHypocrealesMagnetic Resonance SpectroscopyMutationOligosaccharidesRecombinant ProteinsSaccharomyces cerevisiaeSpectrometry, Mass, Electrospray IonizationTrichodermabeta-Glucans

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

11811978

Citation

Magnelli, Paula, et al. "A Refined Method for the Determination of Saccharomyces Cerevisiae Cell Wall Composition and Beta-1,6-glucan Fine Structure." Analytical Biochemistry, vol. 301, no. 1, 2002, pp. 136-50.
Magnelli P, Cipollo JF, Abeijon C. A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure. Anal Biochem. 2002;301(1):136-50.
Magnelli, P., Cipollo, J. F., & Abeijon, C. (2002). A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure. Analytical Biochemistry, 301(1), 136-50.
Magnelli P, Cipollo JF, Abeijon C. A Refined Method for the Determination of Saccharomyces Cerevisiae Cell Wall Composition and Beta-1,6-glucan Fine Structure. Anal Biochem. 2002 Feb 1;301(1):136-50. PubMed PMID: 11811978.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure. AU - Magnelli,Paula, AU - Cipollo,John F, AU - Abeijon,Claudia, PY - 2002/1/29/pubmed PY - 2002/4/26/medline PY - 2002/1/29/entrez SP - 136 EP - 50 JF - Analytical biochemistry JO - Anal Biochem VL - 301 IS - 1 N2 - In yeast and other fungi, cell division, cell shape, and growth depend on the coordinated synthesis and degradation of cell wall polymers. We have developed a reliable and efficient micro method to determine Saccharomyces cerevisiae cell wall composition that distinguishes between beta1,3- and beta1,6-glucan. The method is based on the sequential treatment of cell walls with specific hydrolytic enzymes followed by dialysis. The low molecular weight (MW) products thus separated account for each particular cell wall polymer. The method can be applied to as little as 50-100 mg (wet wt) of radioactively labeled cells. A combination of chitinase and recombinant beta-1,3-glucanase is initially used, releasing all of the chitin and 60-65% of the beta1,3-glucan from the cell walls. Next, recombinant endo-beta-1,6-glucanase from Trichoderma harzianum is utilized to release all the beta-1,6-glucan present in the wall. The chromatographic pattern of endoglucanase digested beta-1,6-glucan provides a characteristic "fingerprint" of beta-1,6-glucan and the fine structure of the oligosaccharides in this pattern was determined by 1H NMR and electrospray ionization mass spectroscopy. The final enzymatic step uses laminarinase and beta-glucosidase to release the remaining beta-1,3-glucan. The cell wall mannan remains as a high MW fraction at the end of the fractionation procedure. Good sensitivity and correlation with cell wall composition determined by traditional methods were observed for wild-type and several cell wall mutants. SN - 0003-2697 UR - https://www.unboundmedicine.com/medline/citation/11811978/A_refined_method_for_the_determination_of_Saccharomyces_cerevisiae_cell_wall_composition_and_beta_16_glucan_fine_structure_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003269701954739 DB - PRIME DP - Unbound Medicine ER -