A gel diffusion assay for quantification of pectin methylesterase activity

Bruce Downie, Lynnette M.A. Dirk, Kristen A. Hadfield, Thea A. Wilkins, Alan B. Bennett, Kent J. Bradford

Research output: Contribution to journalArticlepeer-review

90 Scopus citations

Abstract

Increased binding of ruthenium red to pectin as the number of methyl esters attached to the pectin decreases was used as the basis for a gel diffusion assay for pectin methylesterase (PME, EC 3.1.1.11) activity. The stained zone diameters resulting from the hydrolysis of 0.1% (w/v) 90% esterified pectin in an agarose gel by diffused, commercial PME were log- linear over 4 orders of magnitude, with a minimum detection limit of 3.6 pkatals. Pectin deesterification as the cause for a stained zone after PME incubation was confirmed when only 1 N NaOH, which will chemically deesterify the pectin, and not methanol or acid, the two products formed when PME acts on a methyl ester, resulted in the characteristic stained zone. The stained zone diameters decreased with increasing percentage of substrate esterification, were independent of pH, and were insensitive to simultaneous incubation with two forms of pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), or all combinations. PME extracted from tomato seeds, cotton fibers, and melon fruit showed pH optima of 6, 6, and 8, respectively. Using individual tomato seed parts, the assay was adapted to quantify diffusate activity and to localize activity in tissue prints. The sensitivity, specificity, and simplicity of this PME assay are superior to all others.

Original languageEnglish
Pages (from-to)149-157
Number of pages9
JournalAnalytical Biochemistry
Volume264
Issue number2
DOIs
StatePublished - Nov 15 1998

Bibliographical note

Funding Information:
We are grateful to Dr. John Labavitch, Department of Pomology, UC Davis, for the G12 PGA, and to Carl Greve, Department of Pomology, UC Davis, for the HPLC elution protocol for separating the same. JIM5 and JIM7 antibodies were kindly provided by Dr. Keith Roberts, John Innes Institute, UK. Drs. John Labavitch and Donald Nevins, Department of Vegetable Crops, UC Davis, reviewed a previous version of the manuscript and made many suggestions for its improvement. We thank Ms. Dana Ashford for her excellent assistance and diligence in determining the feasibility of this assay. Grants: T.A.W., Cotton Inc. Grant 92-815; A.B.B., NRICP/USDA Grant 9701534; and K.J.B., NSF Grants 9407264 and 9722798.

Funding Information:
1The work was conducted while B.D. was supported by a Natural Sciences and Engineering Research Council of Canada Post-Doctoral Scholarship. 2Both authors contributed equally to the work. 3To whom correspondence should be addressed. Fax: (530) 752-4554. E-mail: kjbradford@ucdavis.edu.

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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