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Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis

Nature Protocols volume 7, pages 1634–1650 (2012)Cite this article

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Abstract

Most of the glycosyltransferases (GTs) that catalyze the formation of plant cell wall carbohydrates remain to be biochemically characterized. This can be achieved only if specific assays are available for these enzymes. Here we present a protocol for in vitro assays of processive and nonprocessive membrane-bound GTs. The assays are either based on the use of radioactive nucleotide sugars (NDP sugars; e.g., UDP-[U-14C]glucose) and the quantification of the radiolabeled monosaccharides incorporated into soluble or insoluble carbohydrates, or on the coupling of the GT reaction with that of pyruvate kinase (PK) and the oxidation of NADH by lactate dehydrogenase (LDH). The radiometric assays are more suitable for exploratory work on poorly characterized enzymes, whereas the spectrophotometric assays require the availability of highly enriched GTs. Both assays can be performed within 1 d, depending on the number of fractions to be assayed or reaction mixtures to be tested.

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Figure 1: Radiometric assays.
Figure 2: Schematic representation of the protocol used for spectrophotometric assays.
Figure 3: Typical data obtained with a detergent-extracted plant GT using the radiometric assay.

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  1. Division of Glycoscience and Swedish Centre for Biomimetic Fiber Engineering, School of Biotechnology, Royal Institute of Technology, AlbaNova University Centre, Stockholm, Sweden

    Christian Brown, Felicia Leijon & Vincent Bulone

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Contributions

V.B. wrote the manuscript, and C.B. and F.L. performed complementary optimization experiments. C.B. contributed to the writing of the experimental section and the preparation of the figures. All authors checked the accuracy of the experimental details.

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Correspondence to Vincent Bulone.

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Brown, C., Leijon, F. & Bulone, V. Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis. Nat Protoc 7, 1634–1650 (2012). https://doi.org/10.1038/nprot.2012.089

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