Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis.
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-(14)C]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.
Division of Glycoscience, School of Biotechnology, Royal Institute of Technology, AlbaNova University Centre, Stockholm, Sweden.
Christian
C
Brown
Felicia
F
Leijon
Vincent
V
Bulone
D002241:Carbohydrates / Q000096:biosynthesis*; D002473:Cell Wall / Q000201:enzymology*; D016695:Glycosyltransferases / Q000032:analysis* / Q000378:metabolism; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008566:Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D010944:Plants; D011869:Radioligand Assay / Q000379:methods*
D002241:Carbohydrates; D009702:Nucleoside Diphosphate Sugars; D016695:Glycosyltransferases