Successive Glycosyltransfer Activity and Enzymatic Characterization of Pectic Polygalacturonate 4-α-Galacturonosyltransferase Solubilized from Pollen Tubes ofPetunia axillaris Using Pyridylaminated Oligogalacturonates as Substrates

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Abstract
Polygalacturonate 4-α-galacturonosyltransferase (pectin synthase) was solubilized from pollen tubes of Petunia axillaris and characterized. To accomplish this, an assay method using fluorogenic pyridylaminated-oligogalacturonic acids (PA-OGAs) as acceptor substrates was developed. When the pollen tube enzyme was solubilized with 0.5% (v/v) Triton X-100 and was incubated with PA-OGA and UDP-galacturonic acid (UDP-GalUA), successive transfer activity of more than 10 GalUAs from UDP-GalUA to the nonreducing end of PA-OGA was observed by diethylaminoethyl high-performance liquid chromatography. This activity was time- and enzyme concentration-dependent. The optimum enzyme activity was observed at pH 7.0 and 30°C. Among the PA-OGAs investigated, those with a degree of polymerization of more than 10 were preferred as substrates. The crude pollen tube enzyme had an apparentK m value of 13 μm for the PA-OGA with a degree of polymerization 11 and 170 μm for UDP-GalUA. The characteristics of the P. axillarispollen tube enzyme and the usefulness of fluorogenic PA-OGAs for the assay of this enzyme are discussed.