title: Successive glycosyltransfer activity and enzymatic characterization of pectic polygalacturonate 4-alpha-galacturonosyltransferase solubilized from pollen tubes of Petunia axillaris using pyridylaminated oligogalacturonates as substrates.
abstract: Polygalacturonate 4-alpha-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 degrees 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 apparent K(m) value of 13 microM for the PA-OGA with a degree of polymerization 11 and 170 microM for UDP-GalUA. The characteristics of the P. axillaris pollen tube enzyme and the usefulness of fluorogenic PA-OGAs for the assay of this enzyme are discussed.
authors.affiliation: Graduate School of Science, Osaka University, 1-1 Machikaneyamacho, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Akita
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ishimizu
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Tsukamoto
authors.forename: Toshio
authors.initials: T
authors.lastname: Ando
authors.forename: Sumihiro
authors.initials: S
authors.lastname: Hase
mesh_terms: D002412:Cations / Q000494:pharmacology; D014453:Glucuronosyltransferase; D016695:Glycosyltransferases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D006603:Hexuronic Acids / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009844:Oligosaccharides / Q000378:metabolism; D010368:Pectins / Q000096:biosynthesis; D010940:Plant Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011058:Pollen / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D019657:Solanaceae / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism
chemical_list: D002412:Cations; D006603:Hexuronic Acids; D009844:Oligosaccharides; D010940:Plant Proteins; C412763:oligogalacturonic acid; C007819:galacturonic acid; D010368:Pectins; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C438373:alpha-1,4-galacturonosyltransferase