Arabinoxylan biosynthesis in wheat. Characterization of arabinosyltransferase activity in Golgi membranes.
Arabinoxylan arabinosyltransferase (AX-AraT) activity was investigated using microsomes and Golgi vesicles isolated from wheat (Triticum aestivum) seedlings. Incubation of microsomes with UDP-[(14)C]-beta-L-arabinopyranose resulted in incorporation of radioactivity into two different products, although most of the radioactivity was present in xylose (Xyl), indicating a high degree of UDP-arabinose (Ara) epimerization. In isolated Golgi vesicles, the epimerization was negligible, and incubation with UDP-[(14)C]Ara resulted in formation of a product that could be solubilized with proteinase K. In contrast, when Golgi vesicles were incubated with UDP-[(14)C]Ara in the presence of unlabeled UDP-Xyl, the product obtained could be solubilized with xylanase, whereas proteinase K had no effect. Thus, the AX-AraT is dependent on the synthesis of unsubstituted xylan acting as acceptor. Further analysis of the radiolabeled product formed in the presence of unlabeled UDP-Xyl revealed that it had an apparent molecular mass of approximately 500 kD. Furthermore, the total incorporation of [(14)C]Ara was dependent on the time of incubation and the amount of Golgi protein used. AX-AraT activity had a pH optimum at 6, and required the presence of divalent cations, Mn(2+) being the most efficient. In the absence of UDP-Xyl, a single arabinosylated protein with an apparent molecular mass of 40 kD was radiolabeled. The [(14)C]Ara labeling became reversible by adding unlabeled UDP-Xyl to the reaction medium. The possible role of this protein in arabinoxylan biosynthesis is discussed.
Porchia
Andrea Celia
AC
Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, 1871 Frederiksberg C, Copenhagen, Denmark.
Sørensen
Susanne Oxenbøll
SO
Scheller
Henrik Vibe
HV
D002250:Carbon Radioisotopes; D002850:Chromatography, Gel; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D010430:Pentosyltransferases / Q000378:metabolism*; D014908:Triticum / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D014539:Uridine Diphosphate Sugars / Q000494:pharmacology; D014990:Xylans / Q000096:biosynthesis*; D014994:Xylose / Q000378:metabolism
D002250:Carbon Radioisotopes; D014539:Uridine Diphosphate Sugars; D014990:Xylans; C040817:uridine diphosphate arabinose; C085118:arabinoxylan; D014994:Xylose; D010430:Pentosyltransferases; C101967:arabinosyltransferase