Solubilization of an arabinan arabinosyltransferase activity from mung bean hypocotyls.
The biosynthesis of polysaccharides destined for the plant cell wall and the subsequent assembly of the cell wall are poorly understood processes that are currently the focus of much research. Arabinan, a component of the pectic polysaccharide rhamnogalacturonan I, is composed of arabinosyl residues connected via various glycosidic linkages, and therefore, the biosynthesis of arabinan is likely to involve more than one arabinosyltransferase. We have studied the transfer of [(14)C]arabinose (Ara) from UDP-L-arabinopyranose onto polysaccharides using microsomal membranes isolated from mung bean (Vigna radiata) hypocotyls. [(14)C]arabinosyl and [(14)C]xylosyl residues were incorporated into endogenous products due to the presence of UDP-Xyl-4-epimerase activity. Enzymatic digestion of endogenous products with endo-arabinanase released very little radiolabeled sugars, whereas digestion with arabinofuranosidase released some [(14)C]Ara. Microsomal membranes solubilized with the detergent octyl glucoside were able to add a single [(14)C]Ara residue onto (1-->5)-linked alpha-L-arabino-oligosaccharide acceptors. The reaction had a pH optimum of 6.5 and a requirement for manganese ions. However, enzymatic digestion of the radiolabeled oligosaccharides with endo-arabinanase and arabinofuranosidases could not fully release the radiolabeled Ara residue, indicating that the [(14)C]Ara residue was not a (1-->2)-, (1-->3)-, or (1-->5)-linked alpha-L-arabinofuranosyl residue. Rather, mild acid treatment of the product suggested that the radiolabeled Ara residue was in a pyranose conformation, and this result was confirmed by thin-layer chromatography of radiolabeled partially methylated sugars. Using microsomal membranes separated on a discontinuous sucrose gradient, the arabinosyltransferase activity appears to be mainly localized to Golgi membranes.
Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, 1871 Frederiksberg C, Copenhagen, Denmark.
Kylie Joy
KJ
Nunan
Henrik Vibe
HV
Scheller
D001089:Arabinose / Q000378:metabolism; D002250:Carbon Radioisotopes; D002473:Cell Wall / Q000378:metabolism; D004789:Enzyme Activation; D007887:Fabaceae / Q000737:chemistry / Q000201:enzymology*; D006056:Golgi Apparatus / Q000378:metabolism; D006156:Guanosine Diphosphate Sugars / Q000378:metabolism; D018546:Hypocotyl / Q000201:enzymology*; D008861:Microsomes / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D010430:Pentosyltransferases / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis; D014994:Xylose / Q000378:metabolism
D002250:Carbon Radioisotopes; D006156:Guanosine Diphosphate Sugars; D009844:Oligosaccharides; D011134:Polysaccharides; C030080:araban; C088421:guanidine diphosphate-arabinopyranose; D014994:Xylose; D001089:Arabinose; D010430:Pentosyltransferases; C101967:arabinosyltransferase