Characterisation and immunolocation of an 87 kDa polypeptide associated with UDP-glucuronic acid decarboxylase activity from differentiating tobacco cells (Nicotiana tabacum L.).
UDP-glucuronic acid decarboxylase catalyses the reaction responsible for the formation of UDP-xylose and commits assimilate for the biosynthesis of cell wall polysaccharides and glycosylation of proteins. Xylose-rich polymers such as xylans are a feature of dicot secondary walls. Thus a cell culture system of tobacco transformed with the ipt gene from Agrobacterium tumefaciens for cytokinin production and which when manipulated with auxin and sucrose leads to induction of xylogenesis, has been used as a source for purification of the enzyme. UDP-glucuronic acid decarboxylase was purified by ion-exchange, gel filtration and affinity chromatography on Reactive Brown-Agarose. The native enzyme had an apparent M(r) of 220,000 which yielded a single subunit of 87,000 when analysed on SDS-PAGE using silver staining. This appears to be a novel form of the enzyme since a gene family encoding polypeptides around M(r) 40,000 with homology to the fungal enzyme also exists in plants. Using an antibody raised to the native 87 kDa form of the enzyme, this decarboxylase was localised mainly to to cambium and differentiating vascular tissue in tobacco stem, consistent with a role in the provision of UDP-xylose for the synthesis of secondary wall xylan. Further analysis using immunogold electron microscopy localised the 87 kDa UDP-glucuronic acid decarboxylase to the cytosol of developing vascular tissue.
Wheatley
Edward R
ER
School of Biological Sciences, Royal Holloway and Bedford New College, University of London, Surrey, Egham, UK.
Davies
Dewi R
DR
Bolwell
G Paul
GP
D000906:Antibodies / Q000737:chemistry; D002262:Carboxy-Lyases / Q000276:immunology / Q000378:metabolism*; D002461:Cell Line, Transformed; D002852:Chromatography, Ion Exchange; D046508:Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D017979:Gold Colloid; D007150:Immunohistochemistry / Q000379:methods; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011233:Precipitin Tests; D013194:Staining and Labeling; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology* / Q000235:genetics
D000906:Antibodies; D017979:Gold Colloid; D010455:Peptides; D010940:Plant Proteins; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase