Characterization and Properties of Different Glucosyltransferases Isolated from Suspension-Cultured Cells of Daucus carota

Abstract

Particulate enzymes (14,000 g pellet) from suspension-cultured carrot cells (Daucus carota L.) incorporated glucose from UDP-glucose and GDP-glucose into ethanol-insoluble products which were characterized as glucans or glucoprotein. Based on the test system to assay glucansynthe- tases I and II four different enzymatic activities could be distinguished on the basis of their substrate and divalent cation requirements, the influence of active substances such as nucleotides, nucleotide sugars, cellobiose, and in vivo inhibitors of cell wall glucan synthesis, their distribution in linear sucrose gradient and the nature of their products. The enzymatic activities which incor­porated glucose from UDP-glucose or GDP-glucose at low substrate concentrations (10 -6 ᴍ) were both localized in membranes of a density of 1.129 g em-3 (Golgi membranes) and synthesized a β-1,4-glucan chain. Both showed similar properties in most of the characterization experiments. The glucosyltransferase that catalysed the formation of a β-1,3-glucan from UDP-glucose (0.48 mᴍ) was found in membranes which accumulated at a density of 1.170 g · cm-3 (plasma membrane) and differed in its properties from the Golgi-localized glucosyltransferase activities in many aspects. A soluble glucosyltransferase (175,000 × g supernatant) which was also active at low concentrations of UDP-glucose (10-6 ᴍ) but showed enhanced activity under conditions where the other glucosyltransferases were inactive incorporated glucose into a proteinase-sensi­tive product. In linear sucrose gradients this enzyme migrated to different gradient densities depending on conditions.