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Planta

, Volume 210, Issue 4, pp 622–629 | Cite as

In vitro biosynthesis of 1,4-β-galactan attached to rhamnogalacturonan I

  • Naomi Geshi
  • Bodil Jørgensen
  • Henrik V. Scheller
  • Peter Ulvskov
Article

Abstract.

 The biosynthesis of galactan was investigated using microsomal membranes isolated from suspension-cultured cells of potato (Solanum tuberosum L. var. AZY). Incubation of the microsomal membranes in the presence of UDP-[14C]galactose resulted in a radioactive product insoluble in 70% methanol. The product released only [14C]galactose upon acid hydrolysis. Treatment of the product with Aspergillus niger endo-1,4-β-galactanase released 65–70% of the radioactivity to a 70%-methanol-soluble fraction. To a minor extent, [14C]galactose was also incorporated into proteins, however these galactoproteins were not a substrate for Aspergillus niger endo-1,4-β-galactanase. Thus, the majority of the 14C-labelled product was 1,4-β-galactan. Compounds released by the endo-1,4-β-galactanase treatment were mainly [14C]galactose and [14C]galactobiose, indicating that the synthesized 1,4-β-galactan was longer than a trimer. In vitro synthesis of 1,4-β-galactan was most active with 6-d-old cells, which are in the middle of the linear growth phase. The optimal synthesis occurred at pH 6.0 in the presence of 7.5 mM Mn2+. Aspergillus aculeatus rhamnogalacturonase A digested at least 50% of the labelled product to smaller fragments of approx. 14 kDa, suggesting that the synthesized [14C]galactan was attached to the endogenous rhamnogalacturonan I. When rhamnogalacturonase A digests of the labelled product were subsequently treated with endo-1,4-β-galactanase, radioactivity was not only found as [14C]galactose or [14C]galactobiose but also as larger fragments. The larger fragments were likely the [14C]galactose or [14C]galactobiose still attached to the rhamnogalacturonan backbone since treatment with β-galactosidase together with endo-1,4-β-galactanase digested all radioactivity to the fraction eluting as [14C]galactose. The data indicate that the majority of the [14C]galactan was attached directly to the rhamnose residues in rhamnogalacturonan I. Thus, isolated microsomal membranes contain enzyme activities to both initiate and elongate 1,4-β-galactan sidechains in the endogenous pectic rhamnogalacturonan I.

Key words: Galactan – Galactosyltransferase – Pectin – Rhamnogalacturonan I –Solanum tuberosum 

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Copyright information

© Springer-Verlag Berlin Heidelberg 2000

Authors and Affiliations

  • Naomi Geshi
    • 1
  • Bodil Jørgensen
    • 1
  • Henrik V. Scheller
    • 2
  • Peter Ulvskov
    • 1
  1. 1.Biotechnology Group, Danish Institute of Agricultural Sciences, Thorvaldsensvej 40, 1., 1871 Frederiksberg C, DenmarkDK
  2. 2.Department of Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 4., 1871 Frederiksberg C, DenmarkDK

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