In vitro biosynthesis of 1,4-beta-galactan attached to rhamnogalacturonan I.
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-beta-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-beta-galactanase. Thus, the majority of the 14C-labelled product was 1,4-beta-galactan. Compounds released by the endo-1,4-beta-galactanase treatment were mainly [14C]galactose and [14C]galactobiose, indicating that the synthesized 1,4-beta-galactan was longer than a trimer. In vitro synthesis of 1,4-beta-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-beta-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 beta-galactosidase together with endo-1,4-beta-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-beta-galactan sidechains in the endogenous pectic rhamnogalacturonan I.
Geshi
N
N
Biotechnology Group, Danish Institute of Agricultural Sciences, Frekeriksberg. n.geshi@dias.kvl.dk
Jørgensen
B
B
Scheller
H V
HV
Ulvskov
P
P
D002478:Cells, Cultured; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D005685:Galactans / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D010368:Pectins / Q000737:chemistry / Q000378:metabolism*; D011198:Solanum tuberosum / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism*
C411147:1,4-beta-galactan; D005685:Galactans; C042491:rhamnogalacturonan I; D010368:Pectins; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C094192:arabinogalactan endo-1,4-beta-galactosidase