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-[(14)C]galactose resulted in a radioactive product insoluble in 70% methanol. The product released only [(14)C]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, [(14)C]galactose was also incorporated into proteins, however these galactoproteins were not a substrate for Aspergillus niger, endo-l,4-beta-galactanase. Thus, the majority of the (14)C-labelled product was I,4-beta-galactan. Compounds released by the endo-l,4-beta-galactanase treatment were mainly [(14)C]galactose and [(14)C]galactobiose, indicating that the synthesized 1,4-beta-galactan was longer than a trimer. In vitro synthesis of I,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 Mn(2+) Aspergillus aculeatus rhamnogalacturonase A digested at least 50% of the labelled product to smaller fragments of approx. 14 kDa, suggesting that the synthesized [(14)C]galactan was attached to the endogenous rhamnogalacturonan I. When rhamnogalacturonase A digests of the labelled product were subsequently treated with endo-l,4-beta-galactanase, radioactivity was not only found as [(14)C]galactose or [(14)C]galactobiose but also as larger fragments. The larger fragments were likely the [(14)C]galactose or [(14)C]galactobiose still attached to the rhamnogalacturonan backbone since treatment with beta-galactosidase together with endo-l,4-beta-galactanase digested all radioactivity to the fraction eluting as [(14)C]galactose. The data indicate that the majority of the [(14)C]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.