In vitro biosynthesis of 1,4,-beta-galactan attached to rhamnogalacturonanI

The biosynthesis of galactan was investigated using microsomal membranes is olated from suspension-cultured cells of potato (Solanum tuberosum L. var. AZY). Incubation of the microsomal membranes in the presence of UDP-[C-14]g alactose resulted in a radioactive product insoluble in 70% methanol. The p roduct released only [C-14]galactose upon acid hydrolysis. Treatment of the product with Aspergillus niger endo-1,4-beta-galactanase released 65-70% o f the radioactivity to a 70%-methanol-soluble fraction. To a minor extent, [C-14]galactose was also incorporated into proteins, however these galactop roteins were not a substrate for Aspergillus niger, endo-l,4-beta-galactana se. Thus, the majority of the C-14-labelled product was I,4-beta-galactan. Compounds released by the endo-l,4-beta-galactanase treatment were mainly [ C-14]galactose and [C-14]galactobiose, indicating that the synthesized 1,4- beta-galactan was longer than a trimer. In vitro synthesis of I,4-beta-gala ctan was most active with 6-d-old cells, which are in the middle of the lin ear growth phase. The optimal synthesis occurred at pH 6.0 in the presence of 7.5 mM Mn2+ Aspergillus aculeatus rhamnogalacturonase A digested at leas t 50% of the labelled product to smaller fragments of approx. 14 kDa, sugge sting that the synthesized [C-14]galactan was attached to the endogenous rh amnogalacturonan I. When rhamnogalacturonase A digests of the labelled prod uct were subsequently treated with endo-l,4-beta-galactanase, radioactivity was not only found as [C-14]galactose or [C-14]galactobiose but also as la rger fragments. The larger fragments were likely the [C-14]galactose or [C- 14]galactobiose still attached to the rhamnogalacturonan backbone since tre atment with beta-galactosidase together with endo-l,4-beta-galactanase dige sted all radioactivity to the fraction eluting as [C-14]galactose. The data indicate that the majority of the [C-14]galactan was attached directly to the rhamnose residues in rhamnogalacturonan I. Thus, isolated microsomal me mbranes contain enzyme activities to both initiate and elongate 1,4-beta-ga lactan sidechains in the endogenous pectic rhamnogalacturonan I.