Figure 6.

Analysis of the GALS1 Reaction Product.

(A) Radiolabeled product generated with endogenous acceptors and 82 µM UDP-¹⁴C-Gal (6.3 kBq) was treated with 2.4 or 60 milliunits (mU) of endo-β-1,4-galactanase and separated into digested material and polymeric pellet. The graph shows the percentage of total radioactivity (dpm) that was soluble in 70% ethanol after the digestion. A major part of the product is β-1,4-galactan.

(B) Radiolabeled product as in (A) digested with endo-β-1,4-galactanase (+) for 4 h was analyzed by TLC and phosphorimaging. The control reaction was incubated with buffer (−). Galactose, β-1,4-galactobiose (Gal2) and β-1,4-galactopentaose (Gal5) were used as migration standards.

(C) Radiolabeled product was generated with exogenous β-1,4-galactopentaose by incubating for 1 or 3 h with 5 µg microsomal proteins and 0.34 mM UDP-¹⁴C-Gal (7.4 kBq). The products were chromatographed by TLC and phosphorimaging. The amount of Gal incorporated per acceptor molecule was calculated by scintillation counting of replicate samples and is written below each lane.

(D) Radiolabeled product was generated with exogenous β-1,4-galactopentaose by incubation for 1 h with 0.27 mM UDP-¹⁴C-Gal (14.8 kBq) and 2 µg galactopentaose acceptor. Aliquots were digested with endo-β-1,4-galactanase or exo-β-1,4-galactosidase, subjected to TLC, and analyzed by phosphorimaging.