Stable isotope labelling method for studies of saccharide metabolism in Agardhiella subulata

Stable isotopes are preferable in many ways to radioactive isotopes for metabolic studies designed to elucidate biosynthetic pathways. We have developed the methodology to utilize ¹³C-labelled compounds in tracer studies of saccharide metabolism in the red algae. Cultures of Agardhiella subulata were pulse-chase labelled with ¹³C0₂ and ¹²C0₂. Gas chromatography/mass spectrometry (GC-MS) and ¹³C-NMR provided for positive identification of labelled carbohydrate metabolites. In addition, GC-MS can be used to profile the monosaccharide composition of algal species and combined GC-MS and ¹³C-NMR can disclose which carbon(s) is (are) labelled and the extent of labelling. In ¹³C0₂ incubated plants, the label is clearly detected in floridoside and floridean starch. After chasing the labelled alga with ¹²CO₂ for three days or storing the pulse-chase labelled alga in darkness for 6 days, labels disappeared from both floridoside and starch and the contents of these two carbohydrates became very low. More detailed biochemical analysis is being continued to identify labelled cell wall polysaccharides and/or their precursors.