Figure 5
From: Sulfation of sialic acid is ubiquitous and essential for vertebrate development
In vitro SulT-Sia activity of Wscd1 and Wscd2. (a) TLC of the products from the GM1 substrate incubated with the Wscd1, Wscd2, and Mock enzyme fractions, visualized by the orcinol reagent. The product P detected in the Wscd1 lane is marked. The bands denoted by asterisks are GM1. (b) Fluorometric HPLC of the product P in the Wscd1 lane in a (Wscd1 + GM1) and the same area in the Mock lane in a (Mock + GM1). Authentic: Neu5Ac, Neu5Ac8S, and Neu5Ac9S; Wscd1 + GM1 + Neu5Ac8S: Co-injection of the DMB-derivatives in “Wscd1 + GM1” with DMB-Neu5Ac8S. (c) SDS-PAGE/western blotting of the reaction mixtures containing the transferrin (TF) substrate and enzyme fractions using 3G9 (upper panel). Lane 1: TF with Mock + PAPS; lane 2: TF with Wscd1 + PAPS; lane 3: Wscd1 + PAPS; lane 4: TF with Wscd1; lane 5: TF with Wscd2 + PAPS; lane 6: TF with Wscd2; lane 7: Wscd1 + PAPS; lane 8: TF. Lower panel, CBB staining for TF in lanes 1–7 in the upper panel. The arrowhead at 78 kDa indicates TF; (d) Fluorometric HPLC profiles of DMB-derivatives for the TF-derived product by Wscd2 (Wscd2 + TF). Authentic: Neu5Ac, and Neu5Ac8S; Mock + TF: TF incubated with Mock; Wscd2 + TF + Neu5Ac8S: Co-injection of the DMB-derivatives in “WSCD1 + TF” with DMB-Neu5Ac8S. The arrows in (b) and (d) denote the elution position of DMB-Neu5Ac8S.
