Figure 5.

Mannose 6-phosphate receptor mediated delivery and GAG storage reduction. (A) 80 activity units per ml rhIDU with Alexa Fluor 680 label (IDU-AF680) or unlabeled (IDU) were applied to confluent Hurler fibroblasts in six-well plates containing 1 ml of serum free MEM with or without competing 5 mM mannose 6-phosphate (M6P) for 4 hours. Enzymatic activity assays using 4-MUI substrate were performed on cell lysates and percent uptake was plotted relative to maximal uptake observed in the absence of inhibitor. (B) IDU or IDU-AF680 was applied (0.0–0.4 units/ml) to cultured Hurler fibroblasts in the presence of H₂³⁵SO₄ and incubated for 48 h at 37 °C and 5% CO2 as described in the methods and reported in detail previously [11]. Radioactive labeled GAG were isolated from harvested cell pellets by two rounds of ethanol extraction, acid solubilized, and quantified by scintillation counting. Radioactive counts per minute were normalized to total protein concentration in the extracted pellet and plotted as relative percent labeled GAG compared to the untreated samples at 0 units of enzyme applied.