HA synthesis assays

Membranes containing SeHAS were prepared and assayed for HAS activity (Weigel et al. 2013) to determine specific activities either using UDP-[¹⁴C]GlcUA (Tlapak-Simmons et al. 1999b; Kumari and Weigel 2005) or by integration of refractive index values obtained from SEC-MALLS analyses of non-radiolabeled samples (Baggenstoss and Weigel 2006; Weigel and Baggenstoss 2012). Final volumes for the radioactive or MALLS assay samples were 0.1 and 0.5–1.0 mL, respectively. Membranes were suspended by brief sonication at 4°C and added to 2 mL microcentrifuge tubes to a final concentration of ≤2 μg membrane protein per 0.1 mL assay volume (except the nearly inactive ΔCter and K398X mutants, which required 5–20 times more protein to obtain sufficient HA). Assay buffer contained 18.75 mM sodium phosphate and 6.25 mM potassium phosphate, pH 7.0, 75 mM NaCl, 2% (v/v) glycerol, 0.1 mM EDTA, 1.0 mM DTT, 0.1 mM PMSF, 1.0 μM pepstatin, and 2 μM leupeptin. UDP-GlcUA and UDP-GlcNAc were added to 1 mM final concentrations, the mixture was incubated at 30°C for 10 min. For MALLS (HA size) assays, calf intestinal alkaline phosphatase was added (Baggenstoss and Weigel 2006) to a final concentration of 0.02 U/μL. For radiolabel assays, UDP-[¹⁴C]GlcUA was also added to 0.7 μM.

The HA synthesis reactions were then initiated by the addition of MgCl₂ to a final concentration of 20 mM and incubated for either 1 h (for radiolabel assays) or 4 h (for MALLS assays) at 30°C in a vibrating Taitec mixer (San Jose, CA). For MALLS assays, synthesis was terminated by adding EDTA and UDP to final concentrations of 40 mM and 10 mM, respectively, chilling on ice for ~20 min and then heating in a 100°C sand bath (for 1 min/0.1 mL) to inactivate HAS. Radiolabeled assay samples were quenched by adding SDS to 2% (w/v) and then analyzed by descending paper chromatography (DeAngelis and Weigel 1994; Weigel et al. 2013). The synthesis of HA was monitored over time to ensure that the final HA size made by each mutant was not limited by the incubation time. In all cases, steady-state size ranges were reached within 2–4 h in vitro.