In vitro-translated PRN1 has quercetinase activity.

A. in-vitro-translated PRN1 possesses quercetinase activity.. In vitro-translated PRN1 was incubated with a final concentration of 10 μM quercetin in a 500 μL total reaction volume, or as a control, 10 μM quercetin alone, in buffer as described [12]. Reactions were carried out in darkness in a 26°C water bath for 15 min. After incubation at 26°C, absorption spectra of quercetin alone (Quercetin only), Quercetin+PRN1, Quercetin+protein extract, or PRN1 extract only were determined using a Perkin Elmer scanning spectrophotometer (Perkin Elmer; Waltham, MA). n = 3, SD shown. B. The conformational status of GPA1 affects the quercetinase ability of PRN1.. In vitro-translated GPA1 was pre-incubated with non-hydrolyzable analogs (either GTPγS or GDPβS) overnight at 10°C, then was incubated with PRN1, then (PRN1+GPA1+GTPγS or PRN1+GPA1+GDPβS) was added to buffer+quercetin to assess the effects on quercetinase activity as shown. Absorption spectra of quercetin alone (Quercetin only) also shown. n = 3, SD shown.