Glucose 6 phosphate (g6p)

In vitro enzyme activity assays were performed as previously described^{76 (link)}. Briefly, for glucose 6-phosphate dehydrogenase activity, the following substrates were re-constituted in vitro in PPP activity buffer (50 mM Tris, 1 mM MgCl₂): 200 μM glucose 6-phosphate (Sigma) and 100 μM NADP + (Roche). In all, 10 μg of protein lysate from PDZK1IP1-V5 or EV expressing cells were added to the reaction mixture. Reduction of NADP + to NADPH was read by absorption at 340 nm on a spectrophotometer as a readout of glucose 6-phosphate dehydrogenase activity. For 6-phosphogluconate dehydrogenase activity, the following substrates were re-constituted in vitro in. PPP activity buffer: 200 μM 6-phosphogluconate (Sigma) and 100 $\mu$ M NADP + (Roche). 10 $\mu$ g of protein lysate from PDZK1IP1-V5 or EV expressing cells were added to the reaction mixture. Reduction of NADP + to NADPH was read by absorption at 340 nm on a spectrophotometer as a readout of 6-phosphogluconate dehydrogenase activity.

An in vitro heme oxygenase assay was performed according to a previous study (25 (link), 26 (link)). The following reagents were mixed and incubated at 25°C for 8 hours in a final volume of 100 μl: 5 μM recombinant AtHO1, 40 μM hemin (Tokyo Chemical Industry, catalog no. H0008), bovine serum albumin (60 μg/ml; Merck, catalog no. A9647), 4.6 μM ferredoxin (Merck, catalog no. F5875), ferredoxin-NADP⁺ reductase (0.025 U/ml; Merck, catalog no. F0628), 10 μM catalase (Merck, catalog no. 219261), 6.5 mM glucose-6-phosphate (Merck, catalog no. 10127647001), G6PD (Merck, catalog no. G7877), 0.82 mM NADP⁺ (Wako, catalog no. 308-50463), and 5 mM sodium ascorbate (Tokyo Chemical Industry, catalog no. A0539). These reagents were dissolved in 100 mM potassium phosphate buffer (pH 7.2) or DMSO according to the previous study (26 (link)). The reactions were stopped and their proteins removed by mixing water (reaction solution), methanol, and chloroform to a 0.9:1:1 ratio. Following centrifugation at 13,000g for 5 min, the upper layer was collected, dried, and then dissolved in 10 μl of DMSO. In Fig. 2E, biliverdin was added in place of AtHO1, hemin, and the indicated enzymes or coenzyme. The reaction products were mixed with an equal volume of 10 μM apoUnaG, and UnaG florescence was observed under blue light.

All HPLC grade solvents used in the extraction protocol and glucose-6-phosphate were obtained from Merck (Darmstadt, Germany). Ultrapure water was obtained using an Arium^®-pro ultrapure system (Sartorius, Goettingen, Germany). Dimethyl sulfoxide (99.9%), cortisone, hydrocortisone, carbenoxolone disodium salt, and glucose 6-phosphate dehydrogenase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mixed gender pooled human liver microsomes were purchased from Sekisui XenoTech (Kansas, KS, USA). NADP⁺/H was obtained from Cayman Chemical (Ann Arbor, MI, USA).

NADPH activity assay was described in our and other studies [18 (link), 37 ]. Briefly, after treatment of cells, the lysates were incubated with NADP-cycling buffer plus glucose-6-phosphate dehydrogenase (G6PD, Sigma) at 60°C for 30 min [18 (link)]. Afterwards, glucose 6-phosphate (G6P, Sigma) was added to the mixture, and the change in absorbance at 570 nm was measured every 30 s for 4 min at 30°C. The concentration of NADP+ was calculated by subtracting [NADPH] from [total NADP]. NADPH activity was then calculated through NADPH/ NADP+ [37 ].