Hexokinase

C48/80, ketotifen (KET), 3,3',5,5'-tetramethylbenzidine (TMB), methyl serotonin, and amyloglucosidase were purchased from Sigma-Aldrich Japan (Tokyo, Japan); NPC 14686 (NPC) from Funakoshi Co. (Tokyo, Japan); NADP⁺, ATP, glucose 6-phosphate dehydrogenase, and hexokinase from Oriental Yeast Co. (Tokyo, Japan); l-ascorbic acid (reduced form), RRR-α-tocopherol (Toc) and RRR-δ-toc which were used as the authentic standard and the external standard, respectively, glycogen, 2,2'-dipyridyl, 2-thiobarbituric acid (TBA), GSH, ethylendiaminetetraacetic acid (EDTA), gum Arabic, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phthalaldehyde, tetramethoxypropane, trichloroacetic acid (TCA), and other chemicals from Wako Pure Chem. Ind. (Osaka, Japan). All chemicals were used without further purification.

Small-scale β-glucans were measured according to Hirokawa et al. (2008) (link)’s method with a slight modification. Briefly, cells in 2 mL of cultures at 3 days after RNAi were harvested by centrifugation (18,500 × g) and then suspended in 300 μL of the McIlvain buffer (pH6.0). After sonication, methanol was added to the suspension, and the mixture was incubated at 4°C for more than 4 h. After centrifugation, the β-glucan-containing pellet was dried up and then solved in 25 μL of the McIlvain buffer. The β-glucan was converted to glucose by treatment using westase (TaKaRa Bio), and the total glucose content was determined using hexokinase (Oriental Yeast Co., Ltd, Japan) and glucose-6-phosphate dehydrogenase (Roche Diagnostics K.K., Germany), according to Bergmeyer et al. (1974) . westase is an enzyme cocktail, including β-1,6-glucanase and β-1,3-glucanase, for yeast protoplast preparation, and it is demonstrated to completely degrade the β-glucan of P. haptonemofera into glucose (Hirokawa et al., 2008 (link)).

Cells in about 10 ml of a culture were harvested by centrifugation (2,800 × g, 10 min, 4°C) and suspended in 100% ethanol. After vigorous vortexing, the suspension was centrifuged (15,000 × g, 10 min, 4°C), and then the pellet was dried up. The starch-containing pellet was solved in 1 ml of 0.2 M KOH by sonication, and then by boiling (for 30 min). The α-glucans were converted to glucose by treatment with glucoamylase (Seikagakukogyo, Tokyo, Japan), and the total glucose content was determined using hexokinase (Oriental Yeast Co., Ltd., Tokyo, Japan) and glucose-6-phosphate dehydrogenase (Roche Diagnostics K.K., Mannheim, Germany) according to Bergmeyer et al. (1974) . Specifically, the glucose concentration in the glucoamylase-treated suspension was determined as follows: The suspension was centrifuged (15,000 × g, 10 min, 4°C), and then 150 μl each of the supernatant was placed into two holes of a microtiter plate, including 100 μl of NADP⁺-containing buffer [150 mM HEBES-NaOH (pH 7.4), 1 mM MaSO₄, 0.3 mM NADP⁺, 1 mM ATP-Na₂] with and without the enzymes (0.1 μl each). After 30 min-incubation, the absorbance at 340 nm was measured, and the glucose concentration was determined from the difference of the absorbance between with and without the enzymes.