Potassium Hydroxide → (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal

Participants

Reaction Steps

Portions of tissues (200-300 mg) were weighed and homogenized in 500 μl ice-cold 0.5 M perchloric acid using 6 mm ceramic beads on a tissue lyzer. Homogenates were centrifuged (10 min at 10,000), and resultant supernatants were neutralized (pH ˜7.4) with 5 M KOH plus 0.5 M triethanolamine hydrochloride with the help of pH paper and centrifuged (10 min at 10,000 g). Most of the procedures were performed at room temperature unlike suggested in the paper. A portion (200 μl) of the neutralized extract was reconstituted in 1 ml of Optiphase supermix and 200 μl of this radioactive mix was counted on a Microbeta unit to access total radioactivity (2-[3H]DG+2-[3H]DG-6-P). Another aliquot (200 μl) was mixed with 200 μl of 0.3 N Ba(OH)2 and 0.3 N ZnSO4. This mixture was centrifuged, and the supernatant was used to determine unphosphorylated 2-[3H]DG levels by using a portion (200 μl) of the neutralized extract was reconstituted in 1 ml of Optiphase supermix and 200 μl of this radioactive mix was counted on a Microbeta unit. Quench corrections were not made at this time. Tissue 2-[3H]DG-6-P was calculated as the difference between total and 2-[3H]DG values.

Provenance & Evidence