Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans

Yang, H-C; Chen, T-L; Wu, Y-H; Cheng, K-P; Lin, Y-H; Cheng, M-L; Ho, H-Y; Lo, S J; Chiu, D T-Y

G6PD activity assay. The G6PD activity of adult C. elegans was assayed spectrophotometrically at 340 nm by the reduction of NADP þ in the presence of glucose 6-phosphate as previously described with modification. 11 In brief, staged first-day adults were harvested from RNAi NGM plate with washing buffer (1X PBS supplemented with 0.1% Tween 20) followed by washing and pelleting twice to remove bacteria. The worms were resuspended in extraction buffer (20 mM Tris-HCl, pH 8.0, 3 mM magnesium chloride, 1 mM EDTA, 0.02% b-mercaptoethanol, 1 mM e-aminocaproic acid and 0.1% Triton X-100). The worm suspension was chilled immediately on ice and disrupted by sonication (Amplitude: 10%, pulse: 2 s with 5-s intervals for 20 cycles) (VCX400, Sonics and Materials, Danbury, CT, USA). The crude lysates were centrifuged at 12 000r.p.m. for 15 min at 4 1C (Centrifuge 5417R, Eppendorf, Hamburg, Germany) and the supernatants (protein-containing lysate) were collected. Protein concentration of the lysate was determined by Bradford method (Bio-Rad, Hercules, CA, USA). A typical assay mixture consisted of 100 mg of protein lysate in 1 ml of assay buffer (50 mM Tris-HCl pH 8, 50 mM MgCl 2 , 4 mM NADP þ , 4 mM glucose 6-phosphate). The change of absorbance at 340 nm in each sample was measured spectrophotometrically for 15 min at 37 1C.