Changes in Acetyl CoA Levels during the Early Embryonic Development of Xenopus laevis
Figure 2
Validation of the identity and purity of CoASH and acetyl CoA peaks detected in Xenopus embryo extracts.
(A) 30 stage 8/9 embryos were extracted with 150 µl 5% PCA and the PCA soluble fraction was neutralised with TEA/K2CO3. CoA compounds in neutralised PCA extracts were analysed by HPLC before and after KOH treatment as described in the Materials and Methods section. KOH treatment caused conversion of peaks corresponding to succinyl CoA and acetyl CoA, to free CoASH. Retention times (in minutes): CoASH, 5.15; succinyl CoA, 12.13; acetyl CoA, 15.17. (B) 30 stage 8/9 embryos were extracted with 150 µl 5% PCA and the PCA soluble fraction was neutralised with TEA/K2CO3. The neutralised extract was analysed by HPLC without further treatment (top panel), or after incubation for 10 min at 30°C with 0.11 mM oxaloacetate (OA) and citrate synthase (CS) (middel panel), or with 60 mM KCl, 9.4 mM acetyl phosphate (AP), and phosphotransacetylase (PTA) (bottom panel). Incubation of Xenopus extract with citrate synthase, in the presence of oxaloacetate, caused complete disappearance of the peak corresponding to acetyl CoA and an increase in the size of the peak corresponding to CoASH (peak areas before treatment: CoASH, 9337.25; succinyl CoA, 11626; acetyl CoA, 6678. Peak areas after treatment: CoASH, 18217.5; succinyl CoA, 11252.25; acetyl CoA, no peak detected). Conversely, incubation with phosphotransacetylase, in the presence of acetyl phosphate, completely removed the peak corresponding to CoASH and increased the size of the acetyl CoA peak (peak areas after treatment: CoASH, not detected; succinyl CoA, 9278.5; acetyl CoA, 15213.5). These results confirm unequivocally the identity of the CoASH and acetyl CoA peaks and that there are no other compounds co-eluting with these peaks that absorb at 254 nm. Retention times in minutes: top panel; CoASH, 5.17; succinyl CoA, 12.26; acetyl CoA, 15.51; middle panel; CoASH, 5.24; succinyl CoA, 12.43; acetyl CoA, not detected; bottom panel; CoASH, not detected; succinyl CoA, 12.12; acetyl CoA, 15.36.
