Figure 5 | Cell Research

Figure 5

From: Cloning and functional characterization of two cDNAs encoding NADPH-dependent 3-ketoacyl-CoA reductased from developing cotton fibers

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GhKCRs function as NADPH-dependent 3-ketoacyl-CoA reductase in ybr159wΔ mutant cells. (A) Schematic drawings of reactions from palmitoyl-CoA to stearoyl-CoA. (B) ybr159wΔ mutant yeast cells transformed with GhKCR1, 2 or 3 metabolized palmitoyl-CoA at the same rate as wild-type cells in the presence of both NADH and NADPH. (C) When NADPH was not included in the experiment, all cells metabolized palmitoyl-CoA very slowly in the same way as that of ybr159wΔ mutant. (D) 3-ketostearate was accumulated similarly in all cell lines if both NADPH and NADH were not added in the assay. All reactions were initiated by adding 50 μg of proteins extracted from wild-type (wt), ybr159wΔ or ybr159wΔ mutant cells expressing GhKCR1 or 2 (KCR1 or 2 respectively). For TLC analysis, the fatty acyl-CoA thioesters were extracted as free fatty acid. The assays were stopped at the indicated time and the fatty acids were extracted and separated by TLC. Positions labeled as 1, 2, 3, 4 and O denote 3-ketostearate, 3-hydroxystearate, trans-2-stearate, stearate and the origin of TLC respectively. Relative positions were determined by comparing the mobility with authentic standards as described 14.

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