FIG. 7.

The tsc13 mutant cells have normal condensation activity but are deficient in total fatty acid chain elongation. (a) Fatty acid elongation activities in wild-type (wt) and tsc13 mutant microsomes were compared using C₁₆-, C₁₈-, and C₂₀-CoAs as substrates by measuring the incorporation of radiolabeled malonyl-CoA into hexane-extractable fatty acids. The assays were conducted in the absence of NADPH or NADH to measure condensation activity and in the presence of NADPH or NADH to measure total elongation. Activities are normalized to the overall activity measured with wild-type microsomes, using C₂₀-CoA as the substrate. ND, not determined. The assays were conducted in triplicate, and the results were averaged. The variation was less than 7%. (b) The elongation assays were conducted as for panel a, using C₁₆-CoA as the substrate and either wild-type (left) or tsc13 mutant (right) microsomes. The reactions were stopped at the indicated times, and the fatty acids were extracted and separated by TLC. The reactions were conducted with (lanes 1 to 6) or without (lanes 7 to 12) NADPH or NADH. The positions of the 3-ketostearate (3-Keto), stearate, trans-2,3-stearate (Trans-2,3), and 3-hydroxystearate (3-Hydroxy) intermediates were determined by running the standards on the TLC plate and charring after exposure to PhosphorImager screens.