[9] Fatty acid synthase from rabbit mammary gland

This chapter discusses the fatty acid synthase from rabbit mammary gland. The fatty acid synthase complex isolated from lactating rabbit mammary gland catalyzes the synthesis of saturated even-numbered fatty acids from acetyl-CoA, malonyl-CoA, and nicotinamide adenine dinucleotide phosphate (NADPH). The activity of purified fatty acid synthase and of the enzyme during purification can be measured via the oxidation of NADPH. Optimum assay conditions at 37° are 200 mM potassium phosphate buffer, pH 6.6, 1 mM dithiolthreitol, 1 mM EDTA, 0.24 mM NADPH, 30 μM acetyl-CoA, 40-50 μM malonyl-CoA, and enzyme protein to produce an absorbance change of 0.05–0.15 unit/minute in a final volume of 1.0 ml. After measuring NADPH oxidation without added acetyl-CoA and malonyl-CoA, the reaction is started by adding these substrates. The reaction rate should be linear with time for at least 3 minutes. One unit of enzymatic activity oxidizes 1 μmole of NADPH per minute. Mammary tissue (about 100 g wet weight) is obtained from a lactating rabbit 4–20 days postpartum. Fatty acid synthase with maximum specific activity is obtained from animals at 4 days postpartum. From 4 to 20 days postpartum there is a gradual decline in the specific activity of the enzyme isolated. The purified enzyme has a specific activity at 37° of 880 ± 30 mμmoles of NADPH oxidized per minute per milligram of protein (mean of 15 preparations ± S.E.M.).