[7a] Fatty acid synthase from chicken liver

Publisher Summary This chapter describes a procedure routinely used in laboratory for the isolation of fatty acid synthase (FAS) from chicken livers. Spectrophotometric and radioactive assays can be used to measure fatty acid synthase activity based on oxidation of nicotinamide adenine dinucleotide phosphate (NADPH), or incorporation of radioactive acetyl- or malonyl-CoA, respectively. Assay of the enzyme complex in crude homogenates, especially those derived from livers of starved animals or from tissues containing low enzyme activity or high NADPH oxidase levels, are usually performed by the radioactive procedure because of its high sensitivity and direct measure of palmitate synthesis. Values obtained from radioactive assays can be converted to defined units by multiplying by 14 in the case of acetyl-CoA, and by 2 in the case of malonyl-CoA. One unit of enzyme activity is defined as the amount of enzyme required to oxidize 1 nmole of NADPH per minute at 25 °, under the conditions described for the spectrophotometric assay. Specific activity is defined as units of enzyme per milligram of protein. For the purification of the chicken fatty acid synthase, young adult chickens (White Leghorn) weighing about 5 lb are acclimatized for about a week to insure adequate nutritional intake. Then a dozen animals are starved for three days, after which they are fed a 1: 1 mixture of sucrose-bread crumbs (low fat diet). Sucrose solution (1 M ) is substituted for drinking water. The chickens are maintained on this diet for 48 hours, then sacrificed by decapitation and the livers are excised and packed in ice. Typically, livers of starved-refed animals look gray and weigh 30–40 g.