Squalene Synthetase

Squalene synthetase, the first enzyme to synthesize a water-insoluble precursor of cholesterol, has been purified from bakers' yeast to near homogeneity. Key steps in purification are the release of this enzyme from particulate fractions of yeast by treatment with 50% sucrose and the stabilization of enzyme activity with 30% glycerol in 100 to 200 mm phosphate buffer. Enzyme activities of 20 to 40 nmoles of squalene formed per min per mg of protein are obtained with the enzyme purified by the procedure reported herein. Squalene synthetase exists in two forms (termed and protomeric). The first of these forms synthesizes squalene and presqualene pyrophosphate whereas the latter form synthesizes only presqualene pyrophosphate. An equilibrium mixture of the protomeric and polymeric forms of squalene synthetase exists. However, the ratio of these two forms can be affected markedly by the conditions of treatment. At a low glycerol concentration the polymeric form of the enzyme is converted to the protomeric form. In 100 mm potassium phosphate, pH 7.0, containing 20 mm 2-mercaptoethanol and 10% glycerol, the half-life of the polymer is 11.8 hours. Dissociation of the polymeric form is greatly accelerated by detergents. In 10% glycerol and 100 mm potassium phosphate buffer containing 0.4 mg per ml of Tween 80 and 20 mm 2-mercaptoethanol, the half-life of the polymeric form is approximately 3 hours. At a high glycerol concentration (50%) in 100 mm potassium phosphate buffer, pH 7.0, the protomeric form is partially reassociated to the polymer. However, the specific enzyme activity of the reassociated polymer is lower than that of the original enzyme, thereby suggesting that some of the enzyme is denatured in the process of dissociation and subsequent reassociation. The activities of the protomeric and the polymeric forms are not changed by the presence of sterol carrier protein.