Solubilization of fatty acid synthetase, acyl-CoA reductase, and fatty acyl-CoA alcohol transacylase from the microsomes of Euglena gracilis

Abstract

The enzymes involved in the de novo synthesis of wax esters were solubilized from the microsomes of dark-grown Euglena gracilis by the use of deoxycholate or high concentration of sodium chloride. Solubilization with deoxycholate provided a preparation which catalyzed only the synthesis of fatty acids and this activity was stimulated by addition of phospholipids while the fatty acid synthetase from the soluble cytoplasm was not affected by the addition of phospholipids. Detergent Solubilization did not provide active soluble acyl-CoA reductase. Solubilization of the microsomal fatty acid synthetase together with acyl-CoA reductase and acyl-CoA hexadecanol transacylase was achieved upon treatment of the membranes with 3 m sodium chloride. Removal of sodium chloride by dialysis resulted in turbidity and the insoluble material recovered from this turbid solution by centrifugation at 105,000 g contained the bulk of the acyl-CoA reductase activity while the supernatant contained the major portion of the fatty acid synthetase. This aggregation of the reductase and its high detergent sensitivity suggest that this enzyme is highly hydrophobic and probably buried in the membrane matrix. Sucrose density gradient centrifugation of the NaCl solubilized preparation in the presence of 3 m sodium chloride resulted in the resolution of fatty acid synthetase from the acyl-CoA reductase and the transacylase.