Squalene synthase from cell suspension cultures of Tabernaemontana divaricata

Abstract

Squalene synthase (SQS) was partially purified from a membrane-rich fraction obtained from cells of elicitor-treated Tabernaemontana divaricata cell suspension cultures. The enzyme was solubilised using a mixture of the non-ionic detergents n-octyl-β- d-glucopyranoside and Lubrol PX and then purified, always in the presence of the two detergents, by sequential anion-exchange, cation-exchange, and gel filtration chromatography. SDS-PAGE analysis of the partially pure enzyme gave one major band of M r 64 000 and five minor bands with M rs in the range 47–58 000. Gel filtration chromatography indicated a native M r of 55–60 000, while isoelectric focusing of solubilised SQS (ex. microsomal fraction) gave a peak of activity corresponding to pI 7.3 and a minor peak. Throughout the purification procedure, when assayed in the presence of Mg 2+ and NADPH, the enzyme catalysed both the condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP) and the reduction of this intermediate to squalene, with a stoichiometry of close to 1.0. These results indicate that the two partial reactions of squalene synthesis by T. divaricata SQS are tightly coupled. Characterisation of SQS obtained from the cells of control and elicitor-treated T. divaricata cultures indicated differences in the apparent affinity ( K m ) for FPP before solubilisation of the enzyme, while the pH optimum and profile were similar for enzyme obtained from both sources. These results suggest that more than one isoform of SQS may be present in cells of elicitor-treated T. divaricata suspension cultures. © 1997 Elsevier Science Ltd. All rights reserved