SQUALENE SYNTHASE FROM CELL-SUSPENSION CULTURES OF TABERNAEMONTANA-DIVARICATA

Squalene synthase (SQS) was partially purified from a membrane-rich fr action obtained from cells of elicitor-treated Tabernaemontana divaric ata cell suspension cultures. The enzyme was solubilised using a mixtu re of the non-ionic detergents n-octyl-beta-D-glucopyranoside and Lubr ol PX and then purified, always in the presence of the two detergents, by sequential anion-exchange, cation-exchange, and gel filtration chr omatography. SDS-PAGE analysis of the partially pure enzyme gave one m ajor band of M-r 64 000 and five minor bands with M(r)s in the range 4 7-58 000. Gel filtration chromatography indicated a native M-r of 55-6 0 000, while isoelectric focusing of solubilised SQS (ex. microsomal f raction) gave a peak of activity corresponding to pI 7.3 and a minor p eak. Throughout the purification procedure, when assayed in the presen ce of Mg2+ and NADPH, the enzyme catalysed both the condensation of tw o molecules of farnesyl diphosphate (FPP) to form presqualene diphosph ate (PSPP) and the reduction of this intermediate to squalene, with a stoichiometry of close to 1.0. These results indicate that the two par tial reactions of squalene synthesis by T. divaricata SQS are tightly coupled. Characterisation of SQS obtained from the cells of control an d 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. (C) 1997 Elsevier Science Ltd. All rights reserved.