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.