Phyrochemistr)., 1975, Vol. 14, pp. 2407-2409. Pergamon Press. Printed in England zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM CONVERSION OF SQUALENE-2(3) EPOXIDE BY ENZYMES OF ALAWS GLUTINOSA ALAIN NICOLAS, JAQUE~ BASCOUL, ANDRE: CRASTES DE PAULET and PETER D. G. DEAN* Groupe de Recherches sur la Biochimie des Stkroides I.N.S.E.R.M., U.58, Institut de Biologie, Bd Hem? IV, 34000 Montpellier, France. (Receiued 31 January 1975) Key Word Index--Ahus glutinosa; Betulaceae; squalene-2(3) epoxide; squalene-2(3) epoxide cyclases; cycloar- tenol; biosynthesis; backbone rearrangement; cell-free system. Abstract-The incubation of [1-‘4C]-squalene-2(3)epoxide ([1-‘4C]-SO) with a cell-free extract of Alnus glutinosa gave only cycloartenol in 1% yield. The effects of pH, detergent (Triton) and enzyme/ substrate ratio upon cyclase activity were studied in order to determine the optimal conditions for the enzymatic conversion of [l-‘4C]-S0 to cycloartenol. INTRODlJaION Several examples of enzymatic cyclization of [l- “C]-squalene-2(3)epoxide (SO) to triterpenoids by cell-free extracts of both animals and plants are known and it is clear from a comparison of the structures of many triterpenes that several types of cyclase must exist [l], but the formation of any triterpene which results both from biosynthetic backbone rearrangement (BBR) and which gives a backbone rearrangement in acidic medium (BRA) [2] has not been described. Alnus glutinosa is known to contain consider- able quantities of glutinone [3,4], a triterpene which satisfies the above two conditions; thus, we have attempted to label glutinone by enzymatic conversion from [l- ‘“Cl-SO using cell-free extracts of this plant. Under our experimental conditions, neither labelled glutinone nor any other triterpene of the same family was obtained; the only labelled triterpene we could isolate was cycloartenol. RESULTS When [1-‘4C]-S0 was incubated with a cell- free extract of A. glutinosu two radiolabelled frac- * Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX, England. tions F1 and F, were isolated which were less polar and more polar respectively than [ l-14C]- SO. (In the following M1 and Mz refer respect- ively to the total radioactivity of F1 and Fz, M’, and M; refer to the radioactivity of the same frac- tions F’, and F; relative to the control.) F, and F’, had the same radioactivity and were identical on preparative GLC; F,, which probably corres- ponds to a degradation product of [1-‘4C]-S0, was not further studied. Identical aliquots (a and a’) of Fz and F; were analysed by preparative GLC, 5 min fractions were collected and their radioactivity measured; the distribution of radioactivity, given in Fig. 1 shows a conversion only for the fraction F2. The R, of the conversion products (peak C, Fig. 1) is the same as those of glutinol, cycloartenol or taraxerol which are not separated by GLC. However, continuous TLC (15 hr; eluant CHCl,, adsorbent Si gel + 10% AgNO,) gave a successful separation: using 3/3-glutinol as reference (Rf = l), the R,‘s of tar- axerol, cycloartenol and 3cr-glutinol were 0.9; 0.8 and 05 respectively. Under these conditions, frac- tion C (Fig. 1) had an R, = 0.8 on TLC which corresponds therefore to cycloartenol. Further- more, after dilution of 5000 dpm of radioactive cycloartenol fraction isolated from the incubation with 30 mg of non-radioactive cycloartenol, this