Analysis of farnesyl transferase activity during hormone-induced maturation ofXenopus laevis oocytes

Abstract

Preincubation of Xenopus laevis oocytes with insulin or insulin-like growth factor 1 (IGF-1) resulted in inhibition of farnesyl transferase (FTase) activity measured both in vivo (after microinjection of tritiated farnesyl pyrophosphate and Ras-CVIM into oocytes) and in extracts using a filtration assay. FTase activity measured in oocyte extracts was inhibited 55% after a 20 min treatment of oocytes with 1 microM insulin or 10 nM IGF-1. The apparent IC(50) for inhibition of oocyte FTase by IGF-1 is 0.3 nM. The observed decrease in FTase activity was apparently not due to translocation of enzyme from cytosol to membrane, since activities measured both in soluble extracts and resuspended crude pellets displayed comparable levels of inhibition following hormone treatment. Using a hexapeptide (TKCVIM) as substrate, FTase activity was also inhibited 65% when oocytes were pretreated with 10 nM IGF-1. Two FTase inhibitors [(alpha-hydroxyfarnesyl) phosphonic acid (HFPA) and chaetomellic acid A (CA)] effectively inhibited Xenopus oocyte FTase by 80-90% when added to assay mixtures (IC(50) values of 338 +/- 96 nM HFPA and 232 +/- 80 nM CA) or after incubation of oocytes in drug before preparation of soluble extracts for assay (IC(50) values of 7 +/- 6 nM HFPA and 328 +/- 128 nM CA). The farnesyl transferase inhibitors were observed to slow the time course of oocyte maturation but did not block the IGF-1-induced maturation response. J. Exp. Zool. 286:193-203, 2000.