CHARACTERIZATION OF THE PRENYLATED PROTEIN METHYLTRANSFERASE IN HUMANENDOMETRIAL CARCINOMA

The processing of ras and of other GTP-binding proteins includes a fin al reversible step in which the carboxy terminal prenylated cysteine i s methylated by the enzyme prenylated protein methyltransferase (PPMTa se). The significance of this modification and of the role of PPMTase in human tumors has yet to be fully elucidated. Here we characterize t he PPMTase of human endometrial carcinomas (tumors in which the freque ncy of ras gene mutations is relatively high) and compare it to the PP MTase of the normal endometrium. Our results show that in both types o f tissues the enzyme is bound to the membranes. It can utilize synthet ic substrates such as N-acetyl-S-farnesyl-L-cysteine (K-m = 18-20 mu M ) and is blocked by the PPMTase inhibitor S-farnesylthioacetic acid (K -i = 2 mu M). In vitro methylation assays and [alpha-P-32]GTP blot-ove rlay assays showed that the major endogenous PPMTase substrates are sm all GTP-binding proteins. Methylation of these proteins in vitro is bl ocked by farnesylthioacetic acid. The kinetic properties of PPMTase fr om the carcinomas and the normal tissues are very similar. However, le vels of PPMTase activity (but not of its endogenous substrates) are hi gher in the carcinomatous endometrium than in the normal one. The elev ated enzyme activity is restricted to the crude mitochondrial fraction (8.0 +/- 0.4 vs. 5.4 +/- 0.1 pmol N-acetyl farnesylcysteine methyl es ter formed/min/mg protein by the carcinoma and by the normal endometri al preparations, respectively). As this fraction is enriched in plasma membranes, it appears that the elevated enzyme activity could be rela ted to ras protein methylation; if so, selective methylation blockers might inhibit the growth of endometrial carcinomas.