CHARACTERIZATION OF PRENYLCYSTEINE METHYLTRANSFERASE IN INSULIN-SECRETING CELLS

Prenylcysteine carboxymethyltransferase, an enzyme involved in the pos t-translational modification of many signalling proteins, was characte rized in insulin-secreting INS-1 cells and normal rat pancreatic islet s. The activity of this enzyme was monitored by the methylation of an artificial substrate (a prenylated cysteine analogue) with S-adenosyl[ methyl-H-3]methionine as methyl donor. More than 95% of the methyltran sferase activity was associated with the membranes, and high-salt trea tment only partially extracted the enzyme from the membranes. The high est specific activity was in the insulin-granule-enriched 25 000 g pel let obtained by differential centrifugation. However, a highly purifie d insulin-enriched fraction obtained by density centrifugation in Perc oll did not exhibit methyltransferase activity. The analyses of marker enzymes for cellular organelles revealed that-the methyltransferase. was co-localized with the plasma membrane and probably the endoplasmic reticulum, but not with the mitochondria or lysosomes. Guanosine 5'-[ gamma-thio]triphosphate failed to increase methyltransferase activity directly, although it promotes the methylation of GTP-binding proteins . Mastoparan, Ca2+, cAMP and the protein kinase C activator phorbol 12 -myristate 13-acetate did not alter enzyme activity. In addition, meth yltransferase activity was not stably modified by stimulation of intac t cells using glucose or other agents. However, the carboxymethylation of certain low-molecular-mass G-proteins is increased by glucose stim ulation; conversely, treatment of cells with N-acetyl-S-trans,trans-fa rnesyl-L-cysteine inhibited glucose- and forskolin-induced insulin sec retion These results suggest that the membrane-associated prenylcystei ne carboxymethyltransferase may be constitutively active and that the methylation of target proteins in vivo is regulated by the access of t hese proteins to the methyltransferase as well as by their active (GTP -liganded) configuration.