Characterization of prenylcysteine methyltransferase in insulin-secreting cells

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Abstract
Prenylcysteine carboxymethyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, was characterized in insulin-secreting INS-1 cells and normal rat pancreatic islets. The activity of this enzyme was monitored by the methylation of an artificial substrate (a prenylated cysteine analogue) with S-adenosyl[methyl-3H]methionine as methyl donor. More than 95% of the methyltransferase activity was associated with the membranes, and high-salt treatment only partially extracted the enzyme from the membranes. The highest specific activity was in the insulin-granule-enriched 25000 g pellet obtained by differential centrifugation. However, a highly purified insulin-enriched fraction obtained by density centrifugation in Percoll 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′-[γ-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, methyltransferase activity was not stably modified by stimulation of intact cells using glucose or other agents. However, the carboxymethylation of certain low-molecular-mass G-proteins is increased by glucose stimulation; conversely, treatment of cells with N-acetyl-S-trans, trans-farnesyl-L-cysteine inhibited glucose- and forskolin-induced insulin secretion. These results suggest that the membrane-associated prenylcysteine carboxymethyltransferase may be constitutively active and that the methylation of target proteins in vivo is regulated by the access of these proteins to the methyltransferase, as well as by their active (GTP-liganded) configuration.

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