Identification of prenylcysteine carboxymethyltransferase in bovine adrenal chromaffin cells.

Chromaffin cells from bovine adrenal medulla were examined for the presence of a specific prenylcysteine carboxymethyltransferase by using N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine as artificial substrates and a crude cell homogenate as the enzyme source. From Michaelis-Menten kinetics the following constants were calculated: K(m) 90 microM and V(max) 3 pmol/min per mg proteins for N-acetyl-S-farnesyl-L-cysteine; K(m) 52 microM and V(max) 3 pmol/min per mg proteins for N-acetyl-S-geranylgeranyl-L-cysteine. Both substrates were methylated to an optimal extent at the pH range 7. 4-8.0. Methylation activity increased linearly up to 20 min incubation time and was dose dependent up to at least 160 microg of protein. Sinefungin and S-adenosylhomocysteine both caused pronounced inhibition, as also to a lesser extent did farnesylthioacetic acid, deoxymethylthioadenosine and 3-deaza-adenosine. Effector studies showed that the methyltransferase activity varied depending on the concentration and chemical nature of the cations present. Monovalent cations were slightly stimulatory, while divalent metallic ions displayed diverging inhibitory effects. The inhibition by cations was validated by the stimulatory effect of the chelators EDTA and EGTA. Sulphydryl reagents inhibited methylation but to different degrees: Hg(2+)-ions: 100%, N-ethylmaleimide: 30%, dithiothreitol: 0% and mono-iodoacetate: 20%. Due to the hydrophobicity of the substrates dimethyl sulfoxide had to be included in the incubation mixture (<4%; still moderate inhibition at more elevated concentrations). The detergents tested affected the methyltransferase activity to a varying degree. The membrane bound character of the methyltransferase was confirmed.