CYCLIC SOMATOSTATIN ANALOGS BIND SPECIFICALLY TO PI-6.1 CARBOXYLESTERASE OF RAT-LIVER CELLS

The hydrophobic cyclohexapeptide cyclo(Phe-Thr-Lys-Trp-Phe-DPro) (008) , an analog of somatostatin with retro sequence, was previously shown to competitively inhibit the uptake of cholate and taurocholate into i solated rat liver cells. Conversely, the competitive uptake inhibition of 008 into isolated rat hepatocytes by bile acids confirmed the obse rvation of common binding and transport sites by bile acids and cyclos omatostatin. Furthermore the transport characteristics of 008 uptake r evealed a significant and rapid binding to cell membranes. In this con text it was of special interest to investigate the specificity of the binding component since specific binding of the substrate to membrane proteins could be responsible for the low K-m of 008-transport. Theref ore, the cyclohexapeptide 008 could be used as the ligand in affinity chromatography in order to isolate such binding proteins. The gel matr ix used did not interact non-specifically with octylglucoside-solubili zed proteins from isolated rat liver plasma membranes. In affinity chr omatography of octylglucoside-solubilized plasma membranes, two domina nt proteins with apparent molecular masses of 60 and 58 kDa bound spec ifically to the 008 ligand. When used as ligands in affinity chromatog raphy, these membrane-associated 60 and 58 kDa proteins bound exclusiv ely to aromatic cyclopeptides, e.g. cyclosomatostatin 008, but not to linear peptides or taurocholate derivatives. The amino acid sequences of tryptic digests of the 008-affinity-purified 58 kDa protein were id entical to the sequence of a microsomal pI 6.1 carboxylesterase. Immun ofluorescence of intact hepatocytes showed that this xenobiotic metabo lizing enzyme is also located in sinusoidal rat liver plasma membranes and could therefore account for the extensive and specific binding of the cyclosomatostatin to sinusoidal plasma membranes of rat liver.