Azidobenzamido-008, a new photosensitive substrate for the 'multispecific bile acid transporter' of hepatocytes: evidence for a common transport system for bile acids and cyclosomatostatins in basolateral membranes

Cyclo(-Phe(p-NH[1-¹⁴C]Ac)-Trp-Lys-(CO(p-N₃)C₆H₄)-Trp-Phe-dPro), in the following named azidobenzamido-008, was synthesized in order to identify binding sites for c(Phe-Thr-Lys-Trp-Phe-dPro), named 008, (a cyclosomatostatin with retro sequence) in liver cell plasma membranes. In the dark the above photolabel was taken up into isolated hepatocytes, inhibiting the sodium dependent uptake of cholate and taurocholate in a competitive manner (K_i for cholate uptake ihibition = 1 μM; K_i for taurocholate uptake inhibition = 5 μM). When activated by flashed light the inhibition became irreversible (IC₅₀ for cholate uptake inhibition = 2 μM; IC₅₀ for taurocholate uptake inhibition = 9 μM) and the activated cyclopeptide bound chiefly to hepatocellular membrane proteins of 67, 54, 50, 37 kDa. Excess of the initial 008, or of cholate or phalloidin partially protected the above membrane components against labeling with ¹⁴C-labeled azidobenzamido-008. In contrast AS 30 D ascites hepatoma cells, known to be deficient in bile acid and cyclosomatostatin transport, could not be specifically labeled by azidobenzamido-008. The membrane proteins preferentially labeled in hepatocytes (50 and 54 kDa) are integral glycoproteins. The 67 kDa protein is a hydrophilic nonglycosylated membrane component. Independent of labeling with ¹⁴C-labeled azidobenzamido-008 or with ¹⁴C-labeled azidobenzamido-taurocholate, the main radioactive peaks in the pH region of 7, 5.5, 5.25 were identical after solubilization with Nonidet P-40 and subsequent isoelectric focusing. Proteins of 67, 54, 50 and 37 kDa could be enriched by use of 008-containing gels in affinity electrophoresis. Binding sites for 008 were not destroyed by SDS or Nonidet P-40 treatment of plasma membranes.