Cyclo(-Phe(p-NH[1-14C]Ac)-Thr-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 (Ki for
cholate uptake inhibition = 1 microM; Ki for
taurocholate uptake inhibition = 5 microM). When activated by flashed light the inhibition became irreversible (IC50 for
cholate uptake inhibition = 2 microM; IC50 for
taurocholate uptake inhibition = 9 microM) 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.