Abstract
The fluorescent probe l-anilinonaphthalene-8-sulfonate (ANS) has been used to investigate the properties of plasma membranes derived from normal hepatocytes and from hepatoma tissue culture (HTC) cells as well as used to study the effects of Ca 2+ and procaine on these membrane systems. The interaction of ANS with hepatocyte plasma membranes (50 nmol/mg protein; K D = 120, μM) resulted in a marked enhancement of fluorescence and a 20-nm blue shift. Both Ca 2+ and procaine further increased the fluorescence intensity. Binding studies showed no alteration in the number of ANS binding sites but a significant decrease in K D (40–50 μ m). Procaine was also shown to completely displace Ca 2+ from the membrane. The interaction of ANS with HTC cell plasma membranes again resulted in an enhancement in fluorescence intensity but with different binding properties (102 nmol/mg protein; K D = 74 μM) from the hepatocyte system. The addition of Ca +2 resulted in the formation of high and low affinity ANS binding sites as shown by Scatchard plot analysis with K D values of 15 μ m and 50 μ m. The effect of procaine on ANS fluorescence in the normal and transformed cell membranes was indistinguishable; however, in the latter system procaine only displaced 60% of the bound Ca 2+. These studies suggest several structural and binding alterations between plasma membranes derived from hepatocytes and HTC cells.
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