Binding of unconjugated and conjugated sulfobromophthalein to rat liver plasma membrane fractions in vitro.

As part of a study of the mechanism whereby organic anionic dyes such as sulfobromophthalein and bilirubin enter hepatocytes, the binding of [35S]sulfobromophthalein and of its glutathione conjugate to two rat liver plasma membrane fractions were studied in vitro. Both fractions reversibly and saturably bound conjugated and unconjugated sulfobromophthalein. Three classes of binding site were necessary to account for the observed sulfobromophthalein binding, their maximal binding capacities being 3.5 . 10(-11), 1.6 . 10(-7) and 5.4 . 10(-7) mol/mg membrane protein. The corresponding association constants were 5.5 . 10(7), 1.5 . 10(5) and 1.3 . 10(3) M-1. Binding of the glutathione conjugate could be accounted for by two classes of binding site only, their association constants being 2.0 . 10(8) and 1.9 . 10(3) M-1 and their maximal binding capacities 5.0 . 10(-11) and 2.2 . 10(-7) mol/mg protein, respectively. Conjugated and unconjugated sulfobromophthalein mutually competed for binding, KI being 7.8 . 10(-7) and 5.5 . 10(-5) M for conjugated and unconjugated sulfobromophthalein, respectively. Similarly, bilirubin and indocyanine green, but not taurocholate, competitively inhibited sulfobromophthalein binding. Treatment with trypsin and phospholipases reduced, while treatment with neuraminidase did not affect binding. Neither changes in pH nor substitution of other cations for Na+ in the incubation mixture significantly affected sulfobromophthalein binding. Heating the membranes increased binding. This was due to an increase in maximal binding capacity of the low-affinity, high-capacity sites. The description of saturable binding sites on hepatocellular surface membranes, the affinity of one of the sites exceeding the reported affinities for albumin and ligandin, supports the hypothesis that a membrane-located membrane carrier is responsible for hepatic uptake and biliary excretion of organic anionic dyes. Based on the studies with enzyme treatments, it is speculated that this carrier is a phospholipid-dependent, integral membrane protein.