Uptake of Bromosulfophthalein byIsolated Liver Cells

Abstract

Uptake of the hepatodiagnostic dye bromosulfophthalein into isolated hepatocytes was studied with special regard to the kinetics of transport. The following results were obtained. 1 The uptake of bromosulfophthalein follows Michaelis-Menten kinetics only at low substrate concentrations with an apparent K_m= 7 ± 2 μM and V= 2.6 ± 1.7 nmol × mg protein⁻¹× min^−1′. At higher bromosulfophthalein concentrations a second mechanism of uptake is observed as indicated by the deviation from linearity in the Lineweaver-Burk plot. 2 The activation energy of uptake was found to be 11 kcal/mol at 10 μM bromosulfophthalein. 3 Uptake is independent of metabolic energy and of the Na⁺ gradient across the membrane. 4 Taurocholate does not inhibit uptake while indocyanine green inhibits competitively at low bromosulfophthalein concentrations and activates uptake at high bromosulfophthalein concentrations (> 201 μM). 5 Amino acid reagents, such as dinitrofluorobenzene, mersalyl, N-ethylmaleimide, and dithionitrobenzene, which modify specific functional groups, did not affect uptake at a concentration of 100 μM. 6 No pH optimum for bromosulfophthalein uptake was observed in the physiological pH range. 7 Adsorption of bromosulfophthalein to the liver cell membrane has two distinguishable site, with affinities K₁= 5.7 × 10⁻⁶ M and K₂=7 × 10⁻⁵ M and binding capacities n₁= 1.2 nmol/mg protein and n₂= 7 nmol/mg protein. Adsorption is inhibited by indocyanine green. The results do not indicate the mediation of bromosulfophthalein uptake by a carrier protein and are consistent with the hypothesis that bromosulfophthalein is bound in an energy-consuming process to a translocating site, possibly in the undissociated form or as ion pair. The consecutive transfer across the membrane appears to require little additional energy.