Further characterization of membrane proteins involved in the transport of organic anions in hepatocytes. Comparison of two different affinity labels: 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid and brominated taurodehydrocholic acid

4,4'-Diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (H2DIDS) known as an irreversible inhibitor of the anion transport in red blood cells (Cabantchik, Z.I. and Rothstein, A. (1972) J. Membrane Biol. 10, 311-330) blocks also the uptake of bile acids and of some foreign substrates in isolated hepatocytes (Petzinger, E. and Frimmer, M. (1980) Arch. Toxicol. 44, 127-135). [3H]H2DIDS was used for labeling of membrane proteins probably involved in anion transport of rat liver cells. The membrane proteins modified in vitro by [3H]H2DIDS were compared with those labeled by brominated taurodehydrocholic acid. The latter is one of a series of suitable taurocholate derivatives, all able to bind to defined membrane proteins of hepatocytes and also known to block the uptake of bile acids as well as of phallotoxins and of cholecystographic agents (Ziegler, K., Frimmer, M., M?ller, W. and Fasold, H. (1982) Naunyn-Schmiedeberg's Arch. Pharmacol. 319, 254-261). The radiolabeled proteins were compared after SDS-electrophoresis with and without reducing agent present, solubilization by detergents, two-dimensional electrophoresis and after separation of integral and peripheral proteins. Our results suggest that the anion transport system of liver cells cannot distinguish between bile acids and the anionic stilbene derivative (DIDS). The labeling pattern for both kinds of affinity labels was very similar. Various combinations of separation techniques gave evidence that the radiolabeled membrane proteins are not subunits of a single native channel protein.     

Preparation and characterization of nuclear-envelope vesicles from rat liver nuclei.

We describe a procedure for the preparation of sealed nuclear-envelope vesicles from rat liver nuclei. These vesicles are strikingly similar in their polypeptide composition when compared with those of nuclear envelopes prepared conventionally using deoxyribonuclease I. Subfractionation analysis by means of extraction with high salt and urea show that the components of the nuclear envelope, e.g. the pore-complex/lamina fraction, are present. The residual DNA content is only 1.5%, and typical preparations consist of about 80% vesicles, with the vesicular character of these envelopes shown by microscopic and biochemical studies. The vesicles can be obtained in high yield, are tight and stable for at least two days and are enriched in a nucleoside triphosphatase thought to be involved in nucleocytoplasmic transport processes. Because the vesicles are largely free of components of the nuclear interior, but retain properties of intact nuclei, we believe that they are a valuable model system to study nucleocytoplasmic transport. Although in transport studies with isolated nuclei interference from intranuclear events has to be considered, the nuclear-envelope vesicles provide the possibility of studying translocation alone. Furthermore, the less complex nature of these vesicles compared with whole nuclei should facilitate investigation of the components involved in the regulation of nuclear transport processes.     

Chemically tritiated tetrodotoxin: physiological activity and binding to Na-channels

A new method is used to tritiate tetrodotoxin:starting with tetrodotoxin, acetylanhydrotetrodotoxin is formed which is then reacted in $\text{T}{}_2\text{O}\text{TCl}$ to [³H]tetrodotoxin. The formation of the intermediate and of the tritiated product is analytically monitored by bioassay. After purification [³H]tetrodotoxin is obtained at a specific activity of 18 Ci/mmol. No back exchange of tritium was observed under physiological conditions. The binding of [³H]tetrodotoxin to voltage-sensitive Na channels was studied with membrane fragments from Electrophorus electricus electric organ. Binding studies were carried out by variation of the concentration of [³H]tetrodotoxin and by competition between [³H]tetrodotoxin and reference tetrodotoxin. The apparent dissociation constant for binding to Na channels in these membrane fragments is K_D = (20 ± 10) nM. In contrast, [³H]tetrodotoxin blocks Na current in Rana esculenta nodes with an apparent K_D = 3 nM. The difference may be due to a higher density of negative surface charges at the nodal regions.