Identification of cyclosporin binding sites in rat liver plasma membranes, isolated hepatocytes, and hepatoma cells by photoaffinity labeling using [3H]cyclosporin-diaziridine.
[3H]Cyclosporin diaziridine, a new photoaffinity label, enters rat liver cells in the dark. Photoaffinity labeling of isolated rat liver-cell plasma membranes with this probe modifies several polypeptides with molecular mass of 200, 85, 54, 50, 34 kDa. The major labeled protein of 85 kDa represents 2% of the total plasma membrane protein. A 50 kDa protein is heavily labeled in freshly isolated rat hepatocytes at low temperature and after short incubation in the dark. The 85 kDa protein becomes substituted after longer preincubation periods at temperatures above 10 degrees C. This suggests a localisation at the cytoplasmic side of the membrane. Several controls point to a specific interaction with the above mentioned proteins. Comparison of [3H]cyclosporin-diaziridine- and isothiocyanatobenzamido[3H] cholic acid-labeled membrane proteins reveals identity of binding proteins with the exception of the 85 kDa protein. However, the interaction of bile acids with the 85 kDa protein became apparent at higher concentrations as demonstrated by the differential photoaffinity labeling experiments. In the cytosol of rat liver cells, further [3H]cyclosporin-diaziridine binding proteins could be identified. In particular, a 17 kDa polypeptide was found which appears similar to cyclophilin, a protein known to be present in T-lymphocytes (R. Handschumacher et al. (1984) Science 226, 544-547: Cyclophilin. A specific cytosolic binding protein for cyclosporin A). Proteins with molecular mass of 90, 56, 30, 24, 20 kDa are labeled in AS-30D ascites hepatoma cells and those with molecular mass of 200, 150, 80, 70, 42, 25 kDa in Ehrlich ascites tumor cells.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003524:Cyclosporins / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000166:cytology*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D010777:Photochemistry; D051381:Rats; D013696:Temperature
D000345:Affinity Labels; D003524:Cyclosporins; C048004:cyclosporin diaziridine