Direct photoaffinity labeling of leukotriene binding sites.
Due to their conjugated double bonds the leukotrienes themselves are photolabile compounds and may therefore be used directly for photoaffinity labeling of leukotriene binding sites. Cryofixation eliminates unspecific labeling taking place in solution by photoisomers and photodegradation products of leukotrienes. After fixation of receptor ligand interactions by shock-freezing of the samples, irradiation-induced highly reactive excited states and/or intermediates can form covalent bonds with the respective binding site in the frozen state. After cryofixation of a solution of albumin incubated with [3H8]leukotriene E4, irradiation at 300 nm resulted in time-dependent incorporation of radioactivity into the protein. Photoaffinity labeling of rat as well as of human blood serum with [3H8]leukotriene E4 after cryofixation revealed that only one polypeptide with an Mr of 67,000 was labeled. This polypeptide was identified as albumin. Photoaffinity labeling of rat liver membrane subfractions enriched with sinusoidal membranes resulted in the labeling of a polypeptide with an apparent Mr of 48,000, whereas no polypeptide was predominantly labeled in the subfraction enriched with canalicular membranes. Photoaffinity labeling of isolated hepatocytes disclosed different leukotriene E4 binding polypeptides. In the particulate fraction of hepatocytes a polypeptide with an apparent Mr of 48,000 was labeled predominantly, whereas in the soluble fraction several polypeptides were labeled to a similar extent. One of these, with an apparent Mr of 25,000, was identified as subunit 1 of glutathione transferases by immunoprecipitation. The method of direct photoaffinity labeling in the frozen state after cryofixation using leukotrienes as photoactivatable compounds, as exemplified by leukotriene E4, may be most useful for the identification and characterization of various leukotriene binding sites, including receptors, leukotriene-metabolizing enzymes, and transport systems.
Falk
E
E
Institut für Organische Chemie und Biochemie Universität, Freiburg, FRG.
Müller
M
M
Huber
M
M
Keppler
D
D
Kurz
G
G
D000818:Animals; D002462:Cell Membrane / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D017999:Leukotriene E4; D008099:Liver / Q000276:immunology*; D008297:Male; D010782:Photolysis; D051381:Rats; D011919:Rats, Inbred Strains; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000378:metabolism*; D018077:Receptors, Leukotriene; D013189:SRS-A / Q000031:analogs & derivatives / Q000302:isolation & purification / Q000378:metabolism*
D011971:Receptors, Immunologic; D018077:Receptors, Leukotriene; D013189:SRS-A; D017999:Leukotriene E4