Differential degradation of different benzodiazepine binding proteins by incubation of membranes from cerebellum or hippocampus with trypsin.
When rat brain membranes were incubated with [3H]flunitrazepam in the presence of UV light, predominantly one protein (P51) was irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. On digestion of membranes with increasing concentrations of trypsin up to 40% of radioactivity irreversibly bound to proteins was removed from the membranes. In addition, P51 was nearly completely degraded to a peptide with apparent molecular weight 39,000 and this peptide was further degraded to a peptide with apparent molecular weight 25,000. In contrast, protein P55 was only partially degraded by trypsin and yielded two proteolytic peptides with apparent molecular weights 42,000 and 45,000 which seemed to be rather stable against further attack by trypsin. Membranes treated with trypsin still had the capacity to bind [3H]-flunitrazepam reversibly with an affinity similar to that of membranes not previously treated with trypsin. When these membranes were irradiated with UV light, the same proteolytic peptides were detected as in membranes first photolabeled and then digested with trypsin. These results suggest a close association between reversible and irreversible benzodiazepine binding sites and indicate that membrane-associated proteins P51 and P55 are differentially protected against degradation by trypsin.
Eichinger
A
A
Sieghart
W
W
D000345:Affinity Labels; D000731:Androstanes / Q000494:pharmacology; D000818:Animals; D001378:Azasteroids / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005445:Flunitrazepam / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010777:Photochemistry; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D000345:Affinity Labels; D000731:Androstanes; D001378:Azasteroids; D010446:Peptide Fragments; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam; D014357:Trypsin; C032061:RU 5135