Calpain inhibitor I prevents rapid postmortem degradation of benzodiazepine binding proteins: fluorographic and immunological evidence.
Endogenous proteolysis of the major central benzodiazepine (BZ) binding protein of 53K occurs rapidly postmortem and leads to a fragment of 47K. To determine indirectly the protease responsible for this proteolysis, membranes of porcine cortex were prepared from homogenates, which were either frozen immediately or left at room temperature for 12 h in the presence or absence of various representative protease inhibitors. Membranes were subsequently photolabeled with [3H]flunitrazepam, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography or immunoblotted using an alpha-subunit-specific monoclonal antibody bd-24. Both fluorographs and immunoblots revealed that calpain inhibitor I, Ep-459 (E-64 analogue), and EDTA (greater than or equal to 1 mM) prevent endogenous proteolysis. In future studies one of these inhibitors should be added to receptor preparations. The results indicate that calpain is the responsible protease.
Reichelt
R
R
Institut für Humangenetik, Universität Bonn, F.R.G.
Möhler
H
H
Hebebrand
J
J
D000345:Affinity Labels; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors; D002645:Chickens; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005470:Fluorometry; D006023:Glycoproteins / Q000494:pharmacology*; D015151:Immunoblotting; D010447:Peptide Hydrolases / Q000378:metabolism; D011180:Postmortem Changes; D011480:Protease Inhibitors / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000378:metabolism*; D013552:Swine
D000345:Affinity Labels; D006023:Glycoproteins; D011480:Protease Inhibitors; D011963:Receptors, GABA-A; C039304:calpain inhibitors; D004492:Edetic Acid; D010447:Peptide Hydrolases; D002154:Calpain