Calpain Inhibitor I Prevents Rapid Postmortem Degradation of Benzodiazepine Binding Proteins: Fluorographic and Immunological Evidence

Abstract

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.

Specificity of calcium-activated neutral proteinase (CANP) inhibitors for human ?CANP and mCANP

“…A second inhibitor in this group, E64, irreversibly inhibits CANPs and other cysteine proteinases (12,15). Recently, additional CANP inhibitors have been synthesized, including N-acetyl-Leu-Leu-norleucinal (C-I) and N-acetyl-Leu-Leu-methioninal (C-II), which have been used as selective CANP inhibitors (1,3,4,13,18). A newer compound, calpeptin, is considered to be more cell permeable than other CANP inhibitors (22).…”

Ontogeny of the Benzodiazepine Receptor in Human Brain: Fluorographic, Immunochemical, and Reversible Binding Studies

“…In addition, an attempt was made to delineate the development of single benzodiazepine binding proteins between week 8 postconception and 1.75 years postnatally using fluorographs and immunoblots [monoclonal antibody (mAb) bd-241. Because the influence of postmortem proteolysis on labeling patterns and binding parameters has been described extensively (Reichelt et al, , 1990, the fluorographic and immunological pattern of the development of proteins could be evaluated relative to the changes that set in rapidly after death.…”