Proteolysis of N-ethylmaleimide-modified aldolase loaded into erythrocyte ghosts: prevention by inhibitors of calpain.
1. When rabbit muscle aldolase labelled with tritium and inactivated by N-ethylmaleimide (NEM) was loaded into erythrocyte ghosts, significant proteolysis of the loaded protein occurred. The major product of this proteolysis, separated by electrophoresis under dissociating conditions, was found to be approx. 2 kDa smaller than the parent protein. 2. Proteolysis was detectable during erythrocyte ghost loading at 0 degrees C, reaching a plateau after approx. 12 min. Subsequent incubation at 37 degrees C to allow resealing of the ghosts resulted in additional proteolysis, and up to 20% of the loaded protein was converted to the smaller 38 kDa derivative. 3. EDTA, EGTA, leupeptin and chymostatin, each inhibitors of calcium-activated neutral proteinases (calpains), were the most effective inhibitors of the proteolysis of NEM-inactivated aldolase in ghosts. Other proteinase inhibitors were ineffective, while phenylmethanesulphonyl fluoride was only partially effective. 4. Inhibition of the proteolysis by EGTA was prevented by CaCl2, supporting the involvement of erythrocyte calpain. 5. Pretreatment of ghosts with EGTA prior to loading of NEM-modified aldolase followed by microinjection of the protein into HeLa cells did not result in a different rate of its overall breakdown to acid-soluble products. EGTA is suggested as a useful agent for the erythrocyte ghost-mediated microinjection of calpain-sensitive proteins.
Hopgood
M F
MF
CSIRO Division of Human Nutrition, Adelaide, Australia.
Knowles
S E
SE
Ballard
F J
FJ
D002154:Calpain / Q000037:antagonists & inhibitors*; D004533:Egtazic Acid / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology*; D005033:Ethylmaleimide; D005634:Fructose-Bisphosphate Aldolase / Q000097:blood*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008845:Microinjections
D004533:Egtazic Acid; D002154:Calpain; D005634:Fructose-Bisphosphate Aldolase; D005033:Ethylmaleimide