Evidence for membrane-associated calpain I in human erythrocytes. Detection by an immunoelectrophoretic blotting method using monospecific antibody.
Low and high Ca2+-requiring forms of Ca2+-dependent cysteine proteinase are known as calpain I and calpain II, respectively. We have obtained, for the first time, monospecific antibodies for calpain I and for calpain II. Using these antibodies and an electrophoretic blotting method, we have found that a small, but reproducible, amount of calpain I was associated with human erythrocyte membranes while the bulk of the protease was contained in the cytosol. Most of membrane-associated calpain I was extractable with 1% Triton X-100, but not with 0.1% detergent. In the presence of 0.1 mM Ca2+ and 5 mM cysteine, membrane-associated calpain I degraded the membrane protein band 4.1 preferentially and band 3 protein only slowly. The Ca2+-induced autodigestion of the membrane preparation was inhibited by leupeptin but not by a cytosolic calpain inhibitor, calpastatin, added to the incubation medium. No calpain II was detected in either erythrocyte cytosol or membranes when anti-calpain II antibody was used under the same conditions as those for the detection of calpain I.
D000911:Antibodies, Monoclonal; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002154:Calpain; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000097:blood*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007122:Immunoelectrophoresis; D007074:Immunoglobulin G; D007976:Leupeptins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D007074:Immunoglobulin G; D007976:Leupeptins; D011480:Protease Inhibitors; C033668:calpastatin; D010450:Endopeptidases; D002154:Calpain; C032854:leupeptin; D002118:Calcium