Isolation and partial characterization of three acidic proteinases in erythrocyte membranes.
1. The distribution of proteolytic activity in membranes from human erythrocytes and from rabbit reticulocytes and erythrocytes was investigated, after removal of leucocytes and platelets from the cell suspensions. 2. All membrane preparations displayed proteolytic activity in the acidic pH region only. Membranes from human and rabbit mature erythrocytes showed latent activity, which could be increased when extracted with a number of detergents. 3. Three active fractions were resolved either by gel chromatography of solubilized membrane extracts or by standard polyacrylamide-gel electrophoresis. The three proteinase activities (designated proteinases I, II and III) were purified from solubilized extracts of human erythrocyte membranes. 4. The relevant mol.wts. were around 80000, 40000 and 30000, respectively, and each of the three proteinases appeared to be composed of a single polypeptide chain. 5. Distinctive pH optima (in the range pH2.8-3.9) and different saturation profiles with globin as substrate were observed for proteinases I, II and III. 6. Dithioerythritol, Hg(2+) and Cu(2+) inhibited each of the three human enzymes, but more selective inhibitory effects were exerted by other modifiers of proteolytic enzymes and by haemin. Similar effects were observed with the three proteinases from rabbit cells. 7. The activity of the three human proteinases seems to be restricted to naturally occurring protein substrates, although with poor specificity, and none of them was active on synthetic substrates. 8. Digestion of globin by each of the three enzymes yielded similar polypeptide fragments in all cases, this indicating an endopeptidase type of activity.
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000097:blood* / Q000302:isolation & purification; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D005914:Globins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002413:Cations, Divalent; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; D005914:Globins; D004492:Edetic Acid; D010450:Endopeptidases