title: Purification and characterization of acid proteinase from human erythrocyte membranes.
abstract: Two forms of an acid proteinase have been purified from human erythrocyte membranes by a simple method involving selective extraction with 0.5% Brij 35, affinity chromatography on pepstatin A-Sepharose 4B, and chromatography on Sephacryl S-200 and DEAE-Sephadex G-100. One species has an apparent molecular weight of 73 000 as measured by gel filtration and has been purified to apparent homogeneity. This form can be resolved by two-dimensional polyacrylamide gel electrophoresis into two bands with isoelectric points of about 4.5 and 5.0 and with molecular weights of 44 000 and 29 000, respectively. The second form of the enzyme has a molecular weight of about 47 000 and has also been purified to apparent homogeneity. In SDS-polyacrylamide gel electrophoresis the second form showed a single protein band corresponding to a molecular weight of 44 000. Two-dimensional polyacrylamide gel electrophoresis revealed a single band with an isoelectric point of about 4.5 and a molecular weight of 44 000. The high-molecular-weight form represented about a 17 500-fold purification over the erythrocyte membranes and about a 24% recovery, while the low-molecular-weight form represented about a 25 000-fold purification and about a 25% recovery. The two species had similar pH optimum and showed equal sensitivity to a number of ions and proteinase inhibitors. Chymotryptic maps of the 44 000 peptide of high-molecular-weight form and the purified low-molecular-weight form revealed no difference.
mesh_terms: D016282:Aspartic Acid Endopeptidases; D010450:Endopeptidases / Q000097:blood*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011480:Protease Inhibitors; D012995:Solubility; D013696:Temperature
chemical_list: D010446:Peptide Fragments; D011480:Protease Inhibitors; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases