title: Characterization of a novel high molecular mass protein with peptidase activity purified from the human erythrocyte membrane by calmodulin affinity chromatography.
abstract: A previously undescribed high molecular mass protein (HMP) from human erythrocyte membranes was solubilized by Triton X-100 and purified on a calmodulin-agarose column in the presence of Ca2+. It was shown to have a native molecular mass of 522-560 kDa, comprised of a single subunit of a molecular mass of 28 kDa (p28). The protein is associated with the lipid bilayer rather than with the cytoskeletal component of the membrane. The purified HMP showed peptidase-hydrolyzing activity toward substrates containing hydrophobic amino acids at the P1 position of the P2-P1 cleavage site. The activity was inhibited by serine proteinase inhibitors (leupeptin, phenylmethansulfonyl fluoride) and chymotrypsin inhibitors in particular (chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone). The enzyme exhibited maximal activity at slightly alkaline pH (7.5-8.5) and at 37 degrees C and was stimulated over a narrow range of SDS concentrations (maximal at 0.05%). HMP was found to cross-react in Western blots with an antibody raised against the rabbit multicatalytic proteinase. The single subunit of HMP therefore contains both the catalytic activity and a sequence necessary for its association into a multimeric complex. The properties of the human erythrocyte membrane HMP described indicate that it is a novel peptidase related to the ubiquitous multicatalytic proteinase.
authors.affiliation: Department of Pharmacy, University of Sydney, New South Wales, Australia.
authors.forename: M T
authors.initials: MT
authors.lastname: Khan
authors.forename: K K
authors.initials: KK
authors.lastname: Wang
authors.forename: A
authors.initials: A
authors.lastname: Villalobo
authors.forename: B D
authors.initials: BD
authors.lastname: Roufogalis
mesh_terms: D000595:Amino Acid Sequence; D002147:Calmodulin / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000097:blood* / Q000302:isolation & purification*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008051:Lipid Bilayers; D008563:Membrane Lipids / Q000097:blood; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D002147:Calmodulin; D008051:Lipid Bilayers; D008563:Membrane Lipids; D008565:Membrane Proteins; D009842:Oligopeptides; D011480:Protease Inhibitors; D010450:Endopeptidases