CHARACTERIZATION OF A NOVEL HIGH-MOLECULAR-MASS PROTEIN WITH PEPTIDASE ACTIVITY PURIFIED FROM THE HUMAN ERYTHROCYTE-MEMBRANE BY CALMODULIN AFFINITY-CHROMATOGRAPHY

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 h ave a native molecular mass of 522-560 kDa, comprised of a single subu nit of a molecular mass of 28 kDa (p28). The protein is associated wit h the lipid bilayer rather than with the cytoskeletal component of the membrane. The purified HMP showed peptidase-hydrolyzing activity towa rd substrates containing hydrophobic amino acids at the P1 position of the P2-P1 cleavage site. The activity was inhibited by serine protein ase inhibitors (leupeptin, phenylmethanesulfonyl fluoride) and chymotr ypsin inhibitors in particular (chymostatin, N-tosyl-L-phenylalanine c hloromethyl ketone). The enzyme exhibited maximal activity at slightly alkaline pH (7.5-8.5) and at 37-degrees-C and was stimulated over a n arrow range of SDS concentrations (maximal at 0.05%). HMP was found to cross-react in Western blots with an antibody raised against the rabb it multicatalytic proteinase. The single subunit of HMP therefore cont ains both the catalytic activity and a sequence necessary for its asso ciation into a multimeric complex. The properties of the human erythro cyte membrane HMP described indicate that it is a novel peptidase rela ted to the ubiquitous multicatalytic proteinase.