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.
Highlights
( ~ 2 8 )T.he protein is associated with the lipid bilayer peptidase activity, which we term HMP
The MCP activity from T acidophilum body raised against the rabbit multicatalytic proteinase.cleavesbothprotein and chymotrypsin-selectivesubstrates, The single subunit of HMP contains both the e.g. casein, insulin, and Suc-Leu-Leu-Val-Tyr-4-methyl-7-coucatalytic activity and a sequence necessary foirts asso- marylamide [6].While MCP is mainly cytoplasmic or nuclear ciation into a multimeric compleTx.he properties of the human erythrocyte membraneHMP described indicate that it is a novel peptidase related to the ubiquitous multicatalytic proteinase
(51,Kinoshita et a l . ( 8 )first reported the presenocfeMCP in the erythrocyte plasma membrane, which was similar to its cytoplasmic counterpart in its physical as well as enzymological characteristics
Summary
Ferguson plot analysis [12] was used to determine HMP native molecular mass. Threeaction mixture contained 0.05 uersus molecular mass for the standard proteins and HMP. A sample containing pg of HMP was applied to a A zero time point, determined by the addition of trichloroacetic acid. The washed membrane was incubated performed by incubatingthe low ionic strength-treatedmembranes for 2 h with 1:lOOO diluted secondary antibody (alkaline phosphatasewith an equal volumoef 150 mu NaCol n ice for 30 min toremove band conjugated anti-goatI g G ) (wholemolecule) developed in rabbit. The residual membrane containing 4 mM MgCI,, 0.01% nitro blue tetrazolium and 0.05% soluwas solubilized, the normal HMP purification procedure was per- tion of 5-bromo-4-chloro-3-indolyl phosphate dissolved indimethylformed, and the presence of HMP was determined by SDS-PAGE, as formamide. Protein Estimation-Protein concentrations were determined by the dye bindingmethod of Bradford[17].Proteinsamples(50 pl) were
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