MEMBRANE-BOUND HIGH-MOLECULAR-MASS PROTEINASES FROM HUMAN ERYTHROCYTES

Two high molecular mass proteinases, multicatalytic proteinase (MCP) a nd a new high molecular mass proteinase (HMP) with only chymotrypsin-l ike activity (Khan et al. (1994) J. Biol. Chem. 269, 10016-10021) from human erythrocyte membranes, have been compared. For this purpose, MC P was purified from human erythrocyte membranes in the active form tow ards synthetic peptide substrates; it also hydrolysed the protein subs trates [C-14]methyl casein and [C-14]oxidised insulin beta chain at 37 degrees C. MCP from plasma membranes exhibited hollow cylindrical str uctures also typical of cytosolic forms. Radiolabelled diisopropyl flu orophosphate, [H-3]DFP, a serine proteinase inhibitor, labelled a band of M(r) 23000 in membrane MCP. By contrast,no labelling was obtained with HMP. Chymotrypsin-like activity of HMP was also found to be insen sitive to DFP. On the other hand, DFP inhibited chymotrypsin-like and peptidylglutamyl peptide hydrolysing activities of membrane MCP, with no effect on its trypsin-like activity. The inhibition of MCP by DFP w as concentration-dependent. These studies showed that MCP and HMP repr esent two distinct kinds of proteinases with chymotrypsin-like activit ies and can be distinguished by the serine proteinase inhibitor DFP.