Characterisation of the component(s) responsible for the trypsin-like activity of the multicatalytic proteinase

• Main Author: Savory, Peter John
• Published: University of Leicester 1992
• Subjects: 572
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.737399

The multicatalytic proteinase (MCP) is a 700 kDa multisubunit complex which is found in eukaryotic cells. MCP is involved in ubiquitin-dependent non-lysosomal protein degradation and has been implicated in the processing of antigens by the major histocompatability complex class 1 pathway. The MCP purified from rat liver is composed of at least 16 distinct subunits of molecular masses between 22 and 34 kDa. MCP is known to possess at least three distinct peptidase activities, described as the 'trypsin-like', 'chymotrypsin-like' and 'peptidylglutamylpeptide hydrolase' activities. However, it is not known which subunits are responsible for the different peptidase activities. The main aims of my study were (1) to establish which subunit(s) possesses the active site responsible for 'trypsin-like' activity, (2) to identify the catalytic amino acid residues responsible for this activity and (3) to determine whether all MCP particles possess these components. 'Protection' experiments with the peptide aldehyde leupeptin, which is a specific reversible inhibitor of the 'trypsin-like' activity, have identified one or two subunits which possess thiol groups essential for 'trypsin-like' activity. These thiol groups are probably not involved in catalysis but may be located very close to the active site responsible for 'trypsin-like' activity. An active site specific peptidyl chloromethane which specifically inactivates the 'trypsinlike' activity, labels two distinct subunits of 23-24 kDa. One of these polypeptides is also labelled by an active site specific peptidyl diazomethane for the 'chymotrypsin-like' activity, in addition to three other distinct polypeptides of 28-23 kDa. Together these results suggest that one active site is responsible for 'trypsin-like' activity while four distinct active sites are responsible for 'chymotrypsin-like' activity. Affinity chromotography was carried out using an immobilised specific inhibitor of the 'trypsin-like' activity. The results of these studies suggest that all MCP particles purified from rat liver possess the catalytic component responsible for the 'trypsin-like' activity.