Purification and characterization of a protein inhibitor of the 20S proteasome (macropain).
An inhibitory protein for the 20S proteasome (also known as macropain, the multicatalytic proteinase complex and 20S proteinase) has been purified from bovine red blood cells. The inhibitor has an apparent molecular weight of 31,000 on SDS-PAGE and appears to form multimers under nondenaturing conditions. This protein inhibited all three of the putatively distinct catalytic activities of proteasome A (the active form of the proteinase) characterized by the hydrolysis of synthetic peptides such as Z-VLR-MNA, Z-GGL-AMC or Suc-LLVY-AMC and Z-LLE-beta NA. The inhibitor also prevented the hydrolysis of large protein substrates such as casein, lysozyme and bovine serum albumin. Proteasome L (the latent form of the proteinase) does not degrade these large protein substrates, but does hydrolyze the three synthetic peptides at rates similar to those by proteasome A. The inhibitor inhibited only two of these peptidase activities of proteasome L (hydrolysis of Z-GGL-AMC and of Z-LLE-beta NA or Suc-LLVY-AMC); it had no effect on the hydrolysis of Z-VLR-MNA. The inhibitor was specific for inhibition of the proteasome and had no effect on the activity of any other proteinase tested including trypsin, chymotrypsin, papain, subtilisin and both isoforms of calpain. Kinetic analysis indicates that the inhibitor interacted with the proteasome by a mechanism involving tight-binding. Because the proteasome appears to be a key component of the ATP/ubiquitin-dependent pathway of intracellular protein degradation, the inhibitor may represent an important regulatory protein of this pathway.
Chu-Ping
M
M
Department of Physiology, University of Texas Southwestern Medical Center, Dallas.
Slaughter
C A
CA
DeMartino
G N
GN
D000818:Animals; D002417:Cattle; D002845:Chromatography; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004586:Electrophoresis; D007700:Kinetics; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000302:isolation & purification*; D046988:Proteasome Endopeptidase Complex; D014452:Ubiquitins / Q000378:metabolism
D009097:Multienzyme Complexes; D010455:Peptides; D011480:Protease Inhibitors; D014452:Ubiquitins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex