Identification, purification, and characterization of a protein activator (PA28) of the 20 S proteasome (macropain).
A protein that greatly stimulates the multiple peptidase activities of the 20 S proteasome (also known as macropain, the multicatalytic protease complex, and 20 S protease) has been purified from bovine red blood cells and from bovine heart. The activator protein was a single polypeptide with an apparent molecular weight of 28,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had a native molecular weight of approximately 180,000. This protein, which we have termed PA28, regulated all three of the putatively distinct peptidase activities displayed by each of two functionally different forms of the proteasome. This regulation usually included both an increase in the maximal reaction velocity and a decrease in the concentration of substrate required for half-maximal velocity and indicated that PA28 acted as a positive allosteric effector of the proteasome. PA28 failed, however, to stimulate the hydrolysis of large protein substrates such as casein and lysozyme. These results suggested that the hydrolysis of protein substrates occurred at a site or sites distinct from those that hydrolyzed small peptides and that the regulation of the two processes could be uncoupled. Evidence for direct binding of PA28 to the proteasome was obtained by glycerol density gradient centrifugation. PA28 may play an important regulatory role in intracellular proteolytic pathways mediated by the proteasome.
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Department of Physiology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas 75235.
Slaughter
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DeMartino
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D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002853:Chromatography, Liquid; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004912:Erythrocytes / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D011506:Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex