PA28, an activator of the 20 S proteasome, is inactivated by proteolytic modification at its carboxyl terminus

Ma Chu-Ping, Patricia J. Willy, Clive A. Slaughter, George N. DeMartino

Research output: Contribution to journalArticle

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Abstract

PA28, a protein activator of the 20 S proteasome, was previously identified in soluble extracts of bovine red blood cells (Ma, C.-P., Slaughter, C. A., and DeMartino, G. N. (1992) J. Biol. Chem. 267, 10515-10523). To determine whether this regulatory protein is as widely distributed as the proteasome, PA28 content and activity were examined in various eukaryotic tissues by immunoblot analysis and by functional assays of tissue extracts. PA28 protein was present in all sources examined. PA28 activity, however, was not detected in many of these sources, including those with the highest level of PA28 protein. To determine the biochemical basis of this result, PA28 was purified from extracts of rat liver, which had high levels of PA28 protein but no PA28 activity. The resulting purified PA28 had no detectable activity but had native and subunit molecular weights indistinguishable from the active PA28 of bovine red blood cells. Using the inactivation of purified PA28 as an assay, a protein that inactivated PA28 without altering its apparent molecular weight on SDS-polyacrylamide gel electrophoresis was identified, purified, and characterized from bovine liver. It had biochemical and catalytic characteristics similar to those of lysosomal carboxypeptidase B. When leupeptin, an inhibitor of lysosomal carboxypeptidase B, was included in the buffers used for the preparation of PA28, PA28 activity was detected in tissues which otherwise failed to demonstrate this activity. A similar result was obtained when extracts were prepared in a manner that minimized disruption of lysosomes. Other carboxypeptidases such as carboxypeptidase Y and pancreatic carboxypeptidase B also inactivated PA28 without altering its apparent molecular weight. Active PA28 binds to the proteasome to form a protease-activator complex that can be isolated after velocity sedimentation centrifugation through glycerol density gradients. Carboxypeptidase-inactivated PA28 failed to form such a complex, suggesting that the carboxyl terminus of PA28 is required for binding to the proteasome. These results indicate the importance of the carboxyl terminus of PA28 for proteasome activation.

Original languageEnglish (US)
Pages (from-to)22514-22519
Number of pages6
JournalJournal of Biological Chemistry
Volume268
Issue number30
StatePublished - Oct 25 1993

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Proteasome Endopeptidase Complex
Carboxypeptidase B
Carboxypeptidases
Proteins
Molecular Weight
Molecular weight
Liver
Assays
Blood
Erythrocytes
Cells
Cathepsin A
Tissue
Liver Extracts
Tissue Extracts
Centrifugation
Lysosomes
Electrophoresis
Sedimentation
Glycerol

ASJC Scopus subject areas

  • Biochemistry

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PA28, an activator of the 20 S proteasome, is inactivated by proteolytic modification at its carboxyl terminus. / Chu-Ping, Ma; Willy, Patricia J.; Slaughter, Clive A.; DeMartino, George N.

In: Journal of Biological Chemistry, Vol. 268, No. 30, 25.10.1993, p. 22514-22519.

Research output: Contribution to journalArticle

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abstract = "PA28, a protein activator of the 20 S proteasome, was previously identified in soluble extracts of bovine red blood cells (Ma, C.-P., Slaughter, C. A., and DeMartino, G. N. (1992) J. Biol. Chem. 267, 10515-10523). To determine whether this regulatory protein is as widely distributed as the proteasome, PA28 content and activity were examined in various eukaryotic tissues by immunoblot analysis and by functional assays of tissue extracts. PA28 protein was present in all sources examined. PA28 activity, however, was not detected in many of these sources, including those with the highest level of PA28 protein. To determine the biochemical basis of this result, PA28 was purified from extracts of rat liver, which had high levels of PA28 protein but no PA28 activity. The resulting purified PA28 had no detectable activity but had native and subunit molecular weights indistinguishable from the active PA28 of bovine red blood cells. Using the inactivation of purified PA28 as an assay, a protein that inactivated PA28 without altering its apparent molecular weight on SDS-polyacrylamide gel electrophoresis was identified, purified, and characterized from bovine liver. It had biochemical and catalytic characteristics similar to those of lysosomal carboxypeptidase B. When leupeptin, an inhibitor of lysosomal carboxypeptidase B, was included in the buffers used for the preparation of PA28, PA28 activity was detected in tissues which otherwise failed to demonstrate this activity. A similar result was obtained when extracts were prepared in a manner that minimized disruption of lysosomes. Other carboxypeptidases such as carboxypeptidase Y and pancreatic carboxypeptidase B also inactivated PA28 without altering its apparent molecular weight. Active PA28 binds to the proteasome to form a protease-activator complex that can be isolated after velocity sedimentation centrifugation through glycerol density gradients. Carboxypeptidase-inactivated PA28 failed to form such a complex, suggesting that the carboxyl terminus of PA28 is required for binding to the proteasome. These results indicate the importance of the carboxyl terminus of PA28 for proteasome activation.",
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