Assays of proteasome activity in relation to aging.
Proteasomes play a major role in intracellular protein turnover. They exist in cells in several different molecular forms including 20S proteasomes, 26S proteasomes and PA28-20S proteasome complexes. In this study we have compared the properties of these purified proteasome complexes to try to design assays that will distinguish between the different complexes (26S proteasome, 20S proteasome, PA28-20S proteasome) in cell extracts. Although the different purified complexes were found to have differences in stability, and in their sensitivity to low concentrations of SDS and salt, the results suggest that it is not straightforward to assay selectively for each type of complex in cell extracts. The relative contribution of different proteasome complexes varies in different cell types and there may be other proteases present which hydrolyse the chosen substrate. Proteasome assays carried out under defined conditions allow comparisons of activity in cell extracts as a function of age, but separation by gel filtration on a Superose 6 column was found to be a useful method for determining the level of different proteasome related complexes.
Rivett
A Jennifer
AJ
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK. j.rivett@bristol.ac.uk
Bose
Suchira
S
Pemberton
Alexander J
AJ
Brooks
Paul
P
Onion
David
D
Shirley
David
D
Stratford
Fiona L L
FL
Forti
Katia
K
D000375:Aging / Q000502:physiology*; D000818:Animals; D002478:Cells, Cultured; D002850:Chromatography, Gel / Q000379:methods; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism; D051381:Rats
D009097:Multienzyme Complexes; D011506:Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex