title: Assessment of cytokine-modulated proteasome activity.
abstract: This chapter presents two methods for assessment of proteasome function. The first is a modification of the standard fluorogenic peptide cleavage assay which takes into account the effect of ATP on proteasome activity. This method is described in both its macro and high throughput micro-assay forms. The second is the Proteasome Constitutive Immuno-Subunit (active site) ELISA or ProCISE method. ProCISE is a modification of active site directed probe analysis and allows for convenient differentiation between active constitutive and immuno-subunits. While the utility of measuring proteasome activity and its relationship to cytokine action and inflammation are clear, the assessment and interpretation is not always straightforward. Therefore, we also discuss the pitfalls of the standard fluorogenic assay, particularly in the interpretation of results obtained, and the advantages of the newer, ProCISE assay.
authors.affiliation: Onyx Pharmaceuticals, 249 Grand Avenue South, San Francisco, CA, 94080, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Kirk
authors.forename: Saul R
authors.initials: SR
authors.lastname: Powell
authors.forename: Edmund J
authors.initials: EJ
authors.lastname: Miller
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D020134:Catalytic Domain; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry; D016207:Cytokines / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D007118:Immunoassay; D007700:Kinetics; D008163:Luminescent Measurements; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000032:analysis* / Q000378:metabolism; D021122:Protein Subunits / Q000032:analysis* / Q000378:metabolism; D059748:Proteolysis; D013050:Spectrometry, Fluorescence; D014020:Tissue Extracts / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000906:Antibodies; D015853:Cysteine Proteinase Inhibitors; D016207:Cytokines; D005456:Fluorescent Dyes; D010455:Peptides; D021122:Protein Subunits; D014020:Tissue Extracts; C067713:lactacystin; D000255:Adenosine Triphosphate; D006735:Horseradish Peroxidase; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine