title: Purification procedures determine the proteasome activation properties of REG gamma (PA28 gamma).
abstract: The proteasome activation properties of recombinant REG gamma molecules depend on purification procedures. Prior to ammonium sulfate precipitation recombinant REG gamma activates the trypsin-like catalytic subunit of the proteasome; afterwards it activates all three catalytic subunits. The expanded activation specificity is accompanied by reduced stability of the REG gamma heptamer providing support for the idea that a "tight" REG gamma heptamer suppresses the proteasome's chymotrypsin-like and postglutamyl-preferring active sites. In an attempt to determine whether REG gamma synthesized in mammalian cells also exhibits restricted activation properties, extracts were prepared from several mammalian organs and cell lines. Surprisingly, endogenous REG gamma was found to be largely monomeric. In an alternate approach, COS7 cells were cotransfected with plasmids expressing FLAG-REG gamma and REG gamma. The expressed FLAG-REG gamma molecules were shown to form oligomers with untagged REG gamma subunits, and the mixed oligomers preferentially activated the proteasome's trypsin-like subunit. Thus, REG gamma molecules synthesized in mammalian cells also exhibit restricted activation properties.
authors.affiliation: Department of Biochemistry, University of Utah School of Medicine, 50 N Medical Drive, Salt Lake City, UT 84132, USA.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Gao
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Gregory
authors.initials: G
authors.lastname: Pratt
authors.forename: Sherwin
authors.initials: S
authors.lastname: Wilk
authors.forename: Martin
authors.initials: M
authors.lastname: Rechsteiner
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D001324:Autoantigens; D019556:COS Cells; D002522:Chlorocebus aethiops; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004789:Enzyme Activation; D005590:Fractional Precipitation; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D001324:Autoantigens; C099558:Ki antigen; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000645:Ammonium Sulfate