title: Purification and characterization of truncated human AMPK alpha 2 beta 2 gamma 3 heterotrimer from baculovirus-infected insect cells.
abstract: AMP-activated protein kinase (AMPK) is considered an important target for treatment of type II diabetes and the metabolic syndrome. The muscle-specific isoform of the regulatory gamma-subunit, gamma 3, within the context of AMPK alpha 2 beta 2 gamma 3 complex, is involved in glucose and fat metabolism in skeletal muscle. In an effort to identify gamma 3-specific activators of AMPK, we have produced truncated human recombinant AMPK alpha 2 beta 2 gamma 3 (hu alpha 2 beta 2 gamma 3(trunc)) for biochemical characterization. Infection of insect cells with three baculoviral stocks encoding the individual subunits resulted in soluble expression of a stable hu alpha 2 beta 2 gamma 3(trunc) heterotrimeric complex. Co-expression of the three subunits was essential for solubility since the individual protein components, when expressed separately, were almost completely insoluble. The hu alpha 2 beta 2 gamma 3(trunc) heterotrimer was purified to apparent homogeneity from baculovirus-infected insect cells in a 1:1:1 stoichiometric complex. The hu alpha 2 beta 2 gamma 3(trunc) heterotrimer had significant circular dichroism signal that was lost as a function of temperature, implying that the purified protein was folded. The hu alpha 2 beta 2 gamma 3(trunc) complex was capable of binding AMP and ATP, although the heterotrimer showed preference for AMP, in particular, as seen by thermal denaturation circular dichroism analyses. Further experiments showed that the truncated complex bound ZMP (AICAR-monophosphate) albeit with much lower affinity than AMP. To investigate whether there were isoform-specific differences in the nucleotide binding affinities, a well-characterized truncated mammalian alpha 1 beta 2 gamma 1 (m alpha 1 beta 2 gamma 1(trunc)) equivalent of hu alpha 2 beta 2 gamma 3(trunc) was also purified. The gamma 1 and gamma 3 isoforms showed comparable nucleotide binding affinities and solution behavior properties.
authors.affiliation: Protein Science Department, Schering-Plough Research Institute, 320 Bent St, Cambridge, MA 02141, USA. lata.ramanathan@spcorp.com
authors.forename: Lata
authors.initials: L
authors.lastname: Ramanathan
authors.forename: Payal R
authors.initials: PR
authors.lastname: Sheth
authors.forename: Polina
authors.initials: P
authors.lastname: Ogas
authors.forename: Li
authors.initials: L
authors.lastname: Xiao
authors.forename: Hung V
authors.initials: HV
authors.lastname: Le
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000737:chemistry / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011487:Protein Conformation; D016415:Sequence Alignment; D018411:Spodoptera / Q000166:cytology
chemical_list: D007527:Isoenzymes; C526181:PRKAA2 protein, human; C526183:PRKAB2 protein, human; C526225:PRKAG3 protein, human; D055372:AMP-Activated Protein Kinases