WITT, T.L.*; MENZE, M.A.; HAND, S.C.; Louisiana State University, Baton Rouge: Isolation and Characterization of AMP-activated Protein Kinase from Embryos of Artemia franciscana

Metabolic depression during diapause in embryos of the brine shrimp, A. franciscana, is characterized by a high AMP:ATP ratio. AMP-activated protein kinase (AMPK) is known to act as an ultra sensitive sensor of the cellular energy status in mammalian species due to its stimulation allosterically by AMP and covalently via an upstream kinase (LKB1). To explore the potential role of AMPK in cell stasis during diapause, we have enriched a homologue of AMPK from encysted embryos of A. franciscana. Although kinase activity is difficult to detect in crude homogenates, it can be consistently measured in a 2.5-7.0% polyethylene glycol (PEG) fraction (0.063 � 0.012 pmoles Pi incorporated per mg protein per min, mean � SD) as assayed with SAMS peptide, an AMPK-specific substrate. Specific activity is increased 8-fold when the PEG fraction is eluted from a DEAE anion exchange column. Western blots of the PEG fraction and DEAE eluent show enrichment using a phospho-AMPK antibody against the highly conserved catalytic site of human AMPK. Pretreatment of the sample with lambda phosphatase prior to electrophoresis eliminates detection of the AMPK band. The apparent molecular weight of the catalytic subunit for A. franciscana AMPK is 60 kDa, which compares favorably with commercial enzyme from rat liver (63 kDa). Elevated AMPK activity due to phosphorylation can be measured in vitro; AMPK in the PEG fraction is 3.4 times more active than parallel samples dephosphorylated with lambda phosphatase. Efforts are in progress to compare AMPK levels between diapause and post-diapause embryos, as well as to amplify the gene for the catalytic subunit from A. franciscana cDNA by RT-PCR. (supported by NIH grant 1-RO1-GM071345-01)