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The role of eukaryotic initiation factor 2 during the metabolic depression associated with estivation

Julian L. Pakay^1,*, Andrew A. Hobbs¹, Scot R. Kimball² and Michael Guppy¹

¹ Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
² Department of Cellular and Molecular Physiology, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA

View larger version (56K): [in a new window]   Fig. 1. The partial predicted amino acid sequence of Helix aspersa eIF2, deduced from translation of the H. aspersa eIF2 cDNA sequence, aligned with the predicted sequences of yeast (Saccharomyces cerevisiae) and human eIF2. Conserved residues are indicated with an asterisk. The multiple alignment was performed manually after comparing each of the pairwise sequence alignments made using the program ALIGN by FASTA (University of Virginia, Charlottesville, VA, USA).

View larger version (14K): [in a new window]   Fig. 2. (A) Representative western blot showing varying amounts of eIF2–FLAG(P) using the immunoblot procedure described in the Materials and methods. The film of the immunoblot was scanned, and densitometric analysis was performed using the program NIH Image 1.62. (B) Representative standard curve of density versus amount of eIF2–FLAG(P). Similar standards were run on all western blots to aid quantification.

View larger version (36K): [in a new window]   Fig. 3. Representative western immunoblots showing the time course of phosphorylation of endogenous eIF2 with HCR (heme controlled repressor kinase). Post-mitochondrial supernatant was isolated from H. aspersa hepatopancreas (A) or from N. sutor liver (B) and incubated with HCR in the presence of 40 µmol l-1 ATP. Aliquots were removed immediately prior to the addition of HCR (0 min) and at 20 min, 40 min and 60 min after the addition of HCR. Proteins were separated using SDS–PAGE, transferred to nitrocellulose membranes then probed with anti-eIF2(P).

View larger version (22K): [in a new window]   Fig. 4. Representative western immunoblots of post-mitochondrial supernatant either unphosphorylated or phosphorylated with HCR (heme controlled repressor kinase). (A) Helix aspersa hepatopancreas. (B) Neobatrachus sutor liver. Two identical samples from an individual were analysed, one sample was left untreated (–HCR) and the other was phosphorylated (+HCR). Samples from the same individual are shown as pairs on the figure. Proteins were separated using SDS–PAGE, transferred to nitrocellulose membranes and probed with anti-eIF2(P). The comparison between samples incubated with HCR (+HCR) and without HCR (–HCR) was used to determine the ratio of phosphorylated to unphosphorylated eIF2 in both the awake and estivating states. The bands corresponding to eIF2(P) have been quantified and are summarised in Table 1.