Abstract

This project is a collaboration with Prof. Rolf Ziegler of the Institute for Zoology, Martin Luther University, Halle-Wittenberg, Germany, and of the Department of Biochemistry and Center for Insect Science at the University of Arizona, Tucson. The work has been supported by NIH grants AI 26905 and GM 29238, and has resulted in two collaborative publications with the NTLF. This research furthers the understanding of the interactions between ligands and their receptors, specifically as applied to insects. It examines the regulation of energy metabolism in Manduca sexta insects by adipokinetic hormone (M-AKH). Peptides of the AKH family control the mobilization of energy stores in times of need. In molting and in wandering larvae of M. sexta AKH mobilizes fat body lipids and carbohydrates, the latter by activating glycogen phosphorylase, in similarity with glucagon stores in mammals. In initial work, the tritium labelled M. sexta M-AKH (pGlu-Leu-Thr-p-3H-Phe-Thr-Ser-Ser-Trp-Gly-NH2) was synthesized at very high specific activity, and was fully active in a bioassay. It was used in a filtration based binding assay to characterize the M-AKH from the fat body of M. sexta. Recently, single amino acid replacement analogs of M-AKH were tested for activity in receptor binding assays. Amino acids were replaced by Ala and by D-analogs. In addition, an extended M-AKH and analogs containing photoaffinity labels were tested. All analogs had reduced activity. All the peptides that had enough activity to allow a full dose response curve reached the same maximal activity as native M-AKH. The use of analogs, in which L-Phe4 was replaced by Ala or by D-Phe and L-Thr3 replaced by D-Thr, as competitors led to improved binding of M-AKH in our competitive receptor binding assays. In the bioassay an inactive concentration of Ala4 M-AKH increased the activity of a half optimal conc entration of native M-AKH. The well-characterized tritium labelled M-AKH of high specific activity is central to these studies.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
3P41RR001237-16S1
Application #
6220456
Study Section
Project Start
1998-08-01
Project End
2000-07-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
16
Fiscal Year
1999
Total Cost
Indirect Cost

  Institution

Name
Lawrence Berkeley National Laboratory
Department
Type
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720

  Publications

Singh, Navneet; Moody, Alan R; Zhang, Bowen et al. (2017) Age-Specific Sex Differences in Magnetic Resonance Imaging-Depicted Carotid Intraplaque Hemorrhage. Stroke 48:2129-2135
Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Ikemoto, Noriaki et al. (2005) Probing a putative dantrolene-binding site on the cardiac ryanodine receptor. Biochem J 387:905-9
Wang, Huimin; Shimizu, Eiji; Tang, Ya-Ping et al. (2003) Inducible protein knockout reveals temporal requirement of CaMKII reactivation for memory consolidation in the brain. Proc Natl Acad Sci U S A 100:4287-92
Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Bhat, Manjunatha B et al. (2002) Identification of a dantrolene-binding sequence on the skeletal muscle ryanodine receptor. J Biol Chem 277:34918-23
Westler, William M; Frey, Perry A; Lin, Jing et al. (2002) Evidence for a strong hydrogen bond in the catalytic dyad of transition-state analogue inhibitor complexes of chymotrypsin from proton-triton NMR isotope shifts. J Am Chem Soc 124:4196-7
Tomizawa, M; Wen, Z; Chin, H L et al. (2001) Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid. J Neurochem 78:1359-66
Than, C; Morimoto, H; Williams, P G et al. (2001) Preparation, NMR characterization, and labeling reactions of tritiated triacetoxy sodium borohydride. J Org Chem 66:3602-5
Saljoughian, M; Williams, P G (2000) Recent developments in tritium incorporation for radiotracer studies. Curr Pharm Des 6:1029-56
Cianci, C; Yu, K L; Dischino, D D et al. (1999) pH-dependent changes in photoaffinity labeling patterns of the H1 influenza virus hemagglutinin by using an inhibitor of viral fusion. J Virol 73:1785-94
Palnitkar, S S; Bin, B; Jimenez, L S et al. (1999) [3H]Azidodantrolene: synthesis and use in identification of a putative skeletal muscle dantrolene binding site in sarcoplasmic reticulum. J Med Chem 42:1872-80

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