The guanine nucleotide exchange factor (GEF) from rabbit reticulocytes which consists of five subunits will be further purified (Proc. Natl. Acad. Sci. U.S.A. 83, 6746, (1986)) in order to study its interaction with eukaryotic chain initiation factor 2 (eIF-2). By using fluorescent probes covalently attached to eIF- 2, the molecular interaction of eIF-2, GEF and nucleotides will be monitored. The intrinsic fluorescence of GEF-bound NADPH will also be used as a probe for the interaction of GEF with eIF-2, GTP, GDP and GTP analogs and Met-tRNAf. The role of NADP/NADPH in regulating nucleotide exchange and its effect on protein synthesis will be examined. We will also determine which subunits of elF-2 and GEF are responsible for protein: protein interaction and nucleotide binding. The phosphorylation of GEF under conditions of the nucleotide exchange reaction and its effect on GEF activity will be studied. The binding of the eukaryotic polypeptide chain initiation factors 4A, 4B, and 4F to poly(l,N6 -ethenoadenylic acid) will be investigated by fluorescence spectroscopy. Competition experiments will allow us to determine the relative affinity of these proteins for mRNA cap analogs and different triplets. The effect of ATP, Mg2+, and polyamines on these reactions will be determined. We will also label eIF-4F with fluorescent probes in order to monitor the cooperative interactions of eIF-4F, messenger RNA and eIF-4B. The role of eIF-4D and Ca2+ in protein synthesis will be investigated. We will quantitate the binding of Ca2+ to eIF-4D. We will also examine the activation of transglutaminase by elF-4D and calmodulin. A mouse Friend erythroleukemic cell lysate will be utilized to evaluate the roles of elF-4D, Ca2+, and calmodulin in regulating protein synthesis. An Artemia cDNA library which was constructed in the lambda gtll expression vector will be screened with synthetic oligonucleotide probes and antibodies specific for the alpha and beta subunits of Artemia elF-2. The isolated clones will be characterized by restriction mapping, nucleotide sequencing, and their derived amino acid sequence. The sequencing of the genomic clones for

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025451-11
Application #
3273035
Study Section
Biochemistry Study Section (BIO)
Project Start
1978-09-01
Project End
1993-01-31
Budget Start
1990-02-01
Budget End
1991-01-31
Support Year
11
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Mississippi Medical Center
Department
Type
Schools of Medicine
DUNS #
928824473
City
Jackson
State
MS
Country
United States
Zip Code
39216
Singh, L P; Denslow, N D; Wahba, A J (1996) The interaction of rabbit reticulocyte guanine nucleotide exchange factor eIF-2B with chain initiation factor 2: studies with N-ethylmaleimide and trypsin. Biochem Biophys Res Commun 223:604-11
Singh, L P; Denslow, N D; Wahba, A J (1996) Modulation of rabbit reticulocyte guanine nucleotide exchange factor activity by casein kinases 1 and 2 and glycogen synthase kinase 3. Biochemistry 35:3206-12
Aroor, A R; Singh, L P; Wahba, A J (1995) Hexamethylene bisacetamide-induced differentiation of Friend virus-transformed murine erythroleukemia cells is associated with parallel changes in casein kinase II and guanine nucleotide exchange factor activities. Exp Hematol 23:1204-11
Singh, L P; Wahba, A J (1995) Allosteric activation of rabbit reticulocyte guanine nucleotide exchange factor activity by sugar phosphates and inositol phosphates. Biochem Biophys Res Commun 217:616-23
Singh, L P; Aroor, A R; Wahba, A J (1995) Inhibition of rabbit reticulocyte guanine nucleotide exchange factor activity by heparin and its reversal by polyamines. Biochem Biophys Res Commun 212:1007-14
Aroor, A R; Denslow, N D; Singh, L P et al. (1994) Phosphorylation of rabbit reticulocyte guanine nucleotide exchange factor in vivo. Identification of putative casein kinase II phosphorylation sites. Biochemistry 33:3350-7
Singh, L P; Arorr, A R; Wahba, A J (1994) Phosphorylation of the guanine nucleotide exchange factor and eukaryotic initiation factor 2 by casein kinase II regulates guanine nucleotide binding and GDP/GTP exchange. Biochemistry 33:9152-7
Singh, L P; Aroor, A R; Wahba, A J (1994) Translational control of eukaryotic gene expression. Role of the guanine nucleotide exchange factor and chain initiation factor-2. Enzyme Protein 48:61-80
Jung, J E; Karoor, V; Sandbaken, M G et al. (1994) Utilization of selenocysteyl-tRNA[Ser]Sec and seryl-tRNA[Ser]Sec in protein synthesis. J Biol Chem 269:29739-45
Hille, M B; Dholakia, J N; Wahba, A et al. (1990) In-vivo and in-vitro evidence supporting co-regulation of translation in sea-urchin eggs by polypeptide initiation factors, pH optimization, and mRNAs. J Reprod Fertil Suppl 42:235-48

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