Photoaffinity labeling of the rabbit reticulocyte guanine nucleotide exchange factor and eukaryotic initiation factor 2 with 8-azidopurine nucleotides. Identification of GTP- and ATP-binding domains.
We have covalently modified rabbit reticulocyte polypeptide chain initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) with the 8-azido analogs of GTP (8-N3GTP) and ATP (8-N3ATP). Of the five subunits of GEF, the Mr 40,000 polypeptide binds 8-[gamma-32P]N3GTP, and the Mr 55,000 and 65,000 polypeptides bind 8-[gamma-32P]N3ATP. Both 8-N3GTP and 8-N3ATP specifically label the beta-subunit of eIF-2. Covalent binding of 8-azidopurine analogs to the eukaryotic initiation factors is dependent on UV irradiation. Binding of 8-N3GTP and 8-N3ATP is specific for the guanine- and adenine-binding sites on the protein, respectively. GDP and GTP, but not ATP, inhibit the photoinsertion of 8-N3GTP to the protein. Similarly, ATP, but not GTP, inhibits the photoinsertion of 8-N3ATP. The inclusion of NADP+ in the reaction mixtures also interferes with the binding of 8-N3ATP to GEF. Mg2+ inhibits the binding of the 8-azido analogs of GTP and ATP to both eIF-2 and GEF, whereas EDTA stimulates the photoinsertion of these nucleotides. Identical results are obtained when the binding of GTP and ATP to these proteins, in the presence of Mg2+ or EDTA, is estimated by nitrocellulose membranes. In enzymatic assays, 8-N3GTP supports the activity of eIF-2 and GEF, indicating that the interaction of 8-N3GTP is catalytically relevant.
Dholakia
J N
JN
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
Francis
B R
BR
Haley
B E
BE
Wahba
A J
AJ
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000031:analogs & derivatives; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D015852:Eukaryotic Initiation Factor-2 / Q000097:blood*; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism* / Q000031:analogs & derivatives; D007700:Kinetics; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
C021961:8-azidoguanosine triphosphate; D000345:Affinity Labels; D001386:Azides; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; C017040:8-azidoadenosine 5'-triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate