Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5 prime -triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site

Journal Article · Tue Jun 12 04:00:00 EDT 1990 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00475a016· OSTI ID:5229493

Chavan, A J; Rychlik, W; Watt, D S; Rhoads, R E ^[1]; Blaas, D; Kuechler, E ^[2]

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1. Univ. of Kentucky, Lexington (USA)
2. Institut fuer Biochemie der Universitaet Wien, Vienna (Austria)

Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, ({gamma}-{sup 32}P)-{gamma}-(((4-benzoylphenyl)methyl)amido)-7-methyl-GTP. A second probe was synthesized that was an azidophenyltyrosine derivative of m{sup 7}GTP (({sup 125}I)APTM), the monoanhydride of m{sup 7}GDP with ({sup 125}I)-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodophenyl)propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 {mu}M was determined for ({sup 125}I)APTM, which is comparable to the published values for m{sup 7}GTP. A third probe, an azidophenylglycine derivative of m{sup 7}GTP (({sup 32}P)APGM), the monoanhydride of m{sup 7}GDP with ({sup 32}P)-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike ({sup 32}P)BPM and ({sup 125}I)APTM, however, ({sup 32}P)APGM labeled eIF-4E{sup *} approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E{sup *} consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.

• OSTI ID: 5229493
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 29:23; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMALS
AROMATICS
AZAARENES
AZIDES
BENZOPHENONE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CARBOXYLIC ACIDS
CHEMICAL PREPARATION
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DERIVATIZATION
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENZYME INHIBITORS
ERYTHROCYTES
GUANOSINE
HETEROCYCLIC COMPOUNDS
HYDROXY ACIDS
INTERMEDIATE MASS NUCLEI
INTERNAL CONVERSION RADIOISOTOPES
IODINE 125
IODINE ISOTOPES
ISOTOPES
KETONES
LIGHT NUCLEI
MAMMALS
MAN
MATERIALS
MOLECULAR BIOLOGY
NITROGEN COMPOUNDS
NUCLEI
NUCLEOPROTEINS
NUCLEOSIDES
NUCLEOTIDES
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PRIMATES
PROTEIN STRUCTURE
PROTEINS
PURINES
RABBITS
RADIOISOTOPES
RETICULOCYTES
RIBOSIDES
SYNTHESIS
TRANSCRIPTION FACTORS
TYROSINE
VERTEBRATES