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Purification and Characterization of Guanine Nucleotide‐Exchange Factor, eIF‐2B, and p37 Calmodulin‐Binding Protein from Calf Brain
Author(s) -
Alcázar A.,
Martín M. E.,
Soria E.,
Rodríguez S.,
Fando J. L.,
Salinas M.
Publication year - 1995
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1995.65020754.x
Subject(s) - guanine nucleotide exchange factor , calmodulin , biochemistry , guanine , gtp' , chemistry , nucleotide , protein subunit , gel electrophoresis , ternary complex , chromatography , biology , microbiology and biotechnology , signal transduction , gene , enzyme
Eukaryotic initiation factor 2B, or guanine nucleotide‐exchange factor, has been purified for the first time from the brain by a novel procedure that allows the purification of initiation factor 2 as well and uses a salt wash postmicrosomal supernatant as starting material. The procedure includes a three‐part chromatographic step in heparin‐Sepharose and in SP‐5PW and diethylaminoethyl‐5PW ion‐exchange high‐performance chromatographies. The purification of the factors was followed by measuring activity in the guanine nucleotide‐exchange assay and the capacity of initiation factor 2 to form a ternary complex with the initiation form of methionyl‐tRNA and GTP. The method yields guanine nucleotide‐exchange factor (75%) and highly purified initiation factor 2 (>95%), which are separated in the last step. The exchange factor from the brain is a multimeric protein with five subunits of molecular masses of 82, 65, 52, 42, and 30 kDa; it stimulates ternary complex formation in the presence of GDP, and this activity is inhibited by N ‐ethylmaleimide. A 37‐kDa protein that copurifies with initiation factors is characterized in this study as a new calmodulin‐binding protein (p37); it is highly phosphorylated by casein kinase activities and can comigrate with the α subunit of initiation factor 2 under standard sodium dodecyl sulfate electrophoresis conditions.