Purification and characterization of guanine nucleotide-exchange factor, eIF-2B, and p37 calmodulin-binding protein from calf brain.
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 alpha subunit of initiation factor 2 under standard sodium dodecyl sulfate electrophoresis conditions.
Alcázar
A
A
Departamento de Investigación, Hospital Ramón y Cajal, Madrid, Spain.
Martín
M E
ME
Soria
E
E
Rodríguez
S
S
Fando
J L
JL
Salinas
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification*; D002417:Cattle; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D015852:Eukaryotic Initiation Factor-2 / Q000302:isolation & purification; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification*; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D002148:Calmodulin-Binding Proteins; D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D010750:Phosphoproteins; D011506:Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; C094726:p37 calmodulin-binding protein, Bos taurus; D017346:Protein Serine-Threonine Kinases