Partial purification of a novel N-ethylmaleimide-activated translational inhibitor from adult rat brain.
A translational inhibitor that is activated by N-ethylmaleimide treatment can be found in the postmicrosomal fraction prepared from the brain of adult rats, but it is almost undetectable in the same fraction prepared from suckling animals. The inhibitor is thermolabile and remains in the supernatant fraction after precipitation at pH 5. During the purification procedure, the inhibitor in its unactivated state binds to the anion exchanger (diethylaminoethyl-cellulose) but not to the cation exchanger (phosphocellulose). Treatment with N-ethylmaleimide increases inhibitor affinity for the cation exchanger, and this chromatographic step purifies the inhibitor by 143-fold. Both the thermolabile nature and the behavior of the inhibitory activity during the different steps of the purification procedure suggest that this activity is most probably due to a protein. Although the addition of initiation factor 2 reverses the inhibition of protein synthesis in the presence of ATP and Mg2+, the inhibitor does not phosphorylate any of the initiation factor subunits "in vitro," which indicates that it does not contain any intrinsic protein kinase activity. However, its presence in both a crude and a purified preparation of a kinase of the alpha subunit of a brain eukaryotic initiation factor increases the phosphorylation of the alpha subunit of the initiation factor. The mechanism of action of this inhibitor is discussed.
Departamento Investigación, Hospital Ramón y Cajal, Madrid, Spain.
A
A
Alcázar
M E
ME
Martín
A
A
García
J L
JL
Fando
M
M
Salinas
D000375:Aging / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects*; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014020:Tissue Extracts / Q000494:pharmacology*; D019892:eIF-2 Kinase
D015852:Eukaryotic Initiation Factor-2; D014020:Tissue Extracts; D011494:Protein Kinases; D019892:eIF-2 Kinase; D005033:Ethylmaleimide