The two forms of the beta-subunit of initiation factor-2 from reticulocyte lysates arise from proteolytic degradation.
Dholakia and Wahba (J. Biol. Chem. (1987) 262, 10164-10170) have reported that preparations of purified initiation factor-2 (eIF-2) from rabbit reticulocytes contain two forms of the beta-subunit. These forms differ in their apparent molecular weights as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and are accordingly termed beta H (heavy, the slower-migrating species, apparent Mr = 54,300) and beta L (light, the faster-migrating species, apparent Mr = 53,100). We confirm that two forms of eIF-2 beta are present in such preparations, but present evidence that the beta L is generated from beta H during the isolation procedure. Crude reticulocyte lysates contain only the beta H species as judged from immunoblotting of reticulocyte proteins resolved by SDS-PAGE using an antiserum against eIF-2 beta. The beta L species appears after the ammonium sulphate fractionation step used early in the purification procedure, but is not apparent if a cocktail of proteinase inhibitors is included in the buffers used during the purification, indicating that it is a proteolytic degradation product generated during the isolation procedure. Cleveland mapping failed to reveal any differences between the two species. Both the beta H and the beta L forms are phosphorylated by casein kinase-2, and, as judged by one- and two-dimensional peptide mapping, at identical sites in each species. Since casein kinase-2 phosphorylates serine-2 in eIF-2 beta, the beta L form must still contain the N-terminal region and is presumably produced by limited proteolysis at the carboxyl terminus of the beta-subunit.
Price
N T
NT
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Nakielny
S F
SF
Clark
S J
SJ
Proud
C G
CG
D000818:Animals; D015153:Blotting, Western; D047388:Casein Kinases; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011480:Protease Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits
D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D011480:Protease Inhibitors; D011506:Proteins; D011494:Protein Kinases; D047388:Casein Kinases; D010450:Endopeptidases