Purification of free eukaryotic initiation factors eIF-4A and eIF-4D on cibacron blue F3G-A

Mast, C. van der; Voorma, Harry O.

doi:10.1016/0005-2787(80)90161-6

Cited by 12 publications

References 21 publications

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Two Hypusine‐Containing Proteins Identified by Metabolic Labeling in Chick Embryo Fibroblasts

Dou

Chen

1989

J Chinese Chemical Soc

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Incubation of various cultured mammalian cells under growth‐stimulatory condition with [3H]putrescine or [3H]spermidine results in a labeling of an 18, 000‐dalton protein (Cell, 1982, 29, 791; BBA, 1983, 756, 395). The labeling is due to post‐translational conversion of a lysine residue to hypusine residue, using the butylamino moiety derived from spermidine. In order to search for an abundant source for the purification of this protein, we have examined possible existence of this hypusine‐containing 18, 000‐dalton protein (hyp‐18k) in chick embryos by the metabolic labeling method. Metabolic labeling of chick embryo fibroblasts, prepared from the Day 11 embryos, by [3H]putrescine resulted in two prominently labeled protein bands. Mr=18, 000 and 20, 000, as shown by sodiun dodecyl sulfate‐polyacrylamide gel electrophoresis and fluorography. Two‐dimensional gel analysis indicated that the labeled 20, 000‐dalton protein had a pI value of 5.5 and the labeled 18, 000‐dalton protein exhibited isoform structures with pI values ranging from 4.6 to 5.1. Peptide map analysis showed that these two proteins are similar but not identical. Both labeled proteins contained radioactive hypusine residue and exhibited strong binding to Cibacron Blue dye. The time course of the metabolic labeling of these two proteins, however, differed dramatically. The labeling of the 18, 000‐dalton protein appeared early and continued to increase 24 h after serum stimulation. In contrast, the labeling of the 20, 000‐dalton protein became prominent only after much longer period of incubation.

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