Isolation and characterization of an 18 kDa hypusine-containing protein from cultured NB-15 mouse neuroblastoma cells.
An 18 kDa protein can be metabolically labeled by [3H]putrescine or [3H]spermidine in various mammalian cells. The labeling is due to a post-translational modification of one lysine residue to hypusine using the aminobutyl moiety derived from spermidine. In view of the lack of knowledge of the function of this spermidine-modified protein, we decided to use the radioactivity associated with the [3H]spermidine-labeled 18 kDa protein as a tracer to develop a simple procedure for purifying this protein from cultured cells. We first screened more than 15 different affinity adsorbents for their ability to bind the labeled 18 kDa protein. This approach enabled us to develop a four-step procedure to purify the labeled 18 kDa protein from NB-15 mouse neuroblastoma cells. The procedure, including a Cibacron Blue column, an omega-aminooctyl-agarose, a Sepharose G-50, and a Mono Q column, resulted in an 800-fold purification of the labeled 18 kDa protein. Two-dimensional gel analysis of fractions enriched in the labeled 18 kDa protein revealed (i) the presence of isoforms of hypusine-containing 18 kDa protein, with pI values ranging from 4.7 to 5.2, and (ii) the presence of an additional labeled protein with an apparent molecular mass of 22 kDa and a pI value of 5.0. The labeling intensity of the 22 kDa protein, however, was less than 5% of that of the 18 kDa protein. Peptide map analysis, using the V-8 proteinase digestion method, indicated that the 18 kDa hypusine-containing protein obtained from NB-15 cells was similar to eukaryotic initiation factor 4D isolated from rabbit reticulocytes.
D000327:Adsorption; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D008239:Lysine / Q000031:analogs & derivatives* / Q000302:isolation & purification; D051379:Mice; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D009447:Neuroblastoma / Q000032:analysis*; D010448:Peptide Initiation Factors / Q000302:isolation & purification; D011119:Polynucleotides; D016601:RNA-Binding Proteins; D011817:Rabbits; D012156:Reticulocytes / Q000032:analysis; D014407:Tumor Cells, Cultured; D000097574:Eukaryotic Translation Initiation Factor 5A
D008239:Lysine; D009363:Neoplasm Proteins; D010448:Peptide Initiation Factors; D011119:Polynucleotides; D016601:RNA-Binding Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine