Structural analysis of the messenger RNA cap-binding protein. Presence of phosphate, sulfhydryl, and disulfide groups.
The messenger RNA cap-binding protein (CBP) was isolated from human erythrocyte, rabbit erythrocyte, and rabbit reticulocyte lysate by affinity chromatography on 7-methylguanosine 5'-triphosphate-Sepharose. The specific activity of binding to capped oligonucleotides was similar for the human erythrocyte and rabbit reticulocyte CBPs. Isoelectric focusing of human and rabbit preparations revealed that each was composed of up to five species. The pI values of human and rabbit CBPs ranged from 5.7 to 6.5. The predominant form in erythrocytes had a pI of 6.3 while in reticulocytes, two major species, having pI values of 5.9 and 6.3, were present. Labeling of rabbit reticulocytes with [32P]orthophosphate revealed that the pI 5.9 but not the pI 6.3 form contained phosphate. All of the phosphate was found in phosphoserine residues. The amino acid compositions of human erythrocyte and rabbit reticulocyte CBPs were quite similar. Both proteins had 7 tryptophanyl and 6 cysteinyl residues. Labeling with [1-14C]iodoacetic acid under native and denaturing conditions provided evidence that 2 of the cysteinyl residues are present in the reduced form and 4 in disulfide bridges. Species of CBP with faster or slower electrophoretic mobilities could be generated by treatment of the protein either with O2 in the presence of a catalyst or with dithiothreitol. The predominant form of the untreated protein migrated between these two forms.
D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D004220:Disulfides / Q000032:analysis; D006801:Humans; D007525:Isoelectric Focusing; D008722:Methods; D010084:Oxidation-Reduction; D010442:Peptide Chain Initiation, Translational; D010710:Phosphates / Q000032:analysis; D039381:RNA Cap-Binding Proteins; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis
D002352:Carrier Proteins; D004220:Disulfides; D010710:Phosphates; D039381:RNA Cap-Binding Proteins; D013438:Sulfhydryl Compounds